RNAi沉默CXCR4基因抑制胃癌侵袭和转移的实验研究
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摘要
目的：胃癌在我国是最常见的恶性肿瘤之一,其死亡率目前仍居恶性肿瘤首位。因其发病较隐匿,常不能早期发现及诊断,导致其致死率居高不下。手术切除是目前唯一可能根治胃癌的手段,但对于进展期胃癌即使行胃癌根治术,临床五年生存率仍徘徊在50%左右,且术后患者生活质量明显下降。较早出现的癌细胞转移是导致进展期胃癌不良预后的主要原因,但目前对于胃癌转移的分子机制的了解仍然是属于知之甚少的领域。近年来的研究指出,趋化因子CXCL12与其特异性受体CXCR4所构成的CXCR4/CXCL12生物学轴在多种肿瘤的播散和器官特异性转移中发挥着重要的作用。但CXCR4在胃癌侵袭、转移中的作用及机制仍然存在较大争议。为此,本研究首先观察胃癌不同组织中CXCR4/CXCL12的表达及其与胃癌临床病理参数包括患者生存率之间的关系,同时检测四种不同人胃癌细胞系中CXCR4基因mRNA的表达状况；其次,构建CXCR4-shRNA重组腺病毒真核表达载体转染人胃癌细胞系SGC7901,以特异性抑制胃癌细胞SGC7901中CXCR4的表达,观察其阻抑效应；最后,建立裸鼠胃癌原位移植瘤模型,对比观察移植前后的胃癌细胞在动物体内的生物学性状,由此揭示CXCR4在胃癌侵袭和转移中的作用并初步探讨其机制,为胃癌侵袭转移的防治提供新思路。
方法：
1、收集辽宁医学院附属第一医院普通外科2008年2月至2009年2月外科手术切除的85例新鲜胃癌组织标本及距肿瘤边缘5cm以上的正常胃组织41例。应用免疫组织化学方法检测不同组织内CXCR4和CXCL12的表达情况,系统分析它们与临床病理参数之间的关系,研究二者在胃癌不同组织中的表达与胃癌侵袭转移的关系。用RT-PCR方法分别检测4种人胃癌细胞株中CXCR4mRNA的表达情况。
2、在GENBANK中检索基因CXCR4序列,设计3条短发夹RNA (shRNA)并经同源性检索无误后合成,利用PCR、酶切及DNA连接构建真核表达载体shpDown-hCXCR4-eGFP,挑取阳性质粒送交阳性克隆测序鉴定后,再根据Gateway技术将真核表达载体嵌入到PL-Dest腺病毒骨架载体(pAd/PL-Dest)中,经基因测序、PCR及Pac I酶切鉴定获得重组腺病毒载体shpAd-hCXCR4-eGFP。同理构建对照组shpAd-control-eGFP。去内毒素超纯提取该重组腺病毒质粒,经Pac Ⅰ酶切电泳后凝胶回收大片段,提纯并通过脂质体Lipofectamine2000介导转染HEK293A细胞,通过eGFP基因绿色荧光表达检测转染情况并进行病毒包装,采用终点稀释法进行病毒滴度测定。以最佳感染复数(MOI)转染胃癌细胞SGC7901后,利用RT-PCR和Western-blot方法检测各组沉默效率,筛选出沉默效率最高的一组。
3、分别以最佳MOI转染实验组和阴性对照组,并设立空白对照组。将转染后的胃癌细胞分别利用RT-PCR和Western-blot检测重组腺病毒载体沉默CXCR4基因mRNA和蛋白的表达情况；通过MTT法利用吸光度绘制SGC7901细胞生长曲线,检测重组腺病毒载体对胃癌细胞SGC7901增殖的抑制作用；通过Transwell小室对细胞迁移侵袭能力进行检测,获得重组腺病毒载体对胃癌细胞SGC7901侵袭力的影响参数。
4、构建人胃癌细胞SGC7901裸鼠腹腔原位移植瘤实验动物模型,观察CXCR4-shRNA腺病毒载体对胃癌细胞裸鼠腹腔移植瘤生长及转移的影响；应用Western-blot方法检测腹腔转移瘤组织内的CXCR4、VEGF-C和MMP-2蛋白的表达情况。
结果：
1、正常胃组织中均未检测到CXCR4基因的阳性表达(0/41);CXCR4在胃腺癌组织中的阳性表达率明显增高,85例胃癌组织中CXCR4染色阳性59例,63例淋巴转移癌中CXCR4阳性表达56例,17例肝脏转移癌中CXCR4阳性表达15例,12例腹膜转移癌中CXCR4阳性表达12例,染色阳性率分别为69.4%、88.9%、88.2%和100%,除淋巴与肝脏转移癌外,各组间相比均具有显著性差异(P<0.05)。在胃腺癌原发灶中CXCR4和CXCL12的表达与大体形态(二者P均<0.01)、浸润深度(二者P均<0.05；进展期>早期)、肿瘤对淋巴管(两者P均<0.01)和血管(两者P均<0.01)的侵袭、淋巴结(两者P均<0.01)肝脏(两者P均<0.01)和腹膜(两者P均<0.01)转移有关,且二者表达呈正相关；而与年龄(≤65岁与>65岁)(P分别为0.352和0.417)、性别(P分别为0.55和0.20)、肿瘤位置(P分别为0.65和0.09)和肿瘤大小(P分别为0.10和0.30)无关。CXCR4阳性表达胃癌组患者3年生存率为44.07%,明显低于CXCR4阴性表达组65.38%,两组生存率间差异有显著性意义(P<0.05)。CXCR4mRNA在4株细胞株中的2株(SGC7901和BGC-823细胞株)中高表达,而在SNU-1和NCI-N87细胞株中低表达。
2、经基因测序、PCR检测及酶切鉴定表明,重组腺病毒真核表达载体shpAd-hCXCR4-eGFP构建成功,同时成功构建阴性对照病毒载体shpAd-control-eGFP。
3、荧光倒置显微镜下观察到转染重组腺病毒表达载体及其阳性、阴性对照的HEK293A细胞内有大量绿色荧光表达并有明显的细胞病变效应(cytopathic effect,CPE)腺病毒包装成功,病毒滴度检测为4X109PFU/ml。最佳MOI=100,腺病毒载体最佳转染体积为12.5μ1。
4、将shpAd-hCXCR4-eGFP转染至胃癌细胞SGC7901后,检测RT-PCR及Western blot结果表明：CXCR4-shRNAl组的CXCR4mRNA及蛋白表达量明显低于其他两组,具有显著性差异(P<0.05)；三组实验组与空白组具有显著性差异(P<0.01)。测定CXCR4-shRNAl组沉默效率最高,沉默效率为78.08%。
5、将CXCR4-shRNAl转染至胃癌细胞SGC7901后,MTT法绘制生长曲线,结果表明：空白组与对照组细胞生长迅速,两组之间无显著性差异(P>0.05),实验组经腺病毒载体转染后细胞增殖程度显著减少,与前两组相比有显著性差异(P<0.05)。
