基因调控溶血磷脂酸作用通路抑制卵巢癌细胞生长的实验研究
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摘要
研究背景和目的
卵巢癌由于深居盆底,缺乏早期症状体征和有效的诊治方法,5年生存率一直徘徊在30%左右,是死亡率最高、危害最大的妇科恶性肿瘤。但是,如果卵巢癌能得到早期诊治,Ⅰ期患者5年生存率可达90%以上。因此,研究卵巢癌的早期诊断方法和除手术、化疗及放疗以外的其它有效新疗法是卵巢癌研究的热点和关键。
溶血磷脂酸(lysophosphatidic acid,LPA)是具有细胞间信号转导作用的脂类小分子物质,主要通过G蛋白受体介导的信号转导通路发挥多种生物学效应。近年来研究发现卵巢癌细胞能分泌LPA使患者血浆和腹水中LPA水平明显升高,90%以上Ⅰ期卵巢癌患者血浆中LPA明显高于健康人群,Ⅱ-Ⅳ期患者诊断敏感性达100%,其敏感性和特异性均明显高于目前临床常用的肿瘤标志物CA125,LPA被认为是一个非常有价值的用于卵巢癌诊断、病情检测及预后判断的生物标志物。LPA有3个特异性受体,分别是Edg2、Edg4和Edg7,属于内皮分化基因家族(endothelial differentiation gene family,Edgs)成员。Edg2主要在正常卵巢组织中表达,与卵巢癌关系不大。Edg4和Edg7在卵巢癌细胞中表达水平明显升高,认为与卵巢癌发生发展有关。LPA与受体结合后可活化四条细胞信号转导通路,促进卵巢癌细胞的增殖、生存、迁移和血管生成,抑制细胞凋亡,与卵巢癌的发生、发展、浸润、转移等密切相关。LPA的代谢主要通过人磷脂磷酸水解酶-3(human lipid phosphate phosphohydrolase-3,hLPP3)的降解而失活,增强hLPP3基因表达可以降低细胞外LPA水平,降低卵巢癌细胞克隆形成及抑制癌细胞在动物体内的生长。因此,有理由推测通过影响LPA代谢、封闭LPA受体或干扰LPA信号传导系统及阻断LPA的一系列级联反应有可能成为卵巢癌治疗的新途径。
LPA及其受体与卵巢癌发生发展关系的研究是近年来妇科肿瘤领域的新课题,还有许多未知的问题需要进一步的研究和阐明。比如LPA作为卵巢癌诊断指标尚未被公认和用于临床,LPA及其受体促使卵巢癌发生发展的机制还不十分清楚,阻断LPA信号通路是否能抑制卵巢癌细胞生长的研究还没见报道。本研究拟通过测定LPA及其受体在卵巢癌患者血清和组织中的表达,探讨LPA作为卵巢癌早期诊断指标的可行性,验证LPA及其受体在卵巢癌发生发展中的重要作用;采用基因导入增强卵巢癌细胞中hLPP3基因表达,通过体内外实验探讨hLPP3对LPA的调节及对卵巢癌细胞生长的抑制效应;利用RNAi技术沉默LPA受体Edg4和Edg7基因表达,探讨阻断LPA信号转导通路对卵巢癌细胞生长的抑制作用。本实验通过不同途径调控LPA的作用通路,研究LPA及其受体在卵巢癌的发生发展进程中的作用及机制,为卵巢癌基因治疗寻找有效的方法和治疗靶点。
第一部分LPA及其受体在卵巢癌患者血清和组织中的表达及意义
材料和方法
1.血清LPA测定
1.1研究对象:研究组:卵巢上皮性性癌患者50例,包括原发性卵巢癌患者30例(Ⅰ期10例、中晚期即Ⅱ～Ⅳ期20例)和复发性卵巢癌患者20例,组织学类型为浆液性囊腺癌37例,粘液性囊腺癌13例;良性对照组:良性卵巢上皮性肿瘤20例(浆液性囊腺瘤13例、粘液性囊腺瘤6例、子宫内膜样肿瘤1例);正常对照组:20例正常妇女。所有病例均经术后病理确诊。
1.2研究方法:采用生化法检测各组患者血清中LPA水平,同时用放射免疫方法测定血清中CA125水平,并将两者进行比较,探讨LPA对卵巢上皮性癌早期诊断的意义。
2.卵巢癌组织中Edg4和Edg7蛋白检测
2.1标本来源:为郑州大学第一附属医院病理科的存档腊块。研究组:上皮性癌组织48例(浆液性囊腺癌30例,粘液性囊腺癌11例,子宫内膜样癌7例)。临床分期为Ⅰ期12例,Ⅱ期16例,Ⅲ期18例,Ⅳ期2例;组织学分级为:G_1级14例,G_2级16例,G_3级18例。其中有淋巴结转移者22例,无淋巴结转移者26例;腹水≥500ml的30例,腹水＜500ml的18例。对照组:交界性上皮性肿瘤10例,良性上皮性肿瘤10例,正常卵巢组织12例。所有研究对象术前均未接受放疗或化疗,临床及病理资料完整。
2.2研究方法:采用免疫组化SP法检测卵巢上皮性肿瘤组织和正常卵巢组织中Edg4和Edg7蛋白的表达情况,并探讨这两种蛋白的表达与卵巢上皮性癌临床分期、组织学分级、淋巴转移及腹水量等临床和病理参数的关系。
3.统计学处理
计量资料采用单因素方差分析、最小显著差(LSD)检验;分类资料采用x~2检验和Kruskal-Wallis法秩和检验;相关性分析采用Spearman等级相关分析,以α=0.05为检验水准,在SPSS13.0上完成。
结果
1.各期卵巢上皮性癌患者血清LPA水平均明显升高,与正常妇女和良性卵巢上皮性肿瘤患者相比,其差异均有统计学意义(P＜0.001);Ⅰ期卵巢上皮性癌患者血清LPA水平也明显升高,与正常妇女和良性卵巢上皮性肿瘤患者相比差异有显著性(P＜0.001),其水平接近中晚期患者(P＞0.05);良性卵巢上皮性肿瘤患者血清中LPA表达水平较正常妇女略高,但差别无统计学意义(P＞0.05)。原发性卵巢癌Ⅱ～Ⅳ期组和复发组患者血清CA125水平明显高于正常妇女组和良性卵巢上皮性肿瘤组,其差别有统计学意义(P＜0.001);Ⅰ期卵巢上皮性癌患者血清CA125水平轻度升高,与良性卵巢上皮性肿瘤患者和正常妇女相比无明显差异(P＞0.05)。
2.血清LPA水平升高用于检测卵巢上皮性癌的敏感性为96%、特异性为90%、假阳性率10%、假阴性率4%,阳性预测值96%、阴性预测值90%,血清CA125分别为72%、80%、20%、28%、90%、53.3%,与CA125相比LPA有较高的敏感性、较低的假阴性率及较高的阴性预测值(P＜0.05)。LPA用于检测Ⅰ期卵巢上皮性癌的敏感性为90%、特异性90%、假阳性率10%、假阴性率10%、阳性预测值81.8%,阴性预测值94.7%,而CA125分别为30%、80%、20%、70%、42.9%和69.6%,LPA对Ⅰ期上皮性卵巢癌检测的敏感性、阳性预测值及阴性预测值高于CA125,假阴性率低于CA125(P＜0.05)。LPA检测原发性卵巢上皮性癌Ⅰ期、Ⅱ～Ⅳ期、复发性卵巢癌患者的约登指数分别为:0.8、0.85和0.9,而CA125检测的约登指数分别为:0.1、0.6和0.65,提示LPA是检测早期上皮性卵巢癌的良好指标。
3.溶血磷脂酸受体Edg4、Edg7蛋白在卵巢上皮性肿瘤组织中的阳性表达率分别为恶性91.7%和95.8%、交界性80%和70%、良性20%和30%,而在正常卵巢组织中阳性表达率仅为16.7%和16.7%,提示Edg4、Edg7在正常卵巢组织中呈低表达状态;在良性卵巢上皮性肿瘤组织中的表达较正常组织中稍高,但其差异无统计学意义(P＞0.05),而在交界性卵巢上皮性肿瘤和卵巢上皮性癌组织中Edg4、Edg7的表达均显著高于正常卵巢组织及良性上皮性肿瘤组织(P＜0.001),多数上皮癌组织中着色呈“+++”,提示两种受体蛋白在卵巢上皮性癌组织中呈高表达状态。
4.卵巢上皮性癌组织中Edg4和Edg7蛋白在Ⅲ、Ⅳ期的阳性表达明显高于Ⅰ、Ⅱ期;G_2、G_3级组织中两种蛋白的表达明显高于G_1级组织;有淋巴结转移的癌组织中两种蛋白的表达明显高于无淋巴结转移的癌组织;腹水＞500ml的癌组织中两种蛋白的表达明显高于腹水＜500ml的癌组织,比较结果差异均有统计学意义(P＜0.05)。提示在卵巢上皮性癌组织中,Edg4和Edg7的高表达与癌组织学分级、临床分期、淋巴结转移及腹水量增加有关。
5.Edg7蛋白在卵巢上皮性癌组织中的表达阳性率略高于Edg4蛋白,但无统计学差异(P＞0.05);Edg4和Edg7蛋白的表达状态趋势一致,经相关性分析两者的表达呈正相关(P＜0.05)。
小结
1.卵巢上皮性癌患者血清中LPA水平明显升高,其敏感性高于血清CA125,假阴性率低于CA125,尤其是在早期卵巢癌患者更明显。提示LPA有望成为卵巢癌早期诊断新的敏感标记物。
2.溶血磷脂酸受体蛋白Edg4与Edg7在卵巢上皮性癌组织中的表达水平明显升高,并与上皮性癌的临床分期、组织学分级、淋巴转移和腹水量呈正相关。提示Edg4和Edg7可能在卵巢上皮性癌的发生发展、浸润和转移中具有重要作用。
3.Edg4和Edg7受体蛋白在卵巢上皮性癌组织中表达程度呈正相关,推测二者在卵巢上皮性癌的发生发展中具有协同作用。
4.LPA及其受体Edg4和Edg7在卵巢癌患者血清及组织中的高表达等上述结果为下一步的研究即基因调控LPA作用通路抑制卵巢癌细胞生长的研究提供了理论和实验依据。
第二部分增强人磷脂磷酸水解酶-3基因表达对卵巢癌细胞生长的抑制作用
材料和方法
1.从胎盘组织中逆转录扩增目的基因
以人胎盘组织总RNA为模板,根据GenBank上的hLPP3基因cDNA编码序列(BC009196)设计引物:上游引物LPP3-1 5'TGGATCCTCCACCATGCAAAACT ACAAGTACGAC 3'(372-392);下游引物LPP3-2 5'CCTCGAGCTACATCAT GTTGTGGTGAT 3'(1288-1307)。采用RT-PCR技术扩增合成的双链DNA,凝胶电泳回收鉴定。
2.将目的基因克隆入克隆及表达质粒载体
将目的基因hLPP3克隆入pGEM-T easy质粒载体,利用α互补和T7/SP6 PCR扩增筛选鉴定重组子pGEM-T-hLPP3;用BamHⅠ和XhoⅠ双酶切重组子pGEM-T-hLPP3和真核表达载体pLenExpress质粒;将双粘的hLPP3片段亚克隆入表达载体pLenExpress中;利用PCR扩增及BamHⅠ和XhoⅠ双酶切筛选鉴定重组子pLenExpress-hLPP3;对插入序列进行DNA序列分析。
3.包装产生表达目的基因的慢病毒
将表达hLPP3基因的表达载体及辅助包装载体应用脂质体协助转染293FT细胞包装病毒,产生带有绿色荧光蛋白(GFP)并表达hLPP3基因的慢病毒颗粒(lentivires),测定病毒滴度。
4.表达目的基因慢病毒体外细胞转染实验
用表达hLPP3的慢病毒转染卵巢癌细胞系SKOV3和OVCAR3细胞(实验组),用空载体包装的慢病毒转染细胞及未转染细胞作为对照组。利用荧光显微镜观察转染阳性率,加入G418筛选稳定表达hLPP3的细胞株;生化法测定转染前后细胞培养上清液中LPA含量变化;采用实时荧光定量PCR测定各组细胞中hLPP3mRNA表达水平;流式细胞仪检测各组卵巢癌细胞的细胞周期和凋亡率改变。
5.裸鼠体内移植瘤实验
用SKOV3和OVCAR3细胞及稳定表达hLPP3基因的SKOV3和OVCAR3细胞接种裸鼠,构建卵巢癌裸鼠移植瘤动物模型,观察移植瘤生长情况,并测量移植瘤大小;将移植瘤作病理切片检查,实时荧光定量PCR测定移植瘤组织中hLPP3mRNA表达,流式细胞仪检测肿瘤细胞周期变化和细胞的凋亡率。
结果
1.成功扩增出目的基因
利用RT-PCR从胎盘组织中扩增出目的基因hLPP3,电泳条带约在936bp。
2.成功构建表达目的基因的重组真核表达载体
PCR产物与pGEM-Teasy载体连接后,转化JM109大肠杆菌,筛选鉴定得到正确重组子pGEM-T-hLPP3。进行亚克隆后,用BamHⅠ和XhoⅠ双酶切对重组质粒进行鉴定,得到pLenExpress-hLPP3重组真核表达载体。对重组子pLenExpress-hLPP3插入片段DNA序列测序,结果与设计完全一致。
3.获得高浓度的表达目的基因的慢病毒并对卵巢癌细胞系具有高转染效率。
用重组表达载体和包装载体包装慢病毒,其滴度分别如下:表达hLPP3基因的慢病毒:3.1×10~4 IU/ml;空载体包装的慢病毒:2.7×10~7 IU/ml。表达hLPP3的慢病毒转染卵巢癌SKOV3细胞阳性率为92%,高于转染OVCAR3细胞的阳性率(76%)。
4.实验组细胞中hLPP3mRNA表达水平明显增加
表达hLPP3的慢病毒转染卵巢癌细胞系后,经实时荧光定量PCR检测实验组卵巢癌细胞的hLPP3 mRNA相对表达量比空载体组及无转染对照组明显增加(P＜0.05);而无转染对照组与空载体组比较无统计学意义(P＞0.05)。SKOV3与OVCAR3之间hLPP3 mRNA相对表达量比较无统计差异(P＞0.05)。
5.实验组卵巢癌细胞培养上清液中LPA含量明显降低
慢病毒转染细胞后不同时间测定上清液中LPA水平,结果显示实验组LPA水平较对照组明显下降,且随着转染时间延长,LPA含量有进一步下降趋势(P＜0.05),存在时间-效应关系。两种卵巢癌细胞系SKOV3和OVCAR3之间LPA水平比较无统计学差异(P＞0.05)。
6.实验组细胞凋亡率明显增加
实验组卵巢癌细胞凋亡率明显增加,由转染前0.1±0.034%到转染后增加为30.50±2.12%,两者比较有统计学意义(P＜0.05)。实验组细胞凋亡率明显高于两个对照组(P＜0.05)。实验组卵巢癌细胞周期改变不明显(P＞0.05)。
7.成功构建卵巢癌及转基因卵巢癌裸鼠移植瘤模型
用卵巢癌细胞系及表达目的基因的卵巢癌细胞株皮下注射局部生长成瘤,实验组移植瘤体积及重量均小于空载体组和无转染对照组,差异有统计学意义(P＜0.05)。SKOV3细胞形成的移植瘤生长情况与OVCAR3细胞的相似,两者比较无统计学意义(P＞0.05)。
8.体内移植瘤实验结果
实验组裸鼠移植瘤组织中hLPP3 mRNA的相对表达量明显高于空载体组和无转染对照组,差异有统计学意义(P＜0.05);而对照组与空载体组比较无统计学意义(P＞0.05)。SKOV3与OVCAR3之间hLPP3 mRNA相对表达量比较无统计学意义(P＞0.05)。实验组裸鼠移植瘤细胞的凋亡率为16.5%,而空载体组细胞凋亡率为1.26%,无转染对照组细胞凋亡率则为0.10%,实验组细胞的凋亡率明显高于两个对照组,差异有统计学意义(P＜0.05)。各组细胞周期无明显变化(P＞0.05)。
小结
1.成功构建并产生出表达hLPP3的真核表达载体及慢病毒颗粒,并对卵巢癌细胞系SKOV3和OVCAR3细胞转染效率高且容易鉴定,获得了稳定表达hLPP3基因的卵巢癌细胞株。
2.体外实验,表达hLPP3的慢病毒转染卵巢癌细胞后,细胞内hLPP3 mRNA水平明显升高,细胞上清液的LPA水平明显降低,细胞凋亡率明显增加。提示增强hLPP3基因表达能有效降低LPA水平,促进细胞凋亡,其机制可能通过水解胞外的LPA或/和减少细胞LPA释放,抑制LPA的促肿瘤效应。
3.成功建立了卵巢癌裸鼠移植瘤模型及稳定高表达hLPP3基因的卵巢癌裸鼠移植瘤模型,后者体内移植瘤细胞生长速度明显减慢,hLPP3 mRNA水平和细胞凋亡率增加。提示增强hLPP3基因表达在体内也能有效抑制卵巢癌细胞生长,促进细胞凋亡,其机制可能与体外一样通过降低LPA水平发挥抑瘤效应。
4.体内外实验均证明增强hLPP3基因表达能抑制卵巢癌细胞生长,促进肿瘤细胞凋亡,提示调控hLPP3基因可能成为卵巢癌基因治疗的新靶点。
第三部分RNA干扰沉默溶血磷脂磷酸受体Edg4和Edg7基因表达对卵巢癌生长的抑制效应
材料和方法
1.设计合成Edg4和Edg7siRNA序列及发夹样DNA寡核苷酸单链
依据GenBank中Edg4基因(NM_004720)和Edg7基因(NM_012152)的序列,遵循siRNA设计原则进行设计及筛选,选择确定19个碱基的siRNA靶序列。各设计2对包含BamHⅠ和XhoⅠ酶切位点的靶向Edg4和Edg7发夹样DNA寡核苷酸,并合成2对编码短发夹RNA序列的DNA单链。设计合成无关序列DNA链作为对照。
2.构建目的基因的克隆载体和真核表达载体