6、细胞侵袭实验结果表明：在CXCL12的趋化24h后,空白组、阴性对照组和实验组三组穿膜的细胞数量分别为264.7±15.7、270.5±16.9、113.8±9.2,shRNA-CXCR4腺病毒载体组比较空白组和对照组穿膜细胞数量明显减少,具有显著性差异(P<0.05)。而空白组与对照组相比数值没有明显的减少,无显著性差异(P>0.05)。
7、成功构建三组胃癌细胞裸鼠腹腔原位移植瘤模型,与SGC7901组、空病毒组相比,CXCR4-shRNA组腹水产生时间明显延后且量较少,具有显著性差异(P<0.05)；腹腔各脏器转移瘤的个数、重量、直径均要低于后两者,具有显著性差异(P<0.05)；抑瘤率达到68.1%；裸鼠存活时间延长,存活率较高,生命延长率达到40.4%,具有显著性差异(P<0.05)。而SGC7901组与空病毒组对比,以上结果均无显著性差异(P>0.05)。Western Blot检测三组转移瘤组织CXCR4、VEGF-C和MMP-2蛋白表达情况,结果显示CXCR4-shRNA组与SGC7901组、空病毒组相比,CXCR4、VEGF-C和MMP-2蛋白表达低,具有显著性差异(P<0.01); SGC7901组与空病毒组两组相比,CXCR4、VEGF-C和MMP-2蛋白表达无显著性差异(P>0.05)。
结论：
1、研究发现,与正常胃组织相比,CXCR4/CXCL12在胃癌组织中高表达；随着胃癌的发生、进展和转移,CXCR4的表达逐渐上调,提示CXCR4在胃癌细胞中的阳性表达可能促进胃癌的发生、增殖、生长及转移。CXCR4/CXCL12过度表达的胃癌细胞具有更强的侵袭性和转移性,该生物轴也很可能成为协同胃癌进展、侵袭、转移并提示预后不良的一对重要的分子标志物。胃癌细胞株SGC7901能够高表达CXCR4,可以作为进一步细胞及分子领域研究的实验对象。
2、成功构建CXCR4-shRNA腺病毒表达载体并筛选出沉默效率最高的一组。
3、成功将CXCR4-shRNA重组腺病毒载体转染至胃癌细胞SGC7901并证明重组腺病毒载体CXCR4-shRNA可抑制胃癌细胞SGC7901中CXCR4基因的mRNA和蛋白的表达。
4、重组腺病毒载体CXCR4-shRNA能显著抑制胃癌细胞SGC7901的增殖。
5、重组腺病毒载体CXCR4-shRNA能降低胃癌细胞SGC7901的侵袭力。
6、CXCR4-shRNA腺病毒载体可以有效地抑制原位移植瘤裸鼠腹水的产生,抑制肿瘤的生长,降低肿瘤的体积及重量,抑制胃癌细胞向各个腹腔脏器的转移；同时显著延长裸鼠的生存时间,降低死亡率。CXCR4-shRNA腺病毒载体下调CXCR4表达后,VEGF-C、MMP-2蛋白的表达下降,胃癌组织的生长、转移受到抑制。VEGF-C、MMP-2表达下调可能是CXCR4-shRNA腺病毒载体抑制胃癌生长转移的机制之一。
Objective
The gastric cancer is one of the most common malignant tumor in China, the mortality rate is still the first in malignant tumor. Because of its dormant morbilidy, that can not be often discoveried and diagnosised in time, which bright about its high fatality rate cannot be reduced. Operation resection maybe the only way to cure gastric cancer, but even via radical operation, the clinical survival rate in five years still hovers at around50%, and the patients's postoperative quality of life decrease obviously. An earlier onset of cancer cell metastasis is the main reason which due to the poor prognosis of advanced gastric cancer, but the current understanding of the molecular mechanism of gastric cancer is still belong to poorly understood area. Recent research shows that, chemokine factor CXCL12and its specific receptor CXCR4form CXCR4/CXCL12biological axis and the CXCR4/CXCL12biological axis plays an important role in variety of tumor and organ specific metastasis. But the effect and mechanism of CXCR4on invasion,metastasisin of gastric cancer remain controversial. Thereforein this study, the expression of CXCR4/CXCL12in different gastric cancer tissues were observed and relationships between the expression level and gastric carcinoma clinical pathology parameters including patient survival rate were observed firstly. Simultaneous, the expression of CXCR4gene mRNA in four kinds of human gastric cancer cell lines were observed.Secondly, CXCR4-shRNA adenovirus eukaryotic expression vector was transfected into human gastric cancer cell SGC7901to block the expression of CXCR4,and the inhibitory effect was observed. Finally, after the orthotopic transplantation tumor model of human gastric cance was established in nude mice, the biological trait of gastric cancer cell were observed to to reveal the role and possible mechanisms of CXCR4in the course of gastric cancer invading and transferrng and provide new ideas to prevent and control the invasion and metastasis of gastric caner.
Method
1.85cases of fresh gastric cancer tissue specimens and41cases of normal gastric tissue far from the tumor margin of more than5cm were surgically resected from patients with gastric cancer who underwent gastrectomy in The First Affiliated Hospital of Liaoning Medical College from2008February to2009February.the immunohistochemical method was used to detect the CXCR4and CXCL12expression in different tissues, the relationship between CXCR4/CXCL12and clinical pathological parameters of gastric cancer were observed, their expression in different tissues and the relationship with invasion and metastasis of gastric cancer were researched. RT-PCR method was used to detect the CXCR4mRNA's expression in four kinds of gastric cancer cell.
2. CXCR4gene sequence was Retrieved in the GENBANK, three short hairpin RNA (shRNA) were designed and then synthetized after homology search without error.PCR, enzyme cut and DNA connecting method were used to construct eukaryotic expression vector plasmid shpDown-hCXCR4-eGFP, after positive plamsid was picked up and sequenced and authenticated by positive clong, eukaryotic expression vector was embeded into PL-Dest adenovirus skeleton carriers (pAd/PL-Dest) according to the Gateway technology, via gene sequencing, PCR and Pac I endonuclease identification to obtain recombinant adenovirus vector shpAd-hCXCR4-eGFP. shpAd-control-eGFP control group was constructed by the same way. the recombinant adenovirus plasmid was ultra pure extracted by getting rid of endotoxin, large fragments were recovered by gel after Pac I endonuclease electrophoresis, HEK293A cell was transfected by means of liposome Lipofectamine2000, the eGFP gene green flourescent expression was used to detect transfection situation and packaged virus, virus titer was ermined by end-point dilution method. gastric cancer cell SGC7901was transfected with the optimal multiplicity of infection (MOI),then RT-PCR and Western-blot were used to detect silence efficiency of each group,then screened out the highest silence efficiency group.