将发夹样DNA序列退火成双链DNA,克隆入pGEM-T Easy载体,利用α互补和T7/SP6引物经PCR扩增筛选鉴定重组子pGEM-T-siEdg4和pGEM-T-siEdg7;用BamHⅠ和XhoⅠ限制性内切酶双酶切重组子和siRNA真核表达载体pRNAT-U6.1/lenti;将双粘发夹样siRNA片段亚克隆入pRNAT-U6.1/lenti中;利用通用引物PCR筛选及双酶切鉴定重组子;对插入序列进行DNA序列分析。
3.包装产生表达目的基因的慢病毒
将测序正确的siRNA表达载体和辅助包装载体应用脂质体包裹转染293FT细胞包装病毒,产生表达不同siRNA序列的慢病毒颗粒,并测定病毒滴度。
4.体外细胞转染实验
用表达不同目的基因的慢病毒转染入卵巢癌细胞系SKOV3细胞,用G418筛选稳定表达siRNA的细胞株。体外实验分为三个对照组和四个实验组,分别为未转染对照组C1,转染空载体包装的病毒对照组(C2),转染无关序列siRNA包装的病毒对照组(C3),转染siEdg4病毒组(T1),转染siEdg7病毒组(T2),共转染siEdg4病毒和siEdg7病毒组(T3),以及共转染siEdg7病毒和表达hLPP3的病毒组(T4)。利用荧光显微镜观察SKOV3细胞中的转染率;用生化法测定细胞培养上清液中LPA含量变化;用实时荧光定量PCR方法检测细胞Edg4和Edg7mRNA表达水平;用Western Blot测定Edg4和Edg7蛋白表达情况;用流式细胞仪检测转染细胞的细胞周期和凋亡率变化。
5.裸鼠体内移植瘤实验
用SKOV3细胞及稳定表达不同siRNA的SKOV3细胞接种裸鼠,构建表达不同基因的卵巢癌裸鼠移植瘤模型,在体内研究沉默Edg4和Edg7基因表达对卵巢癌细胞生长的影响及其作用机制。测量移植瘤大小和重量,比较各组移植瘤生长情况;实时荧光定量PCR测定瘤细胞中两种受体mRNA表达情况;免疫组化SP法检测移植瘤中Edg4和Edg7蛋白表达情况;流式细胞仪测定移植瘤细胞周期及细胞凋亡率变化。
结果
1.Edg4和Edg7siRNA靶序列筛选及发夹DNA单链设计合成
对候选序列通过同源序列检索和进一步优选,最后确定212-230位(GACCATCGGCTTCTTCTAT)和323-341位(GACCAATCTGCTGGTCATA)为Edg4-siRNA靶序列;277-295位(GCTGGAATTGCCTATGTAT)和697-715位(GTCTTGTCTCCGCATACAA)为Edg7-siRNA靶序列;(TACGCTGACTTGATTGTTC)为无关对照序列。
Edg4发夹样siRNA寡核苷酸序列
Position in gene sequence:212-230
BamHI Target sequence:sense Hairpin Target sequence:antisense XhoIAl 11 5'-GGATCC GACCATCGGCTTCTTCTAT TTCAAGAGA ATAGAAGAAGCCGATGGTC TTTTTT CTCGAGA -3'Al 12 3'-ACCTAGG CTGGTAGCCGAAGAAGATA AAGTTCTCT TATCTTCTTCGGCTACCAG AAAAAA GAGCRC-5'
BamHI Target sequence:sense Hairpin Target sequence:antisense XhoIAl 21 5'-GGATCC GACCAATCTGCTGGTCATA TTCAAGAGA TATGACCAGCAGATTGGTC TTTTTT CTCGAGA -3'Al 22 3'- ACCTAGG CTGGTTAGACGACCAGTAT AAGTTCTTCT ATACTGGTCGTCTAACCAG AAAAAA GAGCTC -5'
Position in gene sequence:323-341
Edg7发夹样siRNA寡核苷酸序列
Position in gene sequence:697-715
BamHI Target sequence:sense Hairpin Target sequence:antisense XhoIA211 5'-GGATCC GTCTTGTCTCCGCATACAA TTCAAGAGA TTGTATGCGGAGACAAGAC TTTTTT CTCGAGA-3'A212 3'-ACCTAGGCAGAACAGAGGCGTATGTT AAGTTCTCT AACATACGCCTCTGTTCTG AAAAAA GAGCTC-5'
BamHI Target sequence:sense Hairpin Target sequence:antisense XhoIA221 5'-GGATCC GCTGGAATTGCCTATGTAT TTCAAGAGA ATACATAGGCAATTCCAGC TTTTTT CTCGAGA-3'A222 3'-ACCTAGGCGACCTTAACGGATACATA AAGTTCTCT TATGTATCCGTTAAGGTCG AAAAAA GAGCTC -5'
Position in gene sequence:277-295
无关序列对照发夹样siRNA寡核苷酸序列
BamHI位点Target sequence:sense Hairpin Target sequence:antisense XhoI位点C1 5'- GGATCC TACGCTGACTTGATTGTTC TTCAAGAGA GAACAATCAAGTCAGCGTA TTTTTT CTCGAGA-3'C2 3'-ACCTAGG ATGCGACTGAACTCAAG AAGTTCICT CTTGTTAGTTCAGTCGCAT AAAAAA GAGCTC-5'
2.成功克隆表达目的siRNA的克隆载体和真核表达载体
不同序列的发夹DNA退火产物与pGEM-T Easy连接后,转化JM109,筛选鉴定后得到重组子pGEM-T-siEdg4、pGEM-T-siEdg7和pGEM-T-siC。亚克隆入表达质粒pRNAT-U6.1/lenti后,用通用引物对重组表达质粒进行鉴定,得到真核表达载体pRNAT-U6.1/lenti-siEdg4、pRNAT-U6.1/lenti-siEdg7和pRNAT-U6.1/lenti-siC。对重组子进行测序,结果与设计完全一致。
3.表达不同目的基因慢病毒滴度
采用脂质体Lipofectaminc~(TM) 2000包裹构建的siRNA表达载体及包装载体,转染293FT细胞,产生表达不同siRNA的慢病毒颗粒(Lentivirus),测定其滴度如下:针对Edg4基因的siRNA Lentivirus—siEdg4/lenti病毒3.2×10~7 IU/ml;针对Edg7基因的siRNA Lentivirus—siEdg7/lenti病毒4.1×10~7 IU/ml;无关对照的siRNA Lentivirus—siC/lenti病毒4.3×10~7 IU/ml;空载体包装的Lentivirus—pRNAT-U6.1/Lenti病毒2.7×10~7 IU/ml。
4.重组慢病毒能高效转染卵巢癌细胞系
含有不同siRNA的病毒转染卵巢癌SKOV3细胞,培养48h后采用荧光显微镜观察,结果显示除了未转染组细胞,其余各组SKOV3细胞的胞核和胞浆内均有大量绿色荧光信号,细胞转染阳性率均在90%以上(92%～95%),说明重组慢病毒可以高效转染卵巢癌SKOV3细胞,而且目的siRNA的插入不影响慢病毒对细胞的转染力。
5.慢病毒体外细胞转染结果
5.1实时荧光定量PCR方法分别测定各组细胞中Edg4和Edg7 mRNA的表达水平,结果显示:转染siEdg4和siEdg7慢病毒的T1组和T2组卵巢癌SKOV3细胞中Edg4和Edg7的mRNA表达水平明显降低,与对照组之间比较差异有统计学意义(P＜0.05)。而3个对照组卵巢癌细胞中Edg4和Edg7 mRNA表达均较高,相互之间比较差异无统计学意义(P＞0.05)。2个共转染实验组mRNA表达更低,与其它单独转染组比较差异有统计学意义(P＜0.05)。
5.2 Western Blot检测结果显示3个对照组细胞中Edg4和Edg7两种蛋白表达水平较高,分子量约40KD,而实验组中相应Edg4和Edg7蛋白表达水平明显降低。提示siRNA良好的靶向性。
5.3各实验组转染后细胞上清液中LPA含量明显下降,且随着转染时间的延长,细胞上清液中的LPA含量降低更明显(P＜0.05)。而3个对照组转染前后LPA含量变化不明显(P＞0.05),实验组与对照组相比较,差异有统计学意义(P＜0.05)。
5.4转染siRNA慢病毒对卵巢癌细胞SKOV3细胞周期影响主要表现为实验组的细胞S期比例降低,G_2期比例升高,差异有显著性(P＜0.05),而3个对照组的细胞周期无明显变化(P＞0.05)。转染后实验组细胞凋亡率明显增加,与对照组相比差异有统计学意义(P＜0.05);其中两个共转染实验组较单独转染实验组细胞凋亡率更高,3个对照组细胞凋亡率无明显变化(P＞0.05)。
6.裸鼠体内移植瘤实验结果
6.1实验组裸鼠移植瘤生长缓慢:各组卵巢癌细胞接种入裸鼠皮下后生长成瘤。各实验组移植瘤体积小于对照组,差异有统计学意义(P＜0.05),两个共转染组移植瘤体积明显小于单转染组(P＜0.05),但两个共转染组之间比较差异无统计学意义(P＞0.05)。处死裸鼠后的移植瘤重量实验组明显低于对照组,差异有统计学意义(P＜0.05)。
6.2对裸鼠皮下移植瘤组织进行Edg4和Edg7蛋白的免疫组化SP法染色,阳性表达主要表现为胞膜和胞浆着色,实验1、3组Edg4蛋白表达水平明显降低,实验2、3、4组Edg7蛋白表达明显降低,与对照组比较有统计学差异(P＜0.05)。而对照组之间比较无统计学意义(P＞0.05)。
6.3实验组移植瘤组织中两种受体mRNA的相对表达量明显低于对照组,差异有统计学意义(P＜0.05),而对照组之间比较无统计学意义(P＞0.05)。
6.4实验组裸鼠移植瘤细胞的凋亡率分别为(16.75±1.24)%、(15.80±1.35)%、(25.42±3.24)%和(24.63±2.76)%,3个对照组细胞凋亡率分别为(1.68±0.03)%、(1.76±0.04)%和(0.67±0.002),各实验组细胞的凋亡率明显高于3个对照组细胞,比较结果有统计学意义(P＜0.05)。
小结
1.成功设计并构建了真对Edg4和Edg7的真核表达载体pRNAT-U6.1/lenti-siEdg4和pRNA-U6.1/lenti-siEdg7,并包装生产出表达Edg4和Edg7siRNA序列的慢病毒颗粒,两种病毒均能高效转染卵巢癌细胞系SKOV3细胞并持久表达,获得了稳定表达siEdg4和siEdg7的SKOV3细胞株。
2.体外实验表明,RNAi沉默Edg4或Edg7基因表达能靶向抑制卵巢癌细胞内相应的Edg4或Edg7 mRNA及蛋白表达,降低细胞培养上清液中LPA水平,阻滞细胞周期进程,增加癌细胞的凋亡率,同时沉默两个受体作用更明显。提示封闭Edg4或/和Edg7基因表达均能有效抑制LPA的促肿瘤作用,Edg4和Edg7在卵巢癌的发生发展中具有协同作用。
3.沉默Edg7基因表达和增强hLPP3基因表达能更明显的降低细胞外LPA水平,增加肿瘤细胞凋亡,hLPP3对Edg7mRNA表达及细胞周期无明显影响。提示封闭Edg7和增强hLPP3基因表达对降低细胞外LPA水平和促进肿瘤细胞凋亡有协同作用,hLPP3主要通过降低LPA水平发挥作用。
4.成功构建了稳定表达siEdg4和siEdg7的卵巢癌裸鼠移植瘤模型,体内实验表明封闭Edg4或/和Edg7及增强hLPP3基因在体内也能明显抑制两种受体的mRNA和蛋白表达,促进细胞凋亡,抑制肿瘤细胞生长,发挥和体外相似的抑制肿瘤效应。
5.沉默Edg4或/和Edg7基因表达及增强hLPP3基因表达在体内外均能有效抑制卵巢癌细胞系SKOV3细胞中LPA的促肿瘤作用,提示对LPA受体及hLPP3基因的调控可能成为卵巢癌的基因治疗的有效靶点和新方法。该研究为验证LPA及其受体与卵巢癌发生、发展的关系提供了实验和理论依据,也为利用RNAi技术进行卵巢癌基因治疗研究奠定了基础并提供了新思路。
全文结论
1.卵巢上皮性癌息者血清中LPA水平明显升高,其敏感性高于血清CA125,尤其是在早期卵巢癌患者更明显。提示LPA有望成为新的敏感的卵巢上皮性癌早期诊断的生物学标记物。
2.溶血磷脂酸受体蛋白Edg4与Edg7在卵巢上皮性癌组织中的表达明显升高,并与上皮性癌的临床分期、组织学分级、淋巴结转移和腹水量呈正相关。提示LPA及其受体Edg4和Edg7可能在卵巢上皮性癌的发生发展、浸润和转移中具有重要作用。
3.成功包装出表达hLPP3的慢病毒对卵巢癌细胞系SKOV3和OVCAR3细胞转染效率高,转染后在体内外均能增强hLPP3基因表达,有效降低LPA水平,促进细胞凋亡,抑制卵巢癌细胞生长。其机制可能通过水解胞外的LPA或/和减少细胞LPA释放,抑制LPA的促肿瘤效应。
4.成功设计并构建了表达Edg4和Edg7siRNA的慢病毒,能高效转染卵巢癌细胞系SKOV3细胞并持久表达。体内外实验表明,RNA干扰沉默Edg4或Edg7基因表达均能明显抑制癌细胞内相应的Edg4和Edg7 mRNA及蛋白表达,降低细胞培养上清液中LPA水平,阻滞细胞周期进程,增加癌细胞的凋亡率,抑制细胞生长,同时沉默Edg4和Edg7具有协同作用。
5.同时沉默Edg7基因表达及增强hLPP3基因表达在体内外均能有效抑制卵巢癌细胞系SKOV3细胞中LPA的促肿瘤作用,二者具有协同作用。
6.该研究为验证LPA及其受体与卵巢癌发生、发展的关系提供了实验和理论依据,也为利用RNAi技术进行卵巢癌基因治疗研究奠定了基础并提供了新思路。对LPA受体Edg4、Edg7及hLPP3基因的调控可能成为卵巢癌的基因治疗的有效靶点和新方法。
7.有关表达hLPP3基因慢病毒的构建和体内外转染实验、利用RNA干扰技术沉默LPA受体Edg4、Edg7抑制卵巢癌细胞生长实验以及同时沉默Edg7及增强hLPP3基因表达抑制卵巢癌细胞生长的研究内容,国内外未见类似报道。
Background and Aims
Ovarian cancer is one of the most common cancers in females and the leading cause of death from gynecological malignancy in the world.Because the ovaries are located in the deep bottom of pelvis and early-stage ovarian cancer is generally asymptomatic,and lack of effective diagnosis and treatment methods,approximately 75%of patients present in advanced stages(ⅢorⅣ) and lost the chance for radical operation treatment.The ovarian cancer calls also are easy to appear resistance to chemotherapy.The 5-year survival in patients with advanced stage ovarian cancer is only 30%.But,the 5-year survival in patients with stageⅠis more than 90%. Therefore,study on the methods of early diagnosis and new effective therapy is the focus of ovarian cancer research recently.