3. Experiment group and negative control group were transfected with optimal MOI and set up blank control group. RT-PCR and Western-blot were used to detect CXCR4mRNA and protein expression in gastric cancer cells after CXCR4gene was silenced by recombinant adenovirus vector, MTT method and absorbance were used to draw SGC7901cell growth curve, and detect recombinant adenovirus vector's inhibition effect on proliferation of gastric carcinoma cell SGC7901; Transwell chamber was used to detect cancer cell migration and invasion ability, obtain the influence parameter of recombinant adenovirus vector effect on gastric cancer cell SGC7901invasion.
4. Human gastric carcinoma cell SGC7901abdominal cavity orthotopic implantation tumor metastasis model was constructed in nude mice, and then investigated the effect of CXCR4-shRNA adenovirus vector on abdominal cavity metastasis of gastric cancer cells in nude mice; Western Blot was used to detect CXCR4,VEGF-C, MMP-2protein expression in abdominal metastasis tissue.
Result
1. CXCR4gene positive expression in normal gastric tissues were not detected (0/41); the positive expression rate of CXCR4in gastric was significantly higher, in85cases of gastric cancer tissue specimens,59cases' CXCR4dye were positive; in63cases of lymphatic metastases carcinoma,56cases' CXCR4dye were positive; in17cases of liver metastatic carcinoma,15cases' CXCR4dye were positive; in12cases of peritoneal metastatic carcinoma,12cases' CXCR4dye were positive, the dye positive rates were69.4%,88.9%,88.2%and100%respectively, in addition to lymphatic carcinoma and liver carcinoma, it has significant difference among groups (P<0.05). In gastric adenocarcinoma primary foci,the expression of CXCR4and CXCL12not only were correlated with the tumor general morphology (P<0.01), depth of invasion (both P<0.05;advanced>early), but also were correlated with tumor's invasion on lymphatic (both P<0.05) and vascular (both P<0.05)and metastasis of lymph node (both P<0.05)、liver (both P<0.05) and peritoneal (both P<0.05); but had nothing to do with age (<65years and>65years old)(P=0.352and0.417), sex (P were0.55and0.2respectively), location of the tumor (P were0.65and0.02respectively) and tumor size (P were0.10and0.30respectively). The patients of positive expression of CXCR4in gastric cancer, the3year survival rate was44.07%, which significant lower than that CXCR4negative expression group, the survival rate difference between two groups had prominent significance difference(P<0.05).in4cell strains, CXCR4mRNA highly expressed in2strains (SGC7901and BGC-823cell lines), and in NCI-N87and SNU-lcell strains, CXCR4mRNA were low expression.
2. Gene sequencing, PCR detection and enzyme digestion showed that recombinant adenovirus eukaryotic expression vector shpAd-hCXCR4-eGFP was constructed successfully, the negative control virus vector shpAd-control-eGFP was constructed successfully at the same time.
3. Inverted fluorescence microscope found that transfection of recombinant adenovirus expression vector and its positive, negative control in HEK293A cells had a large number of green fluorescent expressed and obvious cytopathic effect (cytopathic effect, CPE). Adenovirus was packaged successfully, virus titers was4x109PFU/ml. The best MOI=100,the optimal transfection volume of adenovirus vector was12.5L.
4. After the shpAd-hCXCR4-eGFP was transfected into gastric cancer cell SGC7901, results of RT-PCR and Western blot detection showed that:in CXCR4-shRNA1group, CXCR4mRNA and protein expression were significantly lower than the other two groups with significant difference (P<0.05); the difference between three experimental groups and the control group, the negative control group was significant (P<0.01).CXCR4-shRNA1group silence efficiency was the highest,about78.08%.
5. After the CXCR4-shRNA1was transfected into gastric cancer cell SGC7901, via MTT method to draw growth curve, the results show that:the blank group and the control group cells grew rapidly, the difference between that two groups had no significant (P>0.05), the degree of cell proliferation decreased significantly after the experimental group was transfected with adenovirus vector, and compared to the former two groups, there was significant difference between them(P<0.05).
6. Cell invasion experiment results showed that:in the CXCL12Chemotaxis24h, the transmembrane cell number in blank group, negative control group and the experimental group were264.7±15.7,270.5±16.9,113.8±9.2, compared shRNA-CXCR4adenovirus vectors to blank group and the control group, the transmembrane cell number significantly decreased, difference between them was significant (P<0.05). And the transmembrane cell number in blank group and control group didn't obviously decrease, no significant difference between that two groups(P>0.05).
7. Three groups of gastric cancer cell abdominal cavity metastasis tumor model were successfully built in nude mice, CXCR4-shRNA group was compared with SGC7901group and empty virus group, ascites produced time was delayed and less capacity; abdominal organs metastasis tumors'number, weight, diameter were lower than the latter two, the tumor inhibition rate reached to68.1%; nude mice's survival time and survival rate was prolonged, life extension rate reached to40.4%. SGC7901group compared with empty virus group, the results had no significant difference (P>0.05). Western Blo was used to detect CXCR4.VEGF-C, MMP-2protein expression in three groups of abdominal metastasis tissue,the results show that CXCR4-shRNA group compared with SGC7901group, empty virus group, CXCR4,VEGF-C, MMP-2protein expression were low with significant difference (P<0.01), SGC7901group was contrasted to empty viral group, the protein expression of CXCR4,VEGF-C, MMP-2had no significant difference (P>0.05).
Conclusion
1. Study found that compared with normal gastric tissues, CXCR4/CXCL12in gastric cancer tissues presented high expression level, alone with occurrence、progression and metastasis of gastric cancer, CXCR4expression gradually increased.it prompt that CXCR4high positive expression level can promote gastric career to occur、reproduct、grow and transfer.gastric cancer cells with CXCR4/CXCL12excessive expression were more aggressive and metastatic, the biological axis may become an important molecular markers to cooperate with gastric cancer progression, invasion, metastasis and indicate poor prognosis.gastric carcinoma cell strain SGC7901was capable of high expression of CXCR4, which can be used as further cell and molecular research subjects.
2. CXCR4-shRNA adenovirus expression vector was constructed successfully and the highest scilence efficiency group was screened out.
3. CXCR4-shRNA recombinant adenovirus vector was transfected into gastric cancer cell SGC7901successfully and it demonstrated that recombinant adenovirus vector CXCR4-shRNA can inhibit CXCR4gene mRNA and protein expression in gastric cancer cell strain SGC7901.
4. Recombinant adenovirus vector CXCR4-shRNA can significantly inhibit the proliferation of gastric cancer cell SGC7901.
5. Recombinant adenovirus vector CXCR4-shRNA can reduce invasion force of gastric cancer cell SGC7901.
6. CXCR4-shRNA adenovirus vector can effectively inhibit orthotopic transplantation tumor nude mice's ascites production, inhibit tumor growth, reduce tumor volume and weight, inhibit gastric cancer cell from transfering to the various organs of the abdominal cavity;at the same time,can significantly prolong nude mice's survival time, reduce mortality. CXCR4-shRNA adenovirus vector downregulated the expression of CXCR4, then VEGF-C, MMP-2protein expression in gastric carcinoma tissue decreased corresponding, the growth and metastasis of gastric cancer tissue was inhibited.VEGF-C, MMP-2expression downregulation may be one of the mechanism of CXCR4-shRNA adenovirus vector inhibits the growth and metastasis of gastric carcinoma.