Lysophosphatidic acid(LPA) is the simplest phospholipid,and mediates multiple biological functions through the LPA receptors which have been proposed to mediate LPA signal transduction pathway for multiple biological actions.Recent studies show that LPA is elevated in ascites and plasma of ovarian cancer patients. More than 90%patients with stageⅠovarian cancer have higher levels of LPA than that of healthy women.The sensitivity of LPA to diagnose ovarian cancer in stagesⅡ～Ⅳis 100%,and both of the sensitivity and specificity are higher than CA125 that is a common tumor biomarker used in clinical practice.Therefore,LPA is proposed as a potential and novel biomarker for ovarian cancer diagnosis and prognosis.LPA has three high affinity receptors,Edg2,Edg4 and Edg7,which belong to the members of the endothelial differentiation gene family(Edgs).Edg2 expresses stably in normal ovarian epithelial cells,and has low expression in ovarian cancer cells.Edg4 and Edg7 are overexpressed in the ovarian cancer epithelial cells.The results above suggest that the high level expression of Edg4 and Edg7 maybe related to tumor-promoting effect of LPA in ovarian cancer development and progression.The combination of LPA with Edg4 and Edg7 can activate four cell signaling transduction pathways and promote ovarian cancer cell proliferation,survival,differentiation, migration,angiogenesis and metastasis,and closely relate to the cancer occurrence, development,progression,invasion,metastasis and resistance to chemotherapeutic agents.
Human lipid phosphate phosphohydrolase-3(hLPP3) is a cell surface protein that exhibits ectoenzyme activity and is a number of lipid phosphate phosphohydrolase family(LPPs) that express extensively in cells.It can hydrolyze LPA and decrease the level of extracellular LPA and inhibit the function of LPA. Janos reported that transfecting the ovarian cancer cell line SKOV3 with the vector containing hLPP3 gene could decrease significantly the clon formation of cancer cells, and inhibit cancer cell growth and increase cell apoptosis in vivo.Therefore,it may be a new and effective way to treat ovarian cancer by affecting the metabolism of LPA, blocking LPA receptors,interfering with the signaling transduction pathway of LPA.
The study on the action and relationship between LPA and ovarian cancer is still a new project,and there are many problems that are unclear.Three objectives will be done in this study:1) to explore the feasibility of LPA as a early diagnostic biomarker for ovarian cancer and verify the important action of LPA and its receptors in promoting ovarian cancer development by detecting the LPA level in serum of patients with ovarian epithelial cancer and examining the expression of Edg4 and Edg7 in epithelial ovarian cancer tissues with immunohistochemical method.2) to explore the regulation effect of hLPP3 on LPA and inhibition on ovarian cancer cells growth in vitro and in vivo by enhancing the gene expression of hLPP3 in cancer cells. 3) to explore the inhibition efficacy of blocking LPA signaling transduction pathway by silencing Edg4 and Edg7 with RNAi technology in vitro and in vivo.The aim is to identify the new and effective way and potential targets for early diagnosis and gene therapy of ovarian cancer.
Part 1 Expression and Significance of LPA and its receptors in serum and tumor tissue of patients with ovarian cancer
Material and Methods
1.Detection of serum LPA level
1.1 Patients:Seventy patients with epithelial ovarian tumor were selected in this study,which included 50 malignant cases(37 cases of serous cystadenocarcinoma,13 cases of mucous cystoadenocarcinoma,WHO,2000),including 10 patients with primary early stage(stageⅠ) group,20 with primary advanced stage(stageⅡ～Ⅳ) group(FIGO,2000) and 20 with relapsed ovarian cancer group,the ages of patients were 30～69 years old,and the average was 51.3±5.6 year;20 benign cases(13 cases of serous cystadenoma,6 cases of mucous cystadenoma and 1 case of endometrioid tumor),the range of ages was 24～63 years,the average was 46.7±8.7 years.Twenty normal women were also selected as controls,the ages were 28～70 years,the average was 47.6±9.7 years.All cases were confirmed by pathology after operation.
1.2 Meehods:Levels of serum LPA were detected by biochemistry assay and those of tumor antigen 125(CA125) were measured by radio-immunoassay.Then the two results were compared to evaluate the value of LPA to diagnosis of ovarian cancer, especially to early stage of ovarian cancer.
2 Examination of Edg4 and Edg7 proteins in epithelial ovarian tumor tissues
2.1 Specimens:Sixty-eight specimens of epithelial ovarian tumor paraffin-imbedded and archival tissues and 12 cases of normal ovarian tissues were resected and collected at department of gynaecology and obstetrics between February of 2000 and October of 2004.All specimens were diagnosed by two pathologists and all patients had not accepted the radiotherapy and chemotherapy before operation with complete clinical and pathological information.Eighty samples included normal ovarian tissues (12 cases),benign epithelial ovarian tumors(10 cases),borderline epithelial ovarian tumors(10 cases) and malignant epithelial ovarian cancers(48 cases),while 48 malignant cases were divided into serous cystadenocarcinoma(30 cases),mucous cystoadenocarcinoma(11 cases) and endometrioid carcinoma(7 cases) according to WHO 2000 standard.Their FIGO staging was stageⅠ(12 cases),stageⅡ(16 cases), stageⅢ(18 cases) and stageⅣ(2 cases),their histological staging was G_1 grade(14 cases),G_2 grade(16 cases),G_3 grade(18 cases) according to histological grade.Their subgroups included lymphoid node metastasis(22 cases) and non-lymphoid node metastasis(26 cases),ascites quantity more than 500ml(30 cases) and ascites quantity less than 500ml(18 cases).The patients' ages were from 31 to 60 years old,mean of age was 42.6±4.7 years,and the ages of women with normal ovaries were 34～59,the average age was 45.4±6.3years.
2.2 Methods:The Edg4 and Edg7 protein expression in epithelial ovarian tumor was detected by immunohistochemical assay(streptavidin-biotin -peroxidase complex staining assay).The relationship between expression of Edg4 and Edg7 proteins and the clinical parameters,such as clinical stage,pathological grade,ascites quantity,lymphoid node metastasis,etc.were investigated and analysed.
3 Statistical analysis
The measurement data were presented as mean+SD((?)±s),the comparison of multi-sample means was dealt with using one-way analysis of variance(ANOVA) and least significant difference(LSD) test;numeration data were expressed as n(%),the comparisons of groups were done using contingency table Chi-square test,Fisher's Exact test and Kruskal-Wallis test.Spearman rank correlation analysis was used to analyze the relevance.Analyses were performed using statistics software SPSS13.0 package,and P＜0.05 represented significance.
Results
1.Levels of serum LPA in patients with different stages(Ⅰ-Ⅳ) epithelial ovarian cancer were significantly higher than those in normal women and benign epithelial ovarian tumor(P＜0.001),but serum LPA level of every epithelial carcinoma group had no statistical significance(P＞0.05),such as patients with stageⅠ(early stage) had no difference compared with the advanced patients(stageⅡ～Ⅳ).The same result was obtained from the comparison of normal women and benign cases(P＞0.05) although there was a slight raise in benign cases.CA125 levels in primary ovarian cancer with advanced and relapsed groups were significantly higher than those in early stage group,benign tumor group and normal women group(P＜0.001),but in early stage it was just a little higher than those in benign tumor group and normal women group, then there was no statistic significance(P＞0.05).
2.Serum LPA increase used to diagnose epithelial ovarian carcinoma had sensitivity of 96%、specificity of 90%、false positive rate of 10%、false negative rate 4%、positive predictive value of 96%and negative predictive value of 90%,while those of CA125 were 72%、80%、20%、28%、90%and 53.3%,respectively.These two indexes had differences in sensitivity,false negatiye rate and negative predictive value when used to diagnose epithelial ovarian carcinoma(P＜0.05),but compared with CA125, LPA had higher sensitivity,lower false negative rate and higher negative predictive value.In early stage patients,LPA used to diagnose epithelial ovarian carcinoma of stageⅠhad sensitivity of 90%,specificity of 90%,false positive rate of 10%、false negative rate 10%、positive predictive value of 81.8%and negative predictive value of 94.7%,while those of CA125 were 30%、80%、20%、70%、42.9%and 69.6%,they had difference in sensitivity,false negative rate、positive predictive value and negative predictive value to diagnose primary epithelial ovarian carcinoma of stageⅠ(P＜0.05),and LPA had higher sensitivity、positive predictive value、negative predictive value and lower false negative rate compare with CA125;but used to diagnose primary epithelial ovarian carcinoma of stageⅡ～Ⅳand relapsed groups,all the indexes of CA125 and LPA had no statistic significance(P＞0.05). Youden's index of LPA used to detect primary epithelial ovarian carcinoma of stageⅠ、stageⅡ～Ⅳand relapsed groups were respectively 0.8、0.85 and 0.9,while whose of CA125 were 0.1、0.6 and 0.65.The result indicated that of the two markers used to detect different groups of epithelial ovarian carcinoma,LPA had higher Youden's index than CA125 in full,especially concerning epithelial ovarian carcinoma of stageⅠthe Youden's index had risen to 0.8,it pointed out that LPA would be a satisfactory biomarker index to detect epithelial ovarian carcinoma of early stage.
3.The positive expression rates of Edg4 and Edg7 proteins were 91.7%and 95.8% (malignant),80%and 70%(borderline),20%and 30%(benign),16.7%and 16.7%(normal),it demonstrated that Edg4 and Edg7 were low-expressed in normal ovarian tissues;in benign epithelial ovarian tumors,Edg4 and Edg7 proteins had a little rise over normal tissues,but there was no statistical significance(P＞0.05);but Edg4 and Edg7 proteins expression in borderline and malignant epithelial ovarian tumor was markedly higher than those of normal tissues and benign tumor(P＜0.001), most of the epithelial ovarian carcinoma tissues had evident coloration of(+++),which indicate that these two proteins were high-expressed in epithelial ovarian carcinoma tissues.
4.The expression of Edg4 and Edg7 proteins in tissues of FIGO stageⅢ～Ⅳwas obviously higher than that of FIGO stageⅠ～Ⅱ;the expression of two proteins in the G_1 tissues was lower than that of G_2,G_3 tissues significantly;the expression of Edg4 and Edg7 proteins in tissues with lymphoid node metastasis was higher than that of without lymphoid node metastasis;the expression of two receptor proteins in tissues with ascites quantity more than 500ml was higher than that of the ascites quantity less than 500ml.These differences all had statistical significance(P＜0.05).According to these results,we concluded that the high-expression of Edg4 and Edg7 proteins in epithelial ovarian carcinoma was significantly associated with FIGO stage, pathological grade,ascites quantity and lymphoid node metastasis.
5.The positive expression of Edg7 protein was a little higher than that of Edg4 protein in epithelial ovarian cancer,but there was no significant difference(P＞0.05). The tendency of two proteins expression was positive correlation.(r=0.978,P＜0.05).
Conclusions
1.Serum LPA levels in patients with primary and relapsed epithelial ovarian cancer were significantly higher.The sensitivity and negative predictive value of LPA in diagnosis of epithelial ovarian cancer were superior to CA125,and false positive rate was inferior to CA125,they were more obvious in early stage of ovarian cancer, especially.LPA may be a new potential biomarker for diagnosis before operation and monitoring prognosis and recurrence of epithelial ovarian cancer after operation.
2.The expression of Edg4 and Edg7 proteins in epithelial ovarian cancer had markedly increased,and was significantly associated with the clinical stage, pathological grade,quantity of ascites and lymphoid node metastasis.The results demonstrated that Edg4 and Edg7 may play an important role in occurrence, development,invasion and metastasis of ovarian cancer.
3.The expression degree of Edg4 and Edg7 receptor proteins was positively correlated,which suggests that the two proteins had synergistic effect in the occurrence and development of epithelial ovarian cancer.LPA and its two receptors would become the potential targets to ovarian cancer gene therapy.
4.Above results that the high expression of LPA and its receptors in patients with ovarian cancer have proved basis of theory and experiment for our next studies that regulation to LPA signaling transduction pathway by gene engineering inhibits ovarian cancer cells growth.
Part 2 The inhibition efficacy on ovarian cancer cells growth by enhancing hLLP3 gene expression
Material and Methods
1.Extraction and amplification hLPP3 gene from placenta tissue with RT-PCR
Extracting total RNA from human placenta and synthesizing the hLPP3 cDNA by RT-PCR.According to the hLPP3 eDNA sequence in GenBank(BC009196),the primers were designed and synthesized as LPP3-1 5' TGGATCCTCCA CCATGCAAAACTACAAGTACGAC 3'(372-392);LPP3-2 5' CCTCGAGCT ACATCATGTTGTGGTGAT 3'(1288-1307).The hLPP3 gene was obtained with the technique of RT-PCR by using human placenta tissues RNA as template.The PCR product was retrieved and identified by agar-gel electrophoresis analysis.
2.Construction of recombinant clone and expression plasmids of hLPP3 gene
The PCR product was cloned into pGEM-T easy plasmid(clone vector),thenα-complementation test and T7/SP6 PCR was used to screen the positive clones(pGEM-T-hLPP3),which was digested by restriction enzyme BamHⅠand XhoⅠ,and finally was subcloned into the pLenExpress vector(expression plasmid). Then the recombinants were identified by PCR and BamHⅠand XhoⅠcutting,and the target DNA in the recombinant was sequenced finally.So the eukaryotic expression vector pLenEx- press-hLPP3 was constructed successfully.
3.Pack and producing lentivirus particles which express hLPP3 gene
The 293FT cells were transfected with the eukaryotic expression vector (pLenEx- press-hLPP3) and another two assisted packing vectors(package kit) assisted by Lipofectamine to pack and produce the lentivirus particles which contain and express hLPP3 gene and green fluorescent protein(GFP),then the titers of lentivirus were detected.
4.Cells transfection experiment in vitra
Transfecting the ovarian cancer cell lines SKOV3 and OVCAR3 with recombinant lentivirus.After SKOV3 and OVCAR3 were transfected with lentivirus. Experiment was divided into three groups:experimental group(the cells were transfected by lentiveruses which express hLPP3 gene);blank vector control group(the cells were transfected by lentiviruses which express pLenExpress vector gene) and non-transfection control group(the cells were not transfected by lentivirus). The transfection efficiency was detected by fluorescent microscopy,and the positive ceils were selected and maintained by adding G418 to the cell culture medium.The LPA levels of call culture supernatant before and after transfection were detected by biochemical method,the expression level changes of hLPP3 mRNA were measured by real-time quantitative PCR,and the changes of cell apoptosis and cell cycle were detected by flow cytometry.The non-transfected cells and the cells which were transfected with the lentivirus packed with blank pLenExpress vectors were arranged as controls.
5.Building ovarian cancer animal models and experiment in vivo
The nude mice were inoculated by subcutaneous injection with SKOV3 and OVCAR3 cell lines and the cell lines which express stably hLPP3 gene,and graft tumor animal models were built.The sizes of tumors were measured,the characteristics of tumor were identified by pathological method,the expression levels of hLPP3 mRNA were detected by real-time quantitative PCR,and the cell apoptosis were determined by flow cytometry.
Results
1.Target hLPP3 gene was amplified successfully
The hLPP3 gene was amplified successfully by RT-PCR from human placenta, and the gone band located at about 936bp by agar-gel electrophoresis analysis,which was target gene hLPP3.
2.The recombinant clone and eukaryotic expression vectors were constructed successfully
PCR product(hLPP3 gene) was linked to pGEM-T easy vector and amplified by transforming into competent E coli.JM109,and then the target gene was subcloned to expression vector(pLenExpress),the recombinant eukaryotic expression vectors (pLenExpress-hLPP3) were selected and identified through PCR and BamHⅠand XhoⅠrestrictive enzyme cutting test,and then sent to Shanghai for sequencing.The results of sequencing showed that the DNA sequence inserted into the recombinant pLenExpress-hLPP3 was conformed completely to the design.
3.High titer of lentivirus and high cell transfecting efficiency were obtained
The 293 FT cells were co-transfected by pLenExpress-hLPP3 vector wrapped by Lipofectamine and another two assistant package genes,and the lentiviral particles (lentivirus) which express hLPP3 gene were produced.The concentrations of lentivirus are:lentivirus-hLPP3,3.1×10~7 IU/ml and containing blank vector lentivirus, 2.7×10~7 IU/ml.The cell transfection efficiency of lentivirus-hLPP3 to SKOV3 was 92%,and that to OVCAR3 was 75%,the former is higher than the latter.And the results also showed that the insertion of hLPP3 gene didn't affect the ability of lentivirus to transfect ovarian cancer cells.
4.The mRNA expression level of hLPP3 in experimental ovarian cancer cells increased significantly
After transfection by different lentiviruses,the hLPP3 mRNA level of SKOV3 and OVCAR3 cells in experimental group was higher than those in the two control groups(P＜0.05),and level of hLPP3 mRNA in two control groups were not different(P＞0.05).The different level of hLPP3 mRNA between SKOV3 and OVCAR3 calls had no statistic significance(P＞0.05).
5.The LPA level in experimental group dropped markedly
The LPA levels in supernatant of SKOV3 and OVCAR3 which were transfected with lentivirus-hLPP3 were lower significantly compared with the controls(P＜0.05), and had the tendency of continual decrease accompanying with time progress,and there was a time-effect relationship.The LPA levels between SKOV3 and OVCAR3 cells had no statistic significance.
6.Apoptosis in experimental group was much higher
The apoptosis rate of cancer cells in experimental group increased significantly after transfection(30.50±2.12%vs 0.1±0.03%,P＜0.05).The apoptosis rate in experimental group was markedly higher than two control groups(P＜0.05).The changes of cell cycle were not significant before and after transfection(P＞0.05).
7.The ovarian cancer animal models and the transgenic models were constructed successfully
SKOV3 and OVCAR3 cells and the two cell lines which express hLPP3 gene were inoculated by subcutaneous injection into nude mice and the graft tumor mass were formed in the injection location.The tumor sizes of experimental group were smaller than those of two control groups(P＜0.05),the tumor sizes between SKOV3 and OVCAR3 cell lines were not different(P＞0.05).The tissues of graft tumor were examined by HE staining and the features in microscopy were similar to those of human ovarian cancer tissue.
8.Experimental results in vivo
The expression level of hLPP3 mRNA in transgenic tumor tissue were higher than those in two control groups(P＜0.05),while the comparison between two control groups had no statistic significance(P＞0.05).The expression level of hLPP3 mRNA in SKOV3 and OVCAR3 cells also had no statistic significance(P＞0.05).The apoptosis rates of transgenic tumor cells were significantly higher than that in control tumors (16.5%vs 1.26%and 0.10%,P＜0.05).The changes of cell cycle in each groups were not significant(P＞0.05).
Conclusions
1.The target gene hLPP3 cDNA was obtained successfully and the recombinant eukaryotic expression vector pLenExpress-hLPP3 and lentivirus expressing hLPP3 gene were constructed and produced successfully.The letivirus carrying green fluorescent protein(GFP) could be identified easily and had high transfection efficiency to SKOV3 and OVCAR3.The cell clones that express hLPP3 gene stably had been built and obtained.
2.In vitro,the levels of hLPP3 mRNA in SKOV3 and OVCAR3 cell lines after transfection were significant higher than those before transfection,the concentrations of LPA in cell supematant were significant lower,and the apoptosis rates of cancer cells were significant higher.The results demonstrated that enhancing the expression of hLPP3 gene in ovarian cancer cell lines could decrease LPA level and induce apoptosis of cancer cells effectively,and inhibit the growth of ovarian cancer cells in vitro.The mechanism might be that hLPP3 can inhibit and block the action of LPA by hydrolyzing the extracellular LPA and/or decreasing LPA secretion of cancer cells.
3.The ovarian cancer animal models(nude mice) and the transgenic animal models in which the tumor cells express hLPP3 gene stably were built successfully.The growth of tumors expressing hLPP3 gene was slower than that of non-gene transfection models.The levels of hLPP3 mRNA and apoptosis rate of cancer cells in transgenic models were also higher.The above results show that enhancing the expression of hLPP3 gene in ovarian cancer cells also could inhibit the growth of cancer cells,and induce apoptosis of cancer cells effectively in vivo.The mechanism might be the same as that in vitro.
4.The effects that enhancing hLPP3 gene expression can inhibit the growth of ovarian cancer cells and promote the apoptosis of cancer cells were testified by our experiments in vitro and in vivo.These results demonstrate that hLPP3 gene might become a new target for ovarian cancer and regulating the expression of hLPP3 gene might be a new way ofgene targeting therapy for ovarian cancer.
5.The construction of lentivirus which expresses hLPP3 gene and transfection experiments to ovarian cancer cell lines in vitro and in vivo were not reported domestically or overseas.
Part 3 The inhibiting efficacy on growth of ovarian cancer cells by silencing Edg 4 and Edg 7 using RNA interference
Materials and methods
1.Design and synthesize Edg4 and Edg7 siRNA sequence and hairpin single strand DNA oligonucleotide
According to the Edg4 sequence in GenBank(NM_004720) and Edg7 sequence in GenBank(NM_012152),following the rules of siRNA design,we used the siRNA design and analysis software which were provided in following webpage(http://www. ambion.com/techlib/misc/siRNA_design.html) to design the target oligonucleotides which contain 19 bp.Then the oligonucleotides of target siRNAs were indexed through American National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/Blast) to compare their homology,in order to identify the best siRNA oligonucleotides of Edg4 and Edg7.Afterwards,two pairs of siRNA hairpin single strand DNA oligonucleotides of Edg4,Edg7 and one pair of irrelevant sequence control siRNA oligonucleotide which have palindrome,loop structure and BamHⅠand XhoⅠrestriction enzyme sites were synthesized.
2.Construct clone and eukaryotic expression vectors which express target gene
The hairpin DNAs were annealed to double strands DNA and were cloned into pGEM-T Easy vector.Then,α-complementation test and T7/SP6 PCR primers were used to screen the positive clones(pGEM-T-siEdg4 and pGEM-T-siEdg7);The target DNAs were cut down from pGEM-T-siEdg4 and pGEM-T-siEdg7 by BamHI and XhoI restriction enzyme,and linked with siRNA expression vector (pRNAT-U6.1/lenti);The positive ones(pRNAT-U6.1/lenti-siEdg4 and pRNAT-U6.1/ lenti-siEdg7) were selected by PCR with general primer.Then the target DNAs were sequenced.
3.Pack and produce lentivirus particles which express target siRNA gene
The 293 FT cells were transfected by pRNAT-U6.1/lenti-siEdg4 or pRNAT-U6.1/lenti-siEdg7 or pRNAT-U6.1/lenti-siC or pRNAT-U6.1/lenti vectors and another two assistant package vectors,and the lentiviral particles(lentivirus) containing and expressing target gene and GFP were produced.The titers of lentivirus were measured:
4.Transfection experiments in vitro
Ovarian cancer cell line SKOV3 cells were transfected with the different lentiviruses,and the SKOV3 cells which express different siRNA stably were selected by G418.The experimental groups were divided as follows.The control groups included three subgroups:non-transfection SKOV3 cells,the cells transfected with the lentivirus packed by blank pRNAT-U6.1/lenti vector,and the cells transfected with the lentivirus packed by the irrelevant sequence siRNA vectors.The experimental groups were divided into four subgroups:cells of groupl were transfected with siEdg4/lenti lentivirus,group 2 with siEdg7/lenti lentivirus,group 3 was co-transfected with siEdg4/lenti and siEdg7/lenti lentiviruses,group 4 was co-transfected with siEdg7/lenti lentivirus and the lentivirus expressing hLPP3 gene.The methods used to detect the transfection efficacy of siRNA lentivirus,the levels of Edg4 and Edg7 mRNA,the LPA concentration in supernatant,and the apoptosis of cancer cells before and after transfection were same as those used in part 2.The expression levels of Edg4 and Edg7 proteins were measured by western blotting.
5.Build animal models and experiments in vivo
Building ovarian cancer animal models with 7 kinds different SKOV3 cells(as same as described in 5) and studying the inhibition efficiency and mechanism of silencing Edg4 and Edg7 on growth of ovarian cancer cells in vivo.The methods were same as those used in part 2.The Edg4 and Edg7 proteins in graft tumor tissues were detected by immunohistochemistry SP method.
Results
1.The results of Edg4 and Edg7 siRNA target sequences selection and hairpin single strand DNA design and synthesis
The 20 target siRNA oligonucleotides were obtained.After BLAST search in NCBI database,two target siRNA sequences with 19 bases were ensured for each receptor.The target sequences of Edg4-siRNA were:GACCATCGGCTTCTTCTAT (212-230) and GACCAATCTGCTGGTCATA(323-341).And the target sequences of Edg7-siRNA were GCTGGAATTGCCTATGTAT(277-295) and GTCTTGTCTCCGCATA C AA(697-715).The irrelevant control sequence was TACGCTGACTTGATTGTTC.
Edg4-1,2 hairpin siRNA oligonucleotide sequences as following:
Position in gene sequence:212-230
BamHI Target sequence:sense Hairpin Target sequence:antisense XhoI Al 11 5'-GGATCC GACCATCGGCTTCTTCTAT TTCAAGAGA ATAGAAGAAGCCGATGGTC TTTTTT CTCGAGA -3' Al 12 3'-ACCTAGG CTGGTTAGCCGAAGAAGATA AAGTTCTCT TATCTTCTTCGGCTACCAG AAAAAA GAGCRC-5'
BamHI Target sequence:sense Hairpin Target sequence:antisense XhoI Al 21 5'-GGATCC GACCAATCTGCTGGTCATA TTCAAGAGA TATGACCAGCAGATTGGTC TTTTTT CTCGAGA -3' Al 22 3'- ACCTAGG CTGGITAGACGACCAGTAT AAGTTCTTCT ATACTGGTCGTCTAACCAG AAAAAA GAGCTC -5' Position in gene sequence:323-341
Edg7-1,2 hairpin siRNA oligonueleotide sequences as following:
Position in gene sequence:697-715
BamHI Target sequence:sense Hairpin Target sequence:antisense XhoI A211 5'-GGATCC GTCTTGTCTCCGCATACAA TTCAAGAGA TTGTATGCGGAGACAAGAC TTTTTT CTCGAGA-3' A212 3'-ACCTAGGCAGAACAGAGGCGTATGTT AAGTTCTCT AACATACGCCTCTGTTCTG AAAAAA GAGCTC-5'
BamHI Target sequence:sense Hairpin Target sequence:antisense XhoI A221 5'-GGATCC GCTGGAATTGCCTATGTAT TTCAAGAGA ATACATAGGCAATTCCAGC TTTTTT CTCGAGA-3' A222 3'-ACCTAGGCGACCTTAACGGATACATA AAGTTCTCT TATGTATCCGTTAAGGTCG AAAAAA GAGCTC -5'
Position in gene sequence:277-295
Irrelevant control sequence siRNA oligonucliotide sequence
BamHI位点Target sequence:sense Hairpin Target sequence:antisense XhoI位点C1 5'- GGATCC TACGCTGACTTGATTGTTC TTCAAGAGA GAACAATCAAGTCAGCGTA TTTTTT CTCGAGA-3' C2 3'-ACCTAGG ATGCGACTGAACTCAAG AAGTTCICT CTTGTTAGTTCAGTCGCAT AAAAAA GAGCTC-5'
2.The recombinant clone vector and eukaryotic expression vector which express siRNA were constructed successfully
The hairpin DNAs of siRNA were annealed to double stranded DNA that the molecular weight band of DNA was identified to locate at about 60bp by agarose gel electrophoresis analysis.They were similar to the ones expected.The annealing products were recombined with pGEM-T Easy vector to form pGEM-T-siEdg4 and pGEM-T-siEdg7,which then were transformed into JM109.After being screened and identified by PCR and,the positive clones(pGEM-T- siEdg4 and pGEM-T-siEdg7) were identified.After sub-cloning,the siRNA expression vectors,pRNAT-U6.1/LentisiEdg4 and pRNAT-U6.1/Lenti-siEdg7 were identified by PCR,BamHI XhoI cutting and sequencing.The results showed that the sequences inserted in recombinant expression vectors were same as those designed.
3.Results of different ientivirus titers
The 293FT cells were transfected by siRNA expression vectors with another two package vectors by Lipofectamine,and the siRNA lentiviroural articles were produced. The titers of lentivirus were as below:
siEdg4-1/lenti lentivirus:3.2×10~7IU/ml,siEdg4-2/lenti lentivirus:2.1×10~7IU/m, siEdg7-1/lenti lentivirus:4.1×10~7IU/ml,siEdg7-2/lentivirus:3.1×10~7IU/ml,irrelevant control sequence siC/lenti lentivirus:4.3×10~7IU/ml,blank vector control pRNAT-U6.1/lenti lentivirus:2.7×10~7IU/ml.
4.Recombinant lentiviruses had high transfection efficiency on ovarian cancer cell line SKOV3
A great quantity of GFP can be observed in all transfected SKOV3 cells by fluorescence microscope 48 hours later except the non-transfection cells.The positive cells of all transfected groups were more than 90%(92%～95%).And the positive cell clones were selected by adding G418.The results demonstrated that the siRNA lentiviral particles we produced could transfect SKOV3 cells in high efficiency and the insertion of target siRNA did not affect the ability of lentivirus to transfect ovarian cancer cell line.
5.The results of transfection experiments in vitro
5.1 The mRNA level of Edg4 and Edg7 in relevant experimental groups transfected by the lentiviruses of siEdg4/lenti and siEdg7/lenti was obviously reduced compared with the controls(P＜0.05).The levels of Edg4 and/or Edg7 mRNA in co-transfected groups were lower than single transfection groups(P＜0.05).The mRNA level of Edg4 and/or Edg7 were still high in three control groups and there was no difference among them(P＞0.05).
5.2 There were Edg4 and Edg7 protein bands in control groups showed by western blotting,and the molecular weight was about 40KD.The protein bands of the cells transfected by related siRNA lentivirus was slighter in different experimental groups.
5.3 The LPA concentration in supernatant of experimental groups after transfection decreased significantly in time-dependent manner comparing with that of control groups(P＜0.05).However,there was no difference in control groups(P＞0.05).