方法：
1、收集辽宁医学院附属第一医院普通外科2008年2月至2009年2月外科手术切除的85例新鲜胃癌组织标本及距肿瘤边缘5cm以上的正常胃组织41例。应用免疫组织化学方法检测不同组织内CXCR4和CXCL12的表达情况,系统分析它们与临床病理参数之间的关系,研究二者在胃癌不同组织中的表达与胃癌侵袭转移的关系。用RT-PCR方法分别检测4种人胃癌细胞株中CXCR4mRNA的表达情况。
2、在GENBANK中检索基因CXCR4序列,设计3条短发夹RNA (shRNA)并经同源性检索无误后合成,利用PCR、酶切及DNA连接构建真核表达载体shpDown-hCXCR4-eGFP,挑取阳性质粒送交阳性克隆测序鉴定后,再根据Gateway技术将真核表达载体嵌入到PL-Dest腺病毒骨架载体(pAd/PL-Dest)中,经基因测序、PCR及Pac I酶切鉴定获得重组腺病毒载体shpAd-hCXCR4-eGFP。同理构建对照组shpAd-control-eGFP。去内毒素超纯提取该重组腺病毒质粒,经Pac Ⅰ酶切电泳后凝胶回收大片段,提纯并通过脂质体Lipofectamine2000介导转染HEK293A细胞,通过eGFP基因绿色荧光表达检测转染情况并进行病毒包装,采用终点稀释法进行病毒滴度测定。以最佳感染复数(MOI)转染胃癌细胞SGC7901后,利用RT-PCR和Western-blot方法检测各组沉默效率,筛选出沉默效率最高的一组。
3、分别以最佳MOI转染实验组和阴性对照组,并设立空白对照组。将转染后的胃癌细胞分别利用RT-PCR和Western-blot检测重组腺病毒载体沉默CXCR4基因mRNA和蛋白的表达情况；通过MTT法利用吸光度绘制SGC7901细胞生长曲线,检测重组腺病毒载体对胃癌细胞SGC7901增殖的抑制作用；通过Transwell小室对细胞迁移侵袭能力进行检测,获得重组腺病毒载体对胃癌细胞SGC7901侵袭力的影响参数。
4、构建人胃癌细胞SGC7901裸鼠腹腔原位移植瘤实验动物模型,观察CXCR4-shRNA腺病毒载体对胃癌细胞裸鼠腹腔移植瘤生长及转移的影响；应用Western-blot方法检测腹腔转移瘤组织内的CXCR4、VEGF-C和MMP-2蛋白的表达情况。
结果：
1、正常胃组织中均未检测到CXCR4基因的阳性表达(0/41);CXCR4在胃腺癌组织中的阳性表达率明显增高,85例胃癌组织中CXCR4染色阳性59例,63例淋巴转移癌中CXCR4阳性表达56例,17例肝脏转移癌中CXCR4阳性表达15例,12例腹膜转移癌中CXCR4阳性表达12例,染色阳性率分别为69.4%、88.9%、88.2%和100%,除淋巴与肝脏转移癌外,各组间相比均具有显著性差异(P<0.05)。在胃腺癌原发灶中CXCR4和CXCL12的表达与大体形态(二者P均<0.01)、浸润深度(二者P均<0.05；进展期>早期)、肿瘤对淋巴管(两者P均<0.01)和血管(两者P均<0.01)的侵袭、淋巴结(两者P均<0.01)肝脏(两者P均<0.01)和腹膜(两者P均<0.01)转移有关,且二者表达呈正相关；而与年龄(≤65岁与>65岁)(P分别为0.352和0.417)、性别(P分别为0.55和0.20)、肿瘤位置(P分别为0.65和0.09)和肿瘤大小(P分别为0.10和0.30)无关。CXCR4阳性表达胃癌组患者3年生存率为44.07%,明显低于CXCR4阴性表达组65.38%,两组生存率间差异有显著性意义(P<0.05)。CXCR4mRNA在4株细胞株中的2株(SGC7901和BGC-823细胞株)中高表达,而在SNU-1和NCI-N87细胞株中低表达。
2、经基因测序、PCR检测及酶切鉴定表明,重组腺病毒真核表达载体shpAd-hCXCR4-eGFP构建成功,同时成功构建阴性对照病毒载体shpAd-control-eGFP。
3、荧光倒置显微镜下观察到转染重组腺病毒表达载体及其阳性、阴性对照的HEK293A细胞内有大量绿色荧光表达并有明显的细胞病变效应(cytopathic effect,CPE)腺病毒包装成功,病毒滴度检测为4X109PFU/ml。最佳MOI=100,腺病毒载体最佳转染体积为12.5μ1。
4、将shpAd-hCXCR4-eGFP转染至胃癌细胞SGC7901后,检测RT-PCR及Western blot结果表明：CXCR4-shRNAl组的CXCR4mRNA及蛋白表达量明显低于其他两组,具有显著性差异(P<0.05)；三组实验组与空白组具有显著性差异(P<0.01)。测定CXCR4-shRNAl组沉默效率最高,沉默效率为78.08%。
5、将CXCR4-shRNAl转染至胃癌细胞SGC7901后,MTT法绘制生长曲线,结果表明：空白组与对照组细胞生长迅速,两组之间无显著性差异(P>0.05),实验组经腺病毒载体转染后细胞增殖程度显著减少,与前两组相比有显著性差异(P<0.05)。
6、细胞侵袭实验结果表明：在CXCL12的趋化24h后,空白组、阴性对照组和实验组三组穿膜的细胞数量分别为264.7±15.7、270.5±16.9、113.8±9.2,shRNA-CXCR4腺病毒载体组比较空白组和对照组穿膜细胞数量明显减少,具有显著性差异(P<0.05)。而空白组与对照组相比数值没有明显的减少,无显著性差异(P>0.05)。
7、成功构建三组胃癌细胞裸鼠腹腔原位移植瘤模型,与SGC7901组、空病毒组相比,CXCR4-shRNA组腹水产生时间明显延后且量较少,具有显著性差异(P<0.05)；腹腔各脏器转移瘤的个数、重量、直径均要低于后两者,具有显著性差异(P<0.05)；抑瘤率达到68.1%；裸鼠存活时间延长,存活率较高,生命延长率达到40.4%,具有显著性差异(P<0.05)。而SGC7901组与空病毒组对比,以上结果均无显著性差异(P>0.05)。Western Blot检测三组转移瘤组织CXCR4、VEGF-C和MMP-2蛋白表达情况,结果显示CXCR4-shRNA组与SGC7901组、空病毒组相比,CXCR4、VEGF-C和MMP-2蛋白表达低,具有显著性差异(P<0.01); SGC7901组与空病毒组两组相比,CXCR4、VEGF-C和MMP-2蛋白表达无显著性差异(P>0.05)。
结论：
1、研究发现,与正常胃组织相比,CXCR4/CXCL12在胃癌组织中高表达；随着胃癌的发生、进展和转移,CXCR4的表达逐渐上调,提示CXCR4在胃癌细胞中的阳性表达可能促进胃癌的发生、增殖、生长及转移。CXCR4/CXCL12过度表达的胃癌细胞具有更强的侵袭性和转移性,该生物轴也很可能成为协同胃癌进展、侵袭、转移并提示预后不良的一对重要的分子标志物。胃癌细胞株SGC7901能够高表达CXCR4,可以作为进一步细胞及分子领域研究的实验对象。
2、成功构建CXCR4-shRNA腺病毒表达载体并筛选出沉默效率最高的一组。
3、成功将CXCR4-shRNA重组腺病毒载体转染至胃癌细胞SGC7901并证明重组腺病毒载体CXCR4-shRNA可抑制胃癌细胞SGC7901中CXCR4基因的mRNA和蛋白的表达。