5.4 The proportion of S stage cells in cell cycle decreased and that of G2 stage cells increased significantly in experimental groups(P＜0.05).The apoptosis rate of cancer cells in experimental groups were higher than that in control groups(P＜0.05),and the apoptosis in co-transfected groups was much higher.However,the cell cycle had no change and apoptosis was very low in three control groups,and there was no differen
卵巢癌由于深居盆底,缺乏早期症状体征和有效的诊治方法,5年生存率一直徘徊在30%左右,是死亡率最高、危害最大的妇科恶性肿瘤。但是,如果卵巢癌能得到早期诊治,Ⅰ期患者5年生存率可达90%以上。因此,研究卵巢癌的早期诊断方法和除手术、化疗及放疗以外的其它有效新疗法是卵巢癌研究的热点和关键。
溶血磷脂酸(lysophosphatidic acid,LPA)是具有细胞间信号转导作用的脂类小分子物质,主要通过G蛋白受体介导的信号转导通路发挥多种生物学效应。近年来研究发现卵巢癌细胞能分泌LPA使患者血浆和腹水中LPA水平明显升高,90%以上Ⅰ期卵巢癌患者血浆中LPA明显高于健康人群,Ⅱ-Ⅳ期患者诊断敏感性达100%,其敏感性和特异性均明显高于目前临床常用的肿瘤标志物CA125,LPA被认为是一个非常有价值的用于卵巢癌诊断、病情检测及预后判断的生物标志物。LPA有3个特异性受体,分别是Edg2、Edg4和Edg7,属于内皮分化基因家族(endothelial differentiation gene family,Edgs)成员。Edg2主要在正常卵巢组织中表达,与卵巢癌关系不大。Edg4和Edg7在卵巢癌细胞中表达水平明显升高,认为与卵巢癌发生发展有关。LPA与受体结合后可活化四条细胞信号转导通路,促进卵巢癌细胞的增殖、生存、迁移和血管生成,抑制细胞凋亡,与卵巢癌的发生、发展、浸润、转移等密切相关。LPA的代谢主要通过人磷脂磷酸水解酶-3(human lipid phosphate phosphohydrolase-3,hLPP3)的降解而失活,增强hLPP3基因表达可以降低细胞外LPA水平,降低卵巢癌细胞克隆形成及抑制癌细胞在动物体内的生长。因此,有理由推测通过影响LPA代谢、封闭LPA受体或干扰LPA信号传导系统及阻断LPA的一系列级联反应有可能成为卵巢癌治疗的新途径。
LPA及其受体与卵巢癌发生发展关系的研究是近年来妇科肿瘤领域的新课题,还有许多未知的问题需要进一步的研究和阐明。比如LPA作为卵巢癌诊断指标尚未被公认和用于临床,LPA及其受体促使卵巢癌发生发展的机制还不十分清楚,阻断LPA信号通路是否能抑制卵巢癌细胞生长的研究还没见报道。本研究拟通过测定LPA及其受体在卵巢癌患者血清和组织中的表达,探讨LPA作为卵巢癌早期诊断指标的可行性,验证LPA及其受体在卵巢癌发生发展中的重要作用;采用基因导入增强卵巢癌细胞中hLPP3基因表达,通过体内外实验探讨hLPP3对LPA的调节及对卵巢癌细胞生长的抑制效应;利用RNAi技术沉默LPA受体Edg4和Edg7基因表达,探讨阻断LPA信号转导通路对卵巢癌细胞生长的抑制作用。本实验通过不同途径调控LPA的作用通路,研究LPA及其受体在卵巢癌的发生发展进程中的作用及机制,为卵巢癌基因治疗寻找有效的方法和治疗靶点。
第一部分LPA及其受体在卵巢癌患者血清和组织中的表达及意义
材料和方法
1.血清LPA测定
1.1研究对象:研究组:卵巢上皮性性癌患者50例,包括原发性卵巢癌患者30例(Ⅰ期10例、中晚期即Ⅱ～Ⅳ期20例)和复发性卵巢癌患者20例,组织学类型为浆液性囊腺癌37例,粘液性囊腺癌13例;良性对照组:良性卵巢上皮性肿瘤20例(浆液性囊腺瘤13例、粘液性囊腺瘤6例、子宫内膜样肿瘤1例);正常对照组:20例正常妇女。所有病例均经术后病理确诊。
1.2研究方法:采用生化法检测各组患者血清中LPA水平,同时用放射免疫方法测定血清中CA125水平,并将两者进行比较,探讨LPA对卵巢上皮性癌早期诊断的意义。
2.卵巢癌组织中Edg4和Edg7蛋白检测
2.1标本来源:为郑州大学第一附属医院病理科的存档腊块。研究组:上皮性癌组织48例(浆液性囊腺癌30例,粘液性囊腺癌11例,子宫内膜样癌7例)。临床分期为Ⅰ期12例,Ⅱ期16例,Ⅲ期18例,Ⅳ期2例;组织学分级为:G_1级14例,G_2级16例,G_3级18例。其中有淋巴结转移者22例,无淋巴结转移者26例;腹水≥500ml的30例,腹水＜500ml的18例。对照组:交界性上皮性肿瘤10例,良性上皮性肿瘤10例,正常卵巢组织12例。所有研究对象术前均未接受放疗或化疗,临床及病理资料完整。
2.2研究方法:采用免疫组化SP法检测卵巢上皮性肿瘤组织和正常卵巢组织中Edg4和Edg7蛋白的表达情况,并探讨这两种蛋白的表达与卵巢上皮性癌临床分期、组织学分级、淋巴转移及腹水量等临床和病理参数的关系。
3.统计学处理
计量资料采用单因素方差分析、最小显著差(LSD)检验;分类资料采用x~2检验和Kruskal-Wallis法秩和检验;相关性分析采用Spearman等级相关分析,以α=0.05为检验水准,在SPSS13.0上完成。
结果
1.各期卵巢上皮性癌患者血清LPA水平均明显升高,与正常妇女和良性卵巢上皮性肿瘤患者相比,其差异均有统计学意义(P＜0.001);Ⅰ期卵巢上皮性癌患者血清LPA水平也明显升高,与正常妇女和良性卵巢上皮性肿瘤患者相比差异有显著性(P＜0.001),其水平接近中晚期患者(P＞0.05);良性卵巢上皮性肿瘤患者血清中LPA表达水平较正常妇女略高,但差别无统计学意义(P＞0.05)。原发性卵巢癌Ⅱ～Ⅳ期组和复发组患者血清CA125水平明显高于正常妇女组和良性卵巢上皮性肿瘤组,其差别有统计学意义(P＜0.001);Ⅰ期卵巢上皮性癌患者血清CA125水平轻度升高,与良性卵巢上皮性肿瘤患者和正常妇女相比无明显差异(P＞0.05)。
2.血清LPA水平升高用于检测卵巢上皮性癌的敏感性为96%、特异性为90%、假阳性率10%、假阴性率4%,阳性预测值96%、阴性预测值90%,血清CA125分别为72%、80%、20%、28%、90%、53.3%,与CA125相比LPA有较高的敏感性、较低的假阴性率及较高的阴性预测值(P＜0.05)。LPA用于检测Ⅰ期卵巢上皮性癌的敏感性为90%、特异性90%、假阳性率10%、假阴性率10%、阳性预测值81.8%,阴性预测值94.7%,而CA125分别为30%、80%、20%、70%、42.9%和69.6%,LPA对Ⅰ期上皮性卵巢癌检测的敏感性、阳性预测值及阴性预测值高于CA125,假阴性率低于CA125(P＜0.05)。LPA检测原发性卵巢上皮性癌Ⅰ期、Ⅱ～Ⅳ期、复发性卵巢癌患者的约登指数分别为:0.8、0.85和0.9,而CA125检测的约登指数分别为:0.1、0.6和0.65,提示LPA是检测早期上皮性卵巢癌的良好指标。
3.溶血磷脂酸受体Edg4、Edg7蛋白在卵巢上皮性肿瘤组织中的阳性表达率分别为恶性91.7%和95.8%、交界性80%和70%、良性20%和30%,而在正常卵巢组织中阳性表达率仅为16.7%和16.7%,提示Edg4、Edg7在正常卵巢组织中呈低表达状态;在良性卵巢上皮性肿瘤组织中的表达较正常组织中稍高,但其差异无统计学意义(P＞0.05),而在交界性卵巢上皮性肿瘤和卵巢上皮性癌组织中Edg4、Edg7的表达均显著高于正常卵巢组织及良性上皮性肿瘤组织(P＜0.001),多数上皮癌组织中着色呈“+++”,提示两种受体蛋白在卵巢上皮性癌组织中呈高表达状态。
4.卵巢上皮性癌组织中Edg4和Edg7蛋白在Ⅲ、Ⅳ期的阳性表达明显高于Ⅰ、Ⅱ期;G_2、G_3级组织中两种蛋白的表达明显高于G_1级组织;有淋巴结转移的癌组织中两种蛋白的表达明显高于无淋巴结转移的癌组织;腹水＞500ml的癌组织中两种蛋白的表达明显高于腹水＜500ml的癌组织,比较结果差异均有统计学意义(P＜0.05)。提示在卵巢上皮性癌组织中,Edg4和Edg7的高表达与癌组织学分级、临床分期、淋巴结转移及腹水量增加有关。
5.Edg7蛋白在卵巢上皮性癌组织中的表达阳性率略高于Edg4蛋白,但无统计学差异(P＞0.05);Edg4和Edg7蛋白的表达状态趋势一致,经相关性分析两者的表达呈正相关(P＜0.05)。
小结
1.卵巢上皮性癌患者血清中LPA水平明显升高,其敏感性高于血清CA125,假阴性率低于CA125,尤其是在早期卵巢癌患者更明显。提示LPA有望成为卵巢癌早期诊断新的敏感标记物。
2.溶血磷脂酸受体蛋白Edg4与Edg7在卵巢上皮性癌组织中的表达水平明显升高,并与上皮性癌的临床分期、组织学分级、淋巴转移和腹水量呈正相关。提示Edg4和Edg7可能在卵巢上皮性癌的发生发展、浸润和转移中具有重要作用。
3.Edg4和Edg7受体蛋白在卵巢上皮性癌组织中表达程度呈正相关,推测二者在卵巢上皮性癌的发生发展中具有协同作用。
4.LPA及其受体Edg4和Edg7在卵巢癌患者血清及组织中的高表达等上述结果为下一步的研究即基因调控LPA作用通路抑制卵巢癌细胞生长的研究提供了理论和实验依据。
第二部分增强人磷脂磷酸水解酶-3基因表达对卵巢癌细胞生长的抑制作用
材料和方法
1.从胎盘组织中逆转录扩增目的基因
以人胎盘组织总RNA为模板,根据GenBank上的hLPP3基因cDNA编码序列(BC009196)设计引物:上游引物LPP3-1 5'TGGATCCTCCACCATGCAAAACT ACAAGTACGAC 3'(372-392);下游引物LPP3-2 5'CCTCGAGCTACATCAT GTTGTGGTGAT 3'(1288-1307)。采用RT-PCR技术扩增合成的双链DNA,凝胶电泳回收鉴定。
2.将目的基因克隆入克隆及表达质粒载体
将目的基因hLPP3克隆入pGEM-T easy质粒载体,利用α互补和T7/SP6 PCR扩增筛选鉴定重组子pGEM-T-hLPP3;用BamHⅠ和XhoⅠ双酶切重组子pGEM-T-hLPP3和真核表达载体pLenExpress质粒;将双粘的hLPP3片段亚克隆入表达载体pLenExpress中;利用PCR扩增及BamHⅠ和XhoⅠ双酶切筛选鉴定重组子pLenExpress-hLPP3;对插入序列进行DNA序列分析。
3.包装产生表达目的基因的慢病毒
将表达hLPP3基因的表达载体及辅助包装载体应用脂质体协助转染293FT细胞包装病毒,产生带有绿色荧光蛋白(GFP)并表达hLPP3基因的慢病毒颗粒(lentivires),测定病毒滴度。
4.表达目的基因慢病毒体外细胞转染实验
用表达hLPP3的慢病毒转染卵巢癌细胞系SKOV3和OVCAR3细胞(实验组),用空载体包装的慢病毒转染细胞及未转染细胞作为对照组。利用荧光显微镜观察转染阳性率,加入G418筛选稳定表达hLPP3的细胞株;生化法测定转染前后细胞培养上清液中LPA含量变化;采用实时荧光定量PCR测定各组细胞中hLPP3mRNA表达水平;流式细胞仪检测各组卵巢癌细胞的细胞周期和凋亡率改变。
5.裸鼠体内移植瘤实验
用SKOV3和OVCAR3细胞及稳定表达hLPP3基因的SKOV3和OVCAR3细胞接种裸鼠,构建卵巢癌裸鼠移植瘤动物模型,观察移植瘤生长情况,并测量移植瘤大小;将移植瘤作病理切片检查,实时荧光定量PCR测定移植瘤组织中hLPP3mRNA表达,流式细胞仪检测肿瘤细胞周期变化和细胞的凋亡率。
结果
1.成功扩增出目的基因
利用RT-PCR从胎盘组织中扩增出目的基因hLPP3,电泳条带约在936bp。
2.成功构建表达目的基因的重组真核表达载体
PCR产物与pGEM-Teasy载体连接后,转化JM109大肠杆菌,筛选鉴定得到正确重组子pGEM-T-hLPP3。进行亚克隆后,用BamHⅠ和XhoⅠ双酶切对重组质粒进行鉴定,得到pLenExpress-hLPP3重组真核表达载体。对重组子pLenExpress-hLPP3插入片段DNA序列测序,结果与设计完全一致。
3.获得高浓度的表达目的基因的慢病毒并对卵巢癌细胞系具有高转染效率。
用重组表达载体和包装载体包装慢病毒,其滴度分别如下:表达hLPP3基因的慢病毒:3.1×10~4 IU/ml;空载体包装的慢病毒:2.7×10~7 IU/ml。表达hLPP3的慢病毒转染卵巢癌SKOV3细胞阳性率为92%,高于转染OVCAR3细胞的阳性率(76%)。
4.实验组细胞中hLPP3mRNA表达水平明显增加
表达hLPP3的慢病毒转染卵巢癌细胞系后,经实时荧光定量PCR检测实验组卵巢癌细胞的hLPP3 mRNA相对表达量比空载体组及无转染对照组明显增加(P＜0.05);而无转染对照组与空载体组比较无统计学意义(P＞0.05)。SKOV3与OVCAR3之间hLPP3 mRNA相对表达量比较无统计差异(P＞0.05)。
5.实验组卵巢癌细胞培养上清液中LPA含量明显降低
慢病毒转染细胞后不同时间测定上清液中LPA水平,结果显示实验组LPA水平较对照组明显下降,且随着转染时间延长,LPA含量有进一步下降趋势(P＜0.05),存在时间-效应关系。两种卵巢癌细胞系SKOV3和OVCAR3之间LPA水平比较无统计学差异(P＞0.05)。
6.实验组细胞凋亡率明显增加
实验组卵巢癌细胞凋亡率明显增加,由转染前0.1±0.034%到转染后增加为30.50±2.12%,两者比较有统计学意义(P＜0.05)。实验组细胞凋亡率明显高于两个对照组(P＜0.05)。实验组卵巢癌细胞周期改变不明显(P＞0.05)。
7.成功构建卵巢癌及转基因卵巢癌裸鼠移植瘤模型
用卵巢癌细胞系及表达目的基因的卵巢癌细胞株皮下注射局部生长成瘤,实验组移植瘤体积及重量均小于空载体组和无转染对照组,差异有统计学意义(P＜0.05)。SKOV3细胞形成的移植瘤生长情况与OVCAR3细胞的相似,两者比较无统计学意义(P＞0.05)。
8.体内移植瘤实验结果
实验组裸鼠移植瘤组织中hLPP3 mRNA的相对表达量明显高于空载体组和无转染对照组,差异有统计学意义(P＜0.05);而对照组与空载体组比较无统计学意义(P＞0.05)。SKOV3与OVCAR3之间hLPP3 mRNA相对表达量比较无统计学意义(P＞0.05)。实验组裸鼠移植瘤细胞的凋亡率为16.5%,而空载体组细胞凋亡率为1.26%,无转染对照组细胞凋亡率则为0.10%,实验组细胞的凋亡率明显高于两个对照组,差异有统计学意义(P＜0.05)。各组细胞周期无明显变化(P＞0.05)。
小结
1.成功构建并产生出表达hLPP3的真核表达载体及慢病毒颗粒,并对卵巢癌细胞系SKOV3和OVCAR3细胞转染效率高且容易鉴定,获得了稳定表达hLPP3基因的卵巢癌细胞株。
2.体外实验,表达hLPP3的慢病毒转染卵巢癌细胞后,细胞内hLPP3 mRNA水平明显升高,细胞上清液的LPA水平明显降低,细胞凋亡率明显增加。提示增强hLPP3基因表达能有效降低LPA水平,促进细胞凋亡,其机制可能通过水解胞外的LPA或/和减少细胞LPA释放,抑制LPA的促肿瘤效应。
3.成功建立了卵巢癌裸鼠移植瘤模型及稳定高表达hLPP3基因的卵巢癌裸鼠移植瘤模型,后者体内移植瘤细胞生长速度明显减慢,hLPP3 mRNA水平和细胞凋亡率增加。提示增强hLPP3基因表达在体内也能有效抑制卵巢癌细胞生长,促进细胞凋亡,其机制可能与体外一样通过降低LPA水平发挥抑瘤效应。
4.体内外实验均证明增强hLPP3基因表达能抑制卵巢癌细胞生长,促进肿瘤细胞凋亡,提示调控hLPP3基因可能成为卵巢癌基因治疗的新靶点。
第三部分RNA干扰沉默溶血磷脂磷酸受体Edg4和Edg7基因表达对卵巢癌生长的抑制效应
材料和方法
1.设计合成Edg4和Edg7siRNA序列及发夹样DNA寡核苷酸单链
依据GenBank中Edg4基因(NM_004720)和Edg7基因(NM_012152)的序列,遵循siRNA设计原则进行设计及筛选,选择确定19个碱基的siRNA靶序列。各设计2对包含BamHⅠ和XhoⅠ酶切位点的靶向Edg4和Edg7发夹样DNA寡核苷酸,并合成2对编码短发夹RNA序列的DNA单链。设计合成无关序列DNA链作为对照。
2.构建目的基因的克隆载体和真核表达载体
将发夹样DNA序列退火成双链DNA,克隆入pGEM-T Easy载体,利用α互补和T7/SP6引物经PCR扩增筛选鉴定重组子pGEM-T-siEdg4和pGEM-T-siEdg7;用BamHⅠ和XhoⅠ限制性内切酶双酶切重组子和siRNA真核表达载体pRNAT-U6.1/lenti;将双粘发夹样siRNA片段亚克隆入pRNAT-U6.1/lenti中;利用通用引物PCR筛选及双酶切鉴定重组子;对插入序列进行DNA序列分析。
3.包装产生表达目的基因的慢病毒
将测序正确的siRNA表达载体和辅助包装载体应用脂质体包裹转染293FT细胞包装病毒,产生表达不同siRNA序列的慢病毒颗粒,并测定病毒滴度。
4.体外细胞转染实验
用表达不同目的基因的慢病毒转染入卵巢癌细胞系SKOV3细胞,用G418筛选稳定表达siRNA的细胞株。体外实验分为三个对照组和四个实验组,分别为未转染对照组C1,转染空载体包装的病毒对照组(C2),转染无关序列siRNA包装的病毒对照组(C3),转染siEdg4病毒组(T1),转染siEdg7病毒组(T2),共转染siEdg4病毒和siEdg7病毒组(T3),以及共转染siEdg7病毒和表达hLPP3的病毒组(T4)。利用荧光显微镜观察SKOV3细胞中的转染率;用生化法测定细胞培养上清液中LPA含量变化;用实时荧光定量PCR方法检测细胞Edg4和Edg7mRNA表达水平;用Western Blot测定Edg4和Edg7蛋白表达情况;用流式细胞仪检测转染细胞的细胞周期和凋亡率变化。
5.裸鼠体内移植瘤实验
用SKOV3细胞及稳定表达不同siRNA的SKOV3细胞接种裸鼠,构建表达不同基因的卵巢癌裸鼠移植瘤模型,在体内研究沉默Edg4和Edg7基因表达对卵巢癌细胞生长的影响及其作用机制。测量移植瘤大小和重量,比较各组移植瘤生长情况;实时荧光定量PCR测定瘤细胞中两种受体mRNA表达情况;免疫组化SP法检测移植瘤中Edg4和Edg7蛋白表达情况;流式细胞仪测定移植瘤细胞周期及细胞凋亡率变化。
结果
1.Edg4和Edg7siRNA靶序列筛选及发夹DNA单链设计合成
对候选序列通过同源序列检索和进一步优选,最后确定212-230位(GACCATCGGCTTCTTCTAT)和323-341位(GACCAATCTGCTGGTCATA)为Edg4-siRNA靶序列;277-295位(GCTGGAATTGCCTATGTAT)和697-715位(GTCTTGTCTCCGCATACAA)为Edg7-siRNA靶序列;(TACGCTGACTTGATTGTTC)为无关对照序列。
Edg4发夹样siRNA寡核苷酸序列
Position in gene sequence:212-230
BamHI Target sequence:sense Hairpin Target sequence:antisense XhoIAl 11 5'-GGATCC GACCATCGGCTTCTTCTAT TTCAAGAGA ATAGAAGAAGCCGATGGTC TTTTTT CTCGAGA -3'Al 12 3'-ACCTAGG CTGGTAGCCGAAGAAGATA AAGTTCTCT TATCTTCTTCGGCTACCAG AAAAAA GAGCRC-5'
BamHI Target sequence:sense Hairpin Target sequence:antisense XhoIAl 21 5'-GGATCC GACCAATCTGCTGGTCATA TTCAAGAGA TATGACCAGCAGATTGGTC TTTTTT CTCGAGA -3'Al 22 3'- ACCTAGG CTGGTTAGACGACCAGTAT AAGTTCTTCT ATACTGGTCGTCTAACCAG AAAAAA GAGCTC -5'
Position in gene sequence:323-341
Edg7发夹样siRNA寡核苷酸序列
Position in gene sequence:697-715
BamHI Target sequence:sense Hairpin Target sequence:antisense XhoIA211 5'-GGATCC GTCTTGTCTCCGCATACAA TTCAAGAGA TTGTATGCGGAGACAAGAC TTTTTT CTCGAGA-3'A212 3'-ACCTAGGCAGAACAGAGGCGTATGTT AAGTTCTCT AACATACGCCTCTGTTCTG AAAAAA GAGCTC-5'
BamHI Target sequence:sense Hairpin Target sequence:antisense XhoIA221 5'-GGATCC GCTGGAATTGCCTATGTAT TTCAAGAGA ATACATAGGCAATTCCAGC TTTTTT CTCGAGA-3'A222 3'-ACCTAGGCGACCTTAACGGATACATA AAGTTCTCT TATGTATCCGTTAAGGTCG AAAAAA GAGCTC -5'
Position in gene sequence:277-295
无关序列对照发夹样siRNA寡核苷酸序列
BamHI位点Target sequence:sense Hairpin Target sequence:antisense XhoI位点C1 5'- GGATCC TACGCTGACTTGATTGTTC TTCAAGAGA GAACAATCAAGTCAGCGTA TTTTTT CTCGAGA-3'C2 3'-ACCTAGG ATGCGACTGAACTCAAG AAGTTCICT CTTGTTAGTTCAGTCGCAT AAAAAA GAGCTC-5'
2.成功克隆表达目的siRNA的克隆载体和真核表达载体
不同序列的发夹DNA退火产物与pGEM-T Easy连接后,转化JM109,筛选鉴定后得到重组子pGEM-T-siEdg4、pGEM-T-siEdg7和pGEM-T-siC。亚克隆入表达质粒pRNAT-U6.1/lenti后,用通用引物对重组表达质粒进行鉴定,得到真核表达载体pRNAT-U6.1/lenti-siEdg4、pRNAT-U6.1/lenti-siEdg7和pRNAT-U6.1/lenti-siC。对重组子进行测序,结果与设计完全一致。
3.表达不同目的基因慢病毒滴度
采用脂质体Lipofectaminc~(TM) 2000包裹构建的siRNA表达载体及包装载体,转染293FT细胞,产生表达不同siRNA的慢病毒颗粒(Lentivirus),测定其滴度如下:针对Edg4基因的siRNA Lentivirus—siEdg4/lenti病毒3.2×10~7 IU/ml;针对Edg7基因的siRNA Lentivirus—siEdg7/lenti病毒4.1×10~7 IU/ml;无关对照的siRNA Lentivirus—siC/lenti病毒4.3×10~7 IU/ml;空载体包装的Lentivirus—pRNAT-U6.1/Lenti病毒2.7×10~7 IU/ml。
4.重组慢病毒能高效转染卵巢癌细胞系
含有不同siRNA的病毒转染卵巢癌SKOV3细胞,培养48h后采用荧光显微镜观察,结果显示除了未转染组细胞,其余各组SKOV3细胞的胞核和胞浆内均有大量绿色荧光信号,细胞转染阳性率均在90%以上(92%～95%),说明重组慢病毒可以高效转染卵巢癌SKOV3细胞,而且目的siRNA的插入不影响慢病毒对细胞的转染力。
5.慢病毒体外细胞转染结果
5.1实时荧光定量PCR方法分别测定各组细胞中Edg4和Edg7 mRNA的表达水平,结果显示:转染siEdg4和siEdg7慢病毒的T1组和T2组卵巢癌SKOV3细胞中Edg4和Edg7的mRNA表达水平明显降低,与对照组之间比较差异有统计学意义(P＜0.05)。而3个对照组卵巢癌细胞中Edg4和Edg7 mRNA表达均较高,相互之间比较差异无统计学意义(P＞0.05)。2个共转染实验组mRNA表达更低,与其它单独转染组比较差异有统计学意义(P＜0.05)。
5.2 Western Blot检测结果显示3个对照组细胞中Edg4和Edg7两种蛋白表达水平较高,分子量约40KD,而实验组中相应Edg4和Edg7蛋白表达水平明显降低。提示siRNA良好的靶向性。
5.3各实验组转染后细胞上清液中LPA含量明显下降,且随着转染时间的延长,细胞上清液中的LPA含量降低更明显(P＜0.05)。而3个对照组转染前后LPA含量变化不明显(P＞0.05),实验组与对照组相比较,差异有统计学意义(P＜0.05)。
5.4转染siRNA慢病毒对卵巢癌细胞SKOV3细胞周期影响主要表现为实验组的细胞S期比例降低,G_2期比例升高,差异有显著性(P＜0.05),而3个对照组的细胞周期无明显变化(P＞0.05)。转染后实验组细胞凋亡率明显增加,与对照组相比差异有统计学意义(P＜0.05);其中两个共转染实验组较单独转染实验组细胞凋亡率更高,3个对照组细胞凋亡率无明显变化(P＞0.05)。
6.裸鼠体内移植瘤实验结果
6.1实验组裸鼠移植瘤生长缓慢:各组卵巢癌细胞接种入裸鼠皮下后生长成瘤。各实验组移植瘤体积小于对照组,差异有统计学意义(P＜0.05),两个共转染组移植瘤体积明显小于单转染组(P＜0.05),但两个共转染组之间比较差异无统计学意义(P＞0.05)。处死裸鼠后的移植瘤重量实验组明显低于对照组,差异有统计学意义(P＜0.05)。
6.2对裸鼠皮下移植瘤组织进行Edg4和Edg7蛋白的免疫组化SP法染色,阳性表达主要表现为胞膜和胞浆着色,实验1、3组Edg4蛋白表达水平明显降低,实验2、3、4组Edg7蛋白表达明显降低,与对照组比较有统计学差异(P＜0.05)。而对照组之间比较无统计学意义(P＞0.05)。
6.3实验组移植瘤组织中两种受体mRNA的相对表达量明显低于对照组,差异有统计学意义(P＜0.05),而对照组之间比较无统计学意义(P＞0.05)。
6.4实验组裸鼠移植瘤细胞的凋亡率分别为(16.75±1.24)%、(15.80±1.35)%、(25.42±3.24)%和(24.63±2.76)%,3个对照组细胞凋亡率分别为(1.68±0.03)%、(1.76±0.04)%和(0.67±0.002),各实验组细胞的凋亡率明显高于3个对照组细胞,比较结果有统计学意义(P＜0.05)。
小结
1.成功设计并构建了真对Edg4和Edg7的真核表达载体pRNAT-U6.1/lenti-siEdg4和pRNA-U6.1/lenti-siEdg7,并包装生产出表达Edg4和Edg7siRNA序列的慢病毒颗粒,两种病毒均能高效转染卵巢癌细胞系SKOV3细胞并持久表达,获得了稳定表达siEdg4和siEdg7的SKOV3细胞株。
2.体外实验表明,RNAi沉默Edg4或Edg7基因表达能靶向抑制卵巢癌细胞内相应的Edg4或Edg7 mRNA及蛋白表达,降低细胞培养上清液中LPA水平,阻滞细胞周期进程,增加癌细胞的凋亡率,同时沉默两个受体作用更明显。提示封闭Edg4或/和Edg7基因表达均能有效抑制LPA的促肿瘤作用,Edg4和Edg7在卵巢癌的发生发展中具有协同作用。
3.沉默Edg7基因表达和增强hLPP3基因表达能更明显的降低细胞外LPA水平,增加肿瘤细胞凋亡,hLPP3对Edg7mRNA表达及细胞周期无明显影响。提示封闭Edg7和增强hLPP3基因表达对降低细胞外LPA水平和促进肿瘤细胞凋亡有协同作用,hLPP3主要通过降低LPA水平发挥作用。
4.成功构建了稳定表达siEdg4和siEdg7的卵巢癌裸鼠移植瘤模型,体内实验表明封闭Edg4或/和Edg7及增强hLPP3基因在体内也能明显抑制两种受体的mRNA和蛋白表达,促进细胞凋亡,抑制肿瘤细胞生长,发挥和体外相似的抑制肿瘤效应。
5.沉默Edg4或/和Edg7基因表达及增强hLPP3基因表达在体内外均能有效抑制卵巢癌细胞系SKOV3细胞中LPA的促肿瘤作用,提示对LPA受体及hLPP3基因的调控可能成为卵巢癌的基因治疗的有效靶点和新方法。该研究为验证LPA及其受体与卵巢癌发生、发展的关系提供了实验和理论依据,也为利用RNAi技术进行卵巢癌基因治疗研究奠定了基础并提供了新思路。
全文结论
1.卵巢上皮性癌息者血清中LPA水平明显升高,其敏感性高于血清CA125,尤其是在早期卵巢癌患者更明显。提示LPA有望成为新的敏感的卵巢上皮性癌早期诊断的生物学标记物。
2.溶血磷脂酸受体蛋白Edg4与Edg7在卵巢上皮性癌组织中的表达明显升高,并与上皮性癌的临床分期、组织学分级、淋巴结转移和腹水量呈正相关。提示LPA及其受体Edg4和Edg7可能在卵巢上皮性癌的发生发展、浸润和转移中具有重要作用。
3.成功包装出表达hLPP3的慢病毒对卵巢癌细胞系SKOV3和OVCAR3细胞转染效率高,转染后在体内外均能增强hLPP3基因表达,有效降低LPA水平,促进细胞凋亡,抑制卵巢癌细胞生长。其机制可能通过水解胞外的LPA或/和减少细胞LPA释放,抑制LPA的促肿瘤效应。
4.成功设计并构建了表达Edg4和Edg7siRNA的慢病毒,能高效转染卵巢癌细胞系SKOV3细胞并持久表达。体内外实验表明,RNA干扰沉默Edg4或Edg7基因表达均能明显抑制癌细胞内相应的Edg4和Edg7 mRNA及蛋白表达,降低细胞培养上清液中LPA水平,阻滞细胞周期进程,增加癌细胞的凋亡率,抑制细胞生长,同时沉默Edg4和Edg7具有协同作用。
5.同时沉默Edg7基因表达及增强hLPP3基因表达在体内外均能有效抑制卵巢癌细胞系SKOV3细胞中LPA的促肿瘤作用,二者具有协同作用。
6.该研究为验证LPA及其受体与卵巢癌发生、发展的关系提供了实验和理论依据,也为利用RNAi技术进行卵巢癌基因治疗研究奠定了基础并提供了新思路。对LPA受体Edg4、Edg7及hLPP3基因的调控可能成为卵巢癌的基因治疗的有效靶点和新方法。
7.有关表达hLPP3基因慢病毒的构建和体内外转染实验、利用RNA干扰技术沉默LPA受体Edg4、Edg7抑制卵巢癌细胞生长实验以及同时沉默Edg7及增强hLPP3基因表达抑制卵巢癌细胞生长的研究内容,国内外未见类似报道。
Background and Aims
Ovarian cancer is one of the most common cancers in females and the leading cause of death from gynecological malignancy in the world.Because the ovaries are located in the deep bottom of pelvis and early-stage ovarian cancer is generally asymptomatic,and lack of effective diagnosis and treatment methods,approximately 75%of patients present in advanced stages(ⅢorⅣ) and lost the chance for radical operation treatment.The ovarian cancer calls also are easy to appear resistance to chemotherapy.The 5-year survival in patients with advanced stage ovarian cancer is only 30%.But,the 5-year survival in patients with stageⅠis more than 90%. Therefore,study on the methods of early diagnosis and new effective therapy is the focus of ovarian cancer research recently.