4、重组腺病毒载体CXCR4-shRNA能显著抑制胃癌细胞SGC7901的增殖。
5、重组腺病毒载体CXCR4-shRNA能降低胃癌细胞SGC7901的侵袭力。
6、CXCR4-shRNA腺病毒载体可以有效地抑制原位移植瘤裸鼠腹水的产生,抑制肿瘤的生长,降低肿瘤的体积及重量,抑制胃癌细胞向各个腹腔脏器的转移；同时显著延长裸鼠的生存时间,降低死亡率。CXCR4-shRNA腺病毒载体下调CXCR4表达后,VEGF-C、MMP-2蛋白的表达下降,胃癌组织的生长、转移受到抑制。VEGF-C、MMP-2表达下调可能是CXCR4-shRNA腺病毒载体抑制胃癌生长转移的机制之一。
Objective
The gastric cancer is one of the most common malignant tumor in China, the mortality rate is still the first in malignant tumor. Because of its dormant morbilidy, that can not be often discoveried and diagnosised in time, which bright about its high fatality rate cannot be reduced. Operation resection maybe the only way to cure gastric cancer, but even via radical operation, the clinical survival rate in five years still hovers at around50%, and the patients's postoperative quality of life decrease obviously. An earlier onset of cancer cell metastasis is the main reason which due to the poor prognosis of advanced gastric cancer, but the current understanding of the molecular mechanism of gastric cancer is still belong to poorly understood area. Recent research shows that, chemokine factor CXCL12and its specific receptor CXCR4form CXCR4/CXCL12biological axis and the CXCR4/CXCL12biological axis plays an important role in variety of tumor and organ specific metastasis. But the effect and mechanism of CXCR4on invasion,metastasisin of gastric cancer remain controversial. Thereforein this study, the expression of CXCR4/CXCL12in different gastric cancer tissues were observed and relationships between the expression level and gastric carcinoma clinical pathology parameters including patient survival rate were observed firstly. Simultaneous, the expression of CXCR4gene mRNA in four kinds of human gastric cancer cell lines were observed.Secondly, CXCR4-shRNA adenovirus eukaryotic expression vector was transfected into human gastric cancer cell SGC7901to block the expression of CXCR4,and the inhibitory effect was observed. Finally, after the orthotopic transplantation tumor model of human gastric cance was established in nude mice, the biological trait of gastric cancer cell were observed to to reveal the role and possible mechanisms of CXCR4in the course of gastric cancer invading and transferrng and provide new ideas to prevent and control the invasion and metastasis of gastric caner.
Method
1.85cases of fresh gastric cancer tissue specimens and41cases of normal gastric tissue far from the tumor margin of more than5cm were surgically resected from patients with gastric cancer who underwent gastrectomy in The First Affiliated Hospital of Liaoning Medical College from2008February to2009February.the immunohistochemical method was used to detect the CXCR4and CXCL12expression in different tissues, the relationship between CXCR4/CXCL12and clinical pathological parameters of gastric cancer were observed, their expression in different tissues and the relationship with invasion and metastasis of gastric cancer were researched. RT-PCR method was used to detect the CXCR4mRNA's expression in four kinds of gastric cancer cell.