Lysophosphatidic acid(LPA) is the simplest phospholipid,and mediates multiple biological functions through the LPA receptors which have been proposed to mediate LPA signal transduction pathway for multiple biological actions.Recent studies show that LPA is elevated in ascites and plasma of ovarian cancer patients. More than 90%patients with stageⅠovarian cancer have higher levels of LPA than that of healthy women.The sensitivity of LPA to diagnose ovarian cancer in stagesⅡ～Ⅳis 100%,and both of the sensitivity and specificity are higher than CA125 that is a common tumor biomarker used in clinical practice.Therefore,LPA is proposed as a potential and novel biomarker for ovarian cancer diagnosis and prognosis.LPA has three high affinity receptors,Edg2,Edg4 and Edg7,which belong to the members of the endothelial differentiation gene family(Edgs).Edg2 expresses stably in normal ovarian epithelial cells,and has low expression in ovarian cancer cells.Edg4 and Edg7 are overexpressed in the ovarian cancer epithelial cells.The results above suggest that the high level expression of Edg4 and Edg7 maybe related to tumor-promoting effect of LPA in ovarian cancer development and progression.The combination of LPA with Edg4 and Edg7 can activate four cell signaling transduction pathways and promote ovarian cancer cell proliferation,survival,differentiation, migration,angiogenesis and metastasis,and closely relate to the cancer occurrence, development,progression,invasion,metastasis and resistance to chemotherapeutic agents.
Human lipid phosphate phosphohydrolase-3(hLPP3) is a cell surface protein that exhibits ectoenzyme activity and is a number of lipid phosphate phosphohydrolase family(LPPs) that express extensively in cells.It can hydrolyze LPA and decrease the level of extracellular LPA and inhibit the function of LPA. Janos reported that transfecting the ovarian cancer cell line SKOV3 with the vector containing hLPP3 gene could decrease significantly the clon formation of cancer cells, and inhibit cancer cell growth and increase cell apoptosis in vivo.Therefore,it may be a new and effective way to treat ovarian cancer by affecting the metabolism of LPA, blocking LPA receptors,interfering with the signaling transduction pathway of LPA.
The study on the action and relationship between LPA and ovarian cancer is still a new project,and there are many problems that are unclear.Three objectives will be done in this study:1) to explore the feasibility of LPA as a early diagnostic biomarker for ovarian cancer and verify the important action of LPA and its receptors in promoting ovarian cancer development by detecting the LPA level in serum of patients with ovarian epithelial cancer and examining the expression of Edg4 and Edg7 in epithelial ovarian cancer tissues with immunohistochemical method.2) to explore the regulation effect of hLPP3 on LPA and inhibition on ovarian cancer cells growth in vitro and in vivo by enhancing the gene expression of hLPP3 in cancer cells. 3) to explore the inhibition efficacy of blocking LPA signaling transduction pathway by silencing Edg4 and Edg7 with RNAi technology in vitro and in vivo.The aim is to identify the new and effective way and potential targets for early diagnosis and gene therapy of ovarian cancer.
Part 1 Expression and Significance of LPA and its receptors in serum and tumor tissue of patients with ovarian cancer
Material and Methods
1.Detection of serum LPA level
1.1 Patients:Seventy patients with epithelial ovarian tumor were selected in this study,which included 50 malignant cases(37 cases of serous cystadenocarcinoma,13 cases of mucous cystoadenocarcinoma,WHO,2000),including 10 patients with primary early stage(stageⅠ) group,20 with primary advanced stage(stageⅡ～Ⅳ) group(FIGO,2000) and 20 with relapsed ovarian cancer group,the ages of patients were 30～69 years old,and the average was 51.3±5.6 year;20 benign cases(13 cases of serous cystadenoma,6 cases of mucous cystadenoma and 1 case of endometrioid tumor),the range of ages was 24～63 years,the average was 46.7±8.7 years.Twenty normal women were also selected as controls,the ages were 28～70 years,the average was 47.6±9.7 years.All cases were confirmed by pathology after operation.
1.2 Meehods:Levels of serum LPA were detected by biochemistry assay and those of tumor antigen 125(CA125) were measured by radio-immunoassay.Then the two results were compared to evaluate the value of LPA to diagnosis of ovarian cancer, especially to early stage of ovarian cancer.
2 Examination of Edg4 and Edg7 proteins in epithelial ovarian tumor tissues
2.1 Specimens:Sixty-eight specimens of epithelial ovarian tumor paraffin-imbedded and archival tissues and 12 cases of normal ovarian tissues were resected and collected at department of gynaecology and obstetrics between February of 2000 and October of 2004.All specimens were diagnosed by two pathologists and all patients had not accepted the radiotherapy and chemotherapy before operation with complete clinical and pathological information.Eighty samples included normal ovarian tissues (12 cases),benign epithelial ovarian tumors(10 cases),borderline epithelial ovarian tumors(10 cases) and malignant epithelial ovarian cancers(48 cases),while 48 malignant cases were divided into serous cystadenocarcinoma(30 cases),mucous cystoadenocarcinoma(11 cases) and endometrioid carcinoma(7 cases) according to WHO 2000 standard.Their FIGO staging was stageⅠ(12 cases),stageⅡ(16 cases), stageⅢ(18 cases) and stageⅣ(2 cases),their histological staging was G_1 grade(14 cases),G_2 grade(16 cases),G_3 grade(18 cases) according to histological grade.Their subgroups included lymphoid node metastasis(22 cases) and non-lymphoid node metastasis(26 cases),ascites quantity more than 500ml(30 cases) and ascites quantity less than 500ml(18 cases).The patients' ages were from 31 to 60 years old,mean of age was 42.6±4.7 years,and the ages of women with normal ovaries were 34～59,the average age was 45.4±6.3years.
2.2 Methods:The Edg4 and Edg7 protein expression in epithelial ovarian tumor was detected by immunohistochemical assay(streptavidin-biotin -peroxidase complex staining assay).The relationship between expression of Edg4 and Edg7 proteins and the clinical parameters,such as clinical stage,pathological grade,ascites quantity,lymphoid node metastasis,etc.were investigated and analysed.
3 Statistical analysis
The measurement data were presented as mean+SD((?)±s),the comparison of multi-sample means was dealt with using one-way analysis of variance(ANOVA) and least significant difference(LSD) test;numeration data were expressed as n(%),the comparisons of groups were done using contingency table Chi-square test,Fisher's Exact test and Kruskal-Wallis test.Spearman rank correlation analysis was used to analyze the relevance.Analyses were performed using statistics software SPSS13.0 package,and P＜0.05 represented significance.
Results
1.Levels of serum LPA in patients with different stages(Ⅰ-Ⅳ) epithelial ovarian cancer were significantly higher than those in normal women and benign epithelial ovarian tumor(P＜0.001),but serum LPA level of every epithelial carcinoma group had no statistical significance(P＞0.05),such as patients with stageⅠ(early stage) had no difference compared with the advanced patients(stageⅡ～Ⅳ).The same result was obtained from the comparison of normal women and benign cases(P＞0.05) although there was a slight raise in benign cases.CA125 levels in primary ovarian cancer with advanced and relapsed groups were significantly higher than those in early stage group,benign tumor group and normal women group(P＜0.001),but in early stage it was just a little higher than those in benign tumor group and normal women group, then there was no statistic significance(P＞0.05).
2.Serum LPA increase used to diagnose epithelial ovarian carcinoma had sensitivity of 96%、specificity of 90%、false positive rate of 10%、false negative rate 4%、positive predictive value of 96%and negative predictive value of 90%,while those of CA125 were 72%、80%、20%、28%、90%and 53.3%,respectively.These two indexes had differences in sensitivity,false negatiye rate and negative predictive value when used to diagnose epithelial ovarian carcinoma(P＜0.05),but compared with CA125, LPA had higher sensitivity,lower false negative rate and higher negative predictive value.In early stage patients,LPA used to diagnose epithelial ovarian carcinoma of stageⅠhad sensitivity of 90%,specificity of 90%,false positive rate of 10%、false negative rate 10%、positive predictive value of 81.8%and negative predictive value of 94.7%,while those of CA125 were 30%、80%、20%、70%、42.9%and 69.6%,they had difference in sensitivity,false negative rate、positive predictive value and negative predictive value to diagnose primary epithelial ovarian carcinoma of stageⅠ(P＜0.05),and LPA had higher sensitivity、positive predictive value、negative predictive value and lower false negative rate compare with CA125;but used to diagnose primary epithelial ovarian carcinoma of stageⅡ～Ⅳand relapsed groups,all the indexes of CA125 and LPA had no statistic significance(P＞0.05). Youden's index of LPA used to detect primary epithelial ovarian carcinoma of stageⅠ、stageⅡ～Ⅳand relapsed groups were respectively 0.8、0.85 and 0.9,while whose of CA125 were 0.1、0.6 and 0.65.The result indicated that of the two markers used to detect different groups of epithelial ovarian carcinoma,LPA had higher Youden's index than CA125 in full,especially concerning epithelial ovarian carcinoma of stageⅠthe Youden's index had risen to 0.8,it pointed out that LPA would be a satisfactory biomarker index to detect epithelial ovarian carcinoma of early stage.
3.The positive expression rates of Edg4 and Edg7 proteins were 91.7%and 95.8% (malignant),80%and 70%(borderline),20%and 30%(benign),16.7%and 16.7%(normal),it demonstrated that Edg4 and Edg7 were low-expressed in normal ovarian tissues;in benign epithelial ovarian tumors,Edg4 and Edg7 proteins had a little rise over normal tissues,but there was no statistical significance(P＞0.05);but Edg4 and Edg7 proteins expression in borderline and malignant epithelial ovarian tumor was markedly higher than those of normal tissues and benign tumor(P＜0.001), most of the epithelial ovarian carcinoma tissues had evident coloration of(+++),which indicate that these two proteins were high-expressed in epithelial ovarian carcinoma tissues.
4.The expression of Edg4 and Edg7 proteins in tissues of FIGO stageⅢ～Ⅳwas obviously higher than that of FIGO stageⅠ～Ⅱ;the expression of two proteins in the G_1 tissues was lower than that of G_2,G_3 tissues significantly;the expression of Edg4 and Edg7 proteins in tissues with lymphoid node metastasis was higher than that of without lymphoid node metastasis;the expression of two receptor proteins in tissues with ascites quantity more than 500ml was higher than that of the ascites quantity less than 500ml.These differences all had statistical significance(P＜0.05).According to these results,we concluded that the high-expression of Edg4 and Edg7 proteins in epithelial ovarian carcinoma was significantly associated with FIGO stage, pathological grade,ascites quantity and lymphoid node metastasis.
5.The positive expression of Edg7 protein was a little higher than that of Edg4 protein in epithelial ovarian cancer,but there was no significant difference(P＞0.05). The tendency of two proteins expression was positive correlation.(r=0.978,P＜0.05).
Conclusions
1.Serum LPA levels in patients with primary and relapsed epithelial ovarian cancer were significantly higher.The sensitivity and negative predictive value of LPA in diagnosis of epithelial ovarian cancer were superior to CA125,and false positive rate was inferior to CA125,they were more obvious in early stage of ovarian cancer, especially.LPA may be a new potential biomarker for diagnosis before operation and monitoring prognosis and recurrence of epithelial ovarian cancer after operation.
2.The expression of Edg4 and Edg7 proteins in epithelial ovarian cancer had markedly increased,and was significantly associated with the clinical stage, pathological grade,quantity of ascites and lymphoid node metastasis.The results demonstrated that Edg4 and Edg7 may play an important role in occurrence, development,invasion and metastasis of ovarian cancer.
3.The expression degree of Edg4 and Edg7 receptor proteins was positively correlated,which suggests that the two proteins had synergistic effect in the occurrence and development of epithelial ovarian cancer.LPA and its two receptors would become the potential targets to ovarian cancer gene therapy.
4.Above results that the high expression of LPA and its receptors in patients with ovarian cancer have proved basis of theory and experiment for our next studies that regulation to LPA signaling transduction pathway by gene engineering inhibits ovarian cancer cells growth.
Part 2 The inhibition efficacy on ovarian cancer cells growth by enhancing hLLP3 gene expression
Material and Methods
1.Extraction and amplification hLPP3 gene from placenta tissue with RT-PCR
Extracting total RNA from human placenta and synthesizing the hLPP3 cDNA by RT-PCR.According to the hLPP3 eDNA sequence in GenBank(BC009196),the primers were designed and synthesized as LPP3-1 5' TGGATCCTCCA CCATGCAAAACTACAAGTACGAC 3'(372-392);LPP3-2 5' CCTCGAGCT ACATCATGTTGTGGTGAT 3'(1288-1307).The hLPP3 gene was obtained with the technique of RT-PCR by using human placenta tissues RNA as template.The PCR product was retrieved and identified by agar-gel electrophoresis analysis.
2.Construction of recombinant clone and expression plasmids of hLPP3 gene
The PCR product was cloned into pGEM-T easy plasmid(clone vector),thenα-complementation test and T7/SP6 PCR was used to screen the positive clones(pGEM-T-hLPP3),which was digested by restriction enzyme BamHⅠand XhoⅠ,and finally was subcloned into the pLenExpress vector(expression plasmid). Then the recombinants were identified by PCR and BamHⅠand XhoⅠcutting,and the target DNA in the recombinant was sequenced finally.So the eukaryotic expression vector pLenEx- press-hLPP3 was constructed successfully.
3.Pack and producing lentivirus particles which express hLPP3 gene
The 293FT cells were transfected with the eukaryotic expression vector (pLenEx- press-hLPP3) and another two assisted packing vectors(package kit) assisted by Lipofectamine to pack and produce the lentivirus particles which contain and express hLPP3 gene and green fluorescent protein(GFP),then the titers of lentivirus were detected.
4.Cells transfection experiment in vitra
Transfecting the ovarian cancer cell lines SKOV3 and OVCAR3 with recombinant lentivirus.After SKOV3 and OVCAR3 were transfected with lentivirus. Experiment was divided into three groups:experimental group(the cells were transfected by lentiveruses which express hLPP3 gene);blank vector control group(the cells were transfected by lentiviruses which express pLenExpress vector gene) and non-transfection control group(the cells were not transfected by lentivirus). The transfection efficiency was detected by fluorescent microscopy,and the positive ceils were selected and maintained by adding G418 to the cell culture medium.The LPA levels of call culture supernatant before and after transfection were detected by biochemical method,the expression level changes of hLPP3 mRNA were measured by real-time quantitative PCR,and the changes of cell apoptosis and cell cycle were detected by flow cytometry.The non-transfected cells and the cells which were transfected with the lentivirus packed with blank pLenExpress vectors were arranged as controls.
5.Building ovarian cancer animal models and experiment in vivo
The nude mice were inoculated by subcutaneous injection with SKOV3 and OVCAR3 cell lines and the cell lines which express stably hLPP3 gene,and graft tumor animal models were built.The sizes of tumors were measured,the characteristics of tumor were identified by pathological method,the expression levels of hLPP3 mRNA were detected by real-time quantitative PCR,and the cell apoptosis were determined by flow cytometry.
Results
1.Target hLPP3 gene was amplified successfully
The hLPP3 gene was amplified successfully by RT-PCR from human placenta, and the gone band located at about 936bp by agar-gel electrophoresis analysis,which was target gene hLPP3.
2.The recombinant clone and eukaryotic expression vectors were constructed successfully
PCR product(hLPP3 gene) was linked to pGEM-T easy vector and amplified by transforming into competent E coli.JM109,and then the target gene was subcloned to expression vector(pLenExpress),the recombinant eukaryotic expression vectors (pLenExpress-hLPP3) were selected and identified through PCR and BamHⅠand XhoⅠrestrictive enzyme cutting test,and then sent to Shanghai for sequencing.The results of sequencing showed that the DNA sequence inserted into the recombinant pLenExpress-hLPP3 was conformed completely to the design.
3.High titer of lentivirus and high cell transfecting efficiency were obtained
The 293 FT cells were co-transfected by pLenExpress-hLPP3 vector wrapped by Lipofectamine and another two assistant package genes,and the lentiviral particles (lentivirus) which express hLPP3 gene were produced.The concentrations of lentivirus are:lentivirus-hLPP3,3.1×10~7 IU/ml and containing blank vector lentivirus, 2.7×10~7 IU/ml.The cell transfection efficiency of lentivirus-hLPP3 to SKOV3 was 92%,and that to OVCAR3 was 75%,the former is higher than the latter.And the results also showed that the insertion of hLPP3 gene didn't affect the ability of lentivirus to transfect ovarian cancer cells.
4.The mRNA expression level of hLPP3 in experimental ovarian cancer cells increased significantly
After transfection by different lentiviruses,the hLPP3 mRNA level of SKOV3 and OVCAR3 cells in experimental group was higher than those in the two control groups(P＜0.05),and level of hLPP3 mRNA in two control groups were not different(P＞0.05).The different level of hLPP3 mRNA between SKOV3 and OVCAR3 calls had no statistic significance(P＞0.05).
5.The LPA level in experimental group dropped markedly
The LPA levels in supernatant of SKOV3 and OVCAR3 which were transfected with lentivirus-hLPP3 were lower significantly compared with the controls(P＜0.05), and had the tendency of continual decrease accompanying with time progress,and there was a time-effect relationship.The LPA levels between SKOV3 and OVCAR3 cells had no statistic significance.
6.Apoptosis in experimental group was much higher
The apoptosis rate of cancer cells in experimental group increased significantly after transfection(30.50±2.12%vs 0.1±0.03%,P＜0.05).The apoptosis rate in experimental group was markedly higher than two control groups(P＜0.05).The changes of cell cycle were not significant before and after transfection(P＞0.05).
7.The ovarian cancer animal models and the transgenic models were constructed successfully
SKOV3 and OVCAR3 cells and the two cell lines which express hLPP3 gene were inoculated by subcutaneous injection into nude mice and the graft tumor mass were formed in the injection location.The tumor sizes of experimental group were smaller than those of two control groups(P＜0.05),the tumor sizes between SKOV3 and OVCAR3 cell lines were not different(P＞0.05).The tissues of graft tumor were examined by HE staining and the features in microscopy were similar to those of human ovarian cancer tissue.
8.Experimental results in vivo
The expression level of hLPP3 mRNA in transgenic tumor tissue were higher than those in two control groups(P＜0.05),while the comparison between two control groups had no statistic significance(P＞0.05).The expression level of hLPP3 mRNA in SKOV3 and OVCAR3 cells also had no statistic significance(P＞0.05).The apoptosis rates of transgenic tumor cells were significantly higher than that in control tumors (16.5%vs 1.26%and 0.10%,P＜0.05).The changes of cell cycle in each groups were not significant(P＞0.05).
Conclusions
1.The target gene hLPP3 cDNA was obtained successfully and the recombinant eukaryotic expression vector pLenExpress-hLPP3 and lentivirus expressing hLPP3 gene were constructed and produced successfully.The letivirus carrying green fluorescent protein(GFP) could be identified easily and had high transfection efficiency to SKOV3 and OVCAR3.The cell clones that express hLPP3 gene stably had been built and obtained.
2.In vitro,the levels of hLPP3 mRNA in SKOV3 and OVCAR3 cell lines after transfection were significant higher than those before transfection,the concentrations of LPA in cell supematant were significant lower,and the apoptosis rates of cancer cells were significant higher.The results demonstrated that enhancing the expression of hLPP3 gene in ovarian cancer cell lines could decrease LPA level and induce apoptosis of cancer cells effectively,and inhibit the growth of ovarian cancer cells in vitro.The mechanism might be that hLPP3 can inhibit and block the action of LPA by hydrolyzing the extracellular LPA and/or decreasing LPA secretion of cancer cells.
3.The ovarian cancer animal models(nude mice) and the transgenic animal models in which the tumor cells express hLPP3 gene stably were built successfully.The growth of tumors expressing hLPP3 gene was slower than that of non-gene transfection models.The levels of hLPP3 mRNA and apoptosis rate of cancer cells in transgenic models were also higher.The above results show that enhancing the expression of hLPP3 gene in ovarian cancer cells also could inhibit the growth of cancer cells,and induce apoptosis of cancer cells effectively in vivo.The mechanism might be the same as that in vitro.
4.The effects that enhancing hLPP3 gene expression can inhibit the growth of ovarian cancer cells and promote the apoptosis of cancer cells were testified by our experiments in vitro and in vivo.These results demonstrate that hLPP3 gene might become a new target for ovarian cancer and regulating the expression of hLPP3 gene might be a new way ofgene targeting therapy for ovarian cancer.
5.The construction of lentivirus which expresses hLPP3 gene and transfection experiments to ovarian cancer cell lines in vitro and in vivo were not reported domestically or overseas.
Part 3 The inhibiting efficacy on growth of ovarian cancer cells by silencing Edg 4 and Edg 7 using RNA interference
Materials and methods
1.Design and synthesize Edg4 and Edg7 siRNA sequence and hairpin single strand DNA oligonucleotide
According to the Edg4 sequence in GenBank(NM_004720) and Edg7 sequence in GenBank(NM_012152),following the rules of siRNA design,we used the siRNA design and analysis software which were provided in following webpage(http://www. ambion.com/techlib/misc/siRNA_design.html) to design the target oligonucleotides which contain 19 bp.Then the oligonucleotides of target siRNAs were indexed through American National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/Blast) to compare their homology,in order to identify the best siRNA oligonucleotides of Edg4 and Edg7.Afterwards,two pairs of siRNA hairpin single strand DNA oligonucleotides of Edg4,Edg7 and one pair of irrelevant sequence control siRNA oligonucleotide which have palindrome,loop structure and BamHⅠand XhoⅠrestriction enzyme sites were synthesized.
2.Construct clone and eukaryotic expression vectors which express target gene
The hairpin DNAs were annealed to double strands DNA and were cloned into pGEM-T Easy vector.Then,α-complementation test and T7/SP6 PCR primers were used to screen the positive clones(pGEM-T-siEdg4 and pGEM-T-siEdg7);The target DNAs were cut down from pGEM-T-siEdg4 and pGEM-T-siEdg7 by BamHI and XhoI restriction enzyme,and linked with siRNA expression vector (pRNAT-U6.1/lenti);The positive ones(pRNAT-U6.1/lenti-siEdg4 and pRNAT-U6.1/ lenti-siEdg7) were selected by PCR with general primer.Then the target DNAs were sequenced.
3.Pack and produce lentivirus particles which express target siRNA gene
The 293 FT cells were transfected by pRNAT-U6.1/lenti-siEdg4 or pRNAT-U6.1/lenti-siEdg7 or pRNAT-U6.1/lenti-siC or pRNAT-U6.1/lenti vectors and another two assistant package vectors,and the lentiviral particles(lentivirus) containing and expressing target gene and GFP were produced.The titers of lentivirus were measured:
4.Transfection experiments in vitro
Ovarian cancer cell line SKOV3 cells were transfected with the different lentiviruses,and the SKOV3 cells which express different siRNA stably were selected by G418.The experimental groups were divided as follows.The control groups included three subgroups:non-transfection SKOV3 cells,the cells transfected with the lentivirus packed by blank pRNAT-U6.1/lenti vector,and the cells transfected with the lentivirus packed by the irrelevant sequence siRNA vectors.The experimental groups were divided into four subgroups:cells of groupl were transfected with siEdg4/lenti lentivirus,group 2 with siEdg7/lenti lentivirus,group 3 was co-transfected with siEdg4/lenti and siEdg7/lenti lentiviruses,group 4 was co-transfected with siEdg7/lenti lentivirus and the lentivirus expressing hLPP3 gene.The methods used to detect the transfection efficacy of siRNA lentivirus,the levels of Edg4 and Edg7 mRNA,the LPA concentration in supernatant,and the apoptosis of cancer cells before and after transfection were same as those used in part 2.The expression levels of Edg4 and Edg7 proteins were measured by western blotting.
5.Build animal models and experiments in vivo
Building ovarian cancer animal models with 7 kinds different SKOV3 cells(as same as described in 5) and studying the inhibition efficiency and mechanism of silencing Edg4 and Edg7 on growth of ovarian cancer cells in vivo.The methods were same as those used in part 2.The Edg4 and Edg7 proteins in graft tumor tissues were detected by immunohistochemistry SP method.
Results
1.The results of Edg4 and Edg7 siRNA target sequences selection and hairpin single strand DNA design and synthesis
The 20 target siRNA oligonucleotides were obtained.After BLAST search in NCBI database,two target siRNA sequences with 19 bases were ensured for each receptor.The target sequences of Edg4-siRNA were:GACCATCGGCTTCTTCTAT (212-230) and GACCAATCTGCTGGTCATA(323-341).And the target sequences of Edg7-siRNA were GCTGGAATTGCCTATGTAT(277-295) and GTCTTGTCTCCGCATA C AA(697-715).The irrelevant control sequence was TACGCTGACTTGATTGTTC.
Edg4-1,2 hairpin siRNA oligonucleotide sequences as following:
Position in gene sequence:212-230
BamHI Target sequence:sense Hairpin Target sequence:antisense XhoI Al 11 5'-GGATCC GACCATCGGCTTCTTCTAT TTCAAGAGA ATAGAAGAAGCCGATGGTC TTTTTT CTCGAGA -3' Al 12 3'-ACCTAGG CTGGTTAGCCGAAGAAGATA AAGTTCTCT TATCTTCTTCGGCTACCAG AAAAAA GAGCRC-5'
BamHI Target sequence:sense Hairpin Target sequence:antisense XhoI Al 21 5'-GGATCC GACCAATCTGCTGGTCATA TTCAAGAGA TATGACCAGCAGATTGGTC TTTTTT CTCGAGA -3' Al 22 3'- ACCTAGG CTGGITAGACGACCAGTAT AAGTTCTTCT ATACTGGTCGTCTAACCAG AAAAAA GAGCTC -5' Position in gene sequence:323-341
Edg7-1,2 hairpin siRNA oligonueleotide sequences as following:
Position in gene sequence:697-715
BamHI Target sequence:sense Hairpin Target sequence:antisense XhoI A211 5'-GGATCC GTCTTGTCTCCGCATACAA TTCAAGAGA TTGTATGCGGAGACAAGAC TTTTTT CTCGAGA-3' A212 3'-ACCTAGGCAGAACAGAGGCGTATGTT AAGTTCTCT AACATACGCCTCTGTTCTG AAAAAA GAGCTC-5'
BamHI Target sequence:sense Hairpin Target sequence:antisense XhoI A221 5'-GGATCC GCTGGAATTGCCTATGTAT TTCAAGAGA ATACATAGGCAATTCCAGC TTTTTT CTCGAGA-3' A222 3'-ACCTAGGCGACCTTAACGGATACATA AAGTTCTCT TATGTATCCGTTAAGGTCG AAAAAA GAGCTC -5'
Position in gene sequence:277-295
Irrelevant control sequence siRNA oligonucliotide sequence
BamHI位点Target sequence:sense Hairpin Target sequence:antisense XhoI位点C1 5'- GGATCC TACGCTGACTTGATTGTTC TTCAAGAGA GAACAATCAAGTCAGCGTA TTTTTT CTCGAGA-3' C2 3'-ACCTAGG ATGCGACTGAACTCAAG AAGTTCICT CTTGTTAGTTCAGTCGCAT AAAAAA GAGCTC-5'
2.The recombinant clone vector and eukaryotic expression vector which express siRNA were constructed successfully
The hairpin DNAs of siRNA were annealed to double stranded DNA that the molecular weight band of DNA was identified to locate at about 60bp by agarose gel electrophoresis analysis.They were similar to the ones expected.The annealing products were recombined with pGEM-T Easy vector to form pGEM-T-siEdg4 and pGEM-T-siEdg7,which then were transformed into JM109.After being screened and identified by PCR and,the positive clones(pGEM-T- siEdg4 and pGEM-T-siEdg7) were identified.After sub-cloning,the siRNA expression vectors,pRNAT-U6.1/LentisiEdg4 and pRNAT-U6.1/Lenti-siEdg7 were identified by PCR,BamHI XhoI cutting and sequencing.The results showed that the sequences inserted in recombinant expression vectors were same as those designed.
3.Results of different ientivirus titers
The 293FT cells were transfected by siRNA expression vectors with another two package vectors by Lipofectamine,and the siRNA lentiviroural articles were produced. The titers of lentivirus were as below:
siEdg4-1/lenti lentivirus:3.2×10~7IU/ml,siEdg4-2/lenti lentivirus:2.1×10~7IU/m, siEdg7-1/lenti lentivirus:4.1×10~7IU/ml,siEdg7-2/lentivirus:3.1×10~7IU/ml,irrelevant control sequence siC/lenti lentivirus:4.3×10~7IU/ml,blank vector control pRNAT-U6.1/lenti lentivirus:2.7×10~7IU/ml.
4.Recombinant lentiviruses had high transfection efficiency on ovarian cancer cell line SKOV3
A great quantity of GFP can be observed in all transfected SKOV3 cells by fluorescence microscope 48 hours later except the non-transfection cells.The positive cells of all transfected groups were more than 90%(92%～95%).And the positive cell clones were selected by adding G418.The results demonstrated that the siRNA lentiviral particles we produced could transfect SKOV3 cells in high efficiency and the insertion of target siRNA did not affect the ability of lentivirus to transfect ovarian cancer cell line.
5.The results of transfection experiments in vitro
5.1 The mRNA level of Edg4 and Edg7 in relevant experimental groups transfected by the lentiviruses of siEdg4/lenti and siEdg7/lenti was obviously reduced compared with the controls(P＜0.05).The levels of Edg4 and/or Edg7 mRNA in co-transfected groups were lower than single transfection groups(P＜0.05).The mRNA level of Edg4 and/or Edg7 were still high in three control groups and there was no difference among them(P＞0.05).
5.2 There were Edg4 and Edg7 protein bands in control groups showed by western blotting,and the molecular weight was about 40KD.The protein bands of the cells transfected by related siRNA lentivirus was slighter in different experimental groups.
5.3 The LPA concentration in supernatant of experimental groups after transfection decreased significantly in time-dependent manner comparing with that of control groups(P＜0.05).However,there was no difference in control groups(P＞0.05).
5.4 The proportion of S stage cells in cell cycle decreased and that of G2 stage cells increased significantly in experimental groups(P＜0.05).The apoptosis rate of cancer cells in experimental groups were higher than that in control groups(P＜0.05),and the apoptosis in co-transfected groups was much higher.However,the cell cycle had no change and apoptosis was very low in three control groups,and there was no differen
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