2. CXCR4gene sequence was Retrieved in the GENBANK, three short hairpin RNA (shRNA) were designed and then synthetized after homology search without error.PCR, enzyme cut and DNA connecting method were used to construct eukaryotic expression vector plasmid shpDown-hCXCR4-eGFP, after positive plamsid was picked up and sequenced and authenticated by positive clong, eukaryotic expression vector was embeded into PL-Dest adenovirus skeleton carriers (pAd/PL-Dest) according to the Gateway technology, via gene sequencing, PCR and Pac I endonuclease identification to obtain recombinant adenovirus vector shpAd-hCXCR4-eGFP. shpAd-control-eGFP control group was constructed by the same way. the recombinant adenovirus plasmid was ultra pure extracted by getting rid of endotoxin, large fragments were recovered by gel after Pac I endonuclease electrophoresis, HEK293A cell was transfected by means of liposome Lipofectamine2000, the eGFP gene green flourescent expression was used to detect transfection situation and packaged virus, virus titer was ermined by end-point dilution method. gastric cancer cell SGC7901was transfected with the optimal multiplicity of infection (MOI),then RT-PCR and Western-blot were used to detect silence efficiency of each group,then screened out the highest silence efficiency group.
3. Experiment group and negative control group were transfected with optimal MOI and set up blank control group. RT-PCR and Western-blot were used to detect CXCR4mRNA and protein expression in gastric cancer cells after CXCR4gene was silenced by recombinant adenovirus vector, MTT method and absorbance were used to draw SGC7901cell growth curve, and detect recombinant adenovirus vector's inhibition effect on proliferation of gastric carcinoma cell SGC7901; Transwell chamber was used to detect cancer cell migration and invasion ability, obtain the influence parameter of recombinant adenovirus vector effect on gastric cancer cell SGC7901invasion.
4. Human gastric carcinoma cell SGC7901abdominal cavity orthotopic implantation tumor metastasis model was constructed in nude mice, and then investigated the effect of CXCR4-shRNA adenovirus vector on abdominal cavity metastasis of gastric cancer cells in nude mice; Western Blot was used to detect CXCR4,VEGF-C, MMP-2protein expression in abdominal metastasis tissue.
Result
1. CXCR4gene positive expression in normal gastric tissues were not detected (0/41); the positive expression rate of CXCR4in gastric was significantly higher, in85cases of gastric cancer tissue specimens,59cases' CXCR4dye were positive; in63cases of lymphatic metastases carcinoma,56cases' CXCR4dye were positive; in17cases of liver metastatic carcinoma,15cases' CXCR4dye were positive; in12cases of peritoneal metastatic carcinoma,12cases' CXCR4dye were positive, the dye positive rates were69.4%,88.9%,88.2%and100%respectively, in addition to lymphatic carcinoma and liver carcinoma, it has significant difference among groups (P<0.05). In gastric adenocarcinoma primary foci,the expression of CXCR4and CXCL12not only were correlated with the tumor general morphology (P<0.01), depth of invasion (both P<0.05;advanced>early), but also were correlated with tumor's invasion on lymphatic (both P<0.05) and vascular (both P<0.05)and metastasis of lymph node (both P<0.05)、liver (both P<0.05) and peritoneal (both P<0.05); but had nothing to do with age (<65years and>65years old)(P=0.352and0.417), sex (P were0.55and0.2respectively), location of the tumor (P were0.65and0.02respectively) and tumor size (P were0.10and0.30respectively). The patients of positive expression of CXCR4in gastric cancer, the3year survival rate was44.07%, which significant lower than that CXCR4negative expression group, the survival rate difference between two groups had prominent significance difference(P<0.05).in4cell strains, CXCR4mRNA highly expressed in2strains (SGC7901and BGC-823cell lines), and in NCI-N87and SNU-lcell strains, CXCR4mRNA were low expression.
2. Gene sequencing, PCR detection and enzyme digestion showed that recombinant adenovirus eukaryotic expression vector shpAd-hCXCR4-eGFP was constructed successfully, the negative control virus vector shpAd-control-eGFP was constructed successfully at the same time.
3. Inverted fluorescence microscope found that transfection of recombinant adenovirus expression vector and its positive, negative control in HEK293A cells had a large number of green fluorescent expressed and obvious cytopathic effect (cytopathic effect, CPE). Adenovirus was packaged successfully, virus titers was4x109PFU/ml. The best MOI=100,the optimal transfection volume of adenovirus vector was12.5L.
4. After the shpAd-hCXCR4-eGFP was transfected into gastric cancer cell SGC7901, results of RT-PCR and Western blot detection showed that:in CXCR4-shRNA1group, CXCR4mRNA and protein expression were significantly lower than the other two groups with significant difference (P<0.05); the difference between three experimental groups and the control group, the negative control group was significant (P<0.01).CXCR4-shRNA1group silence efficiency was the highest,about78.08%.
5. After the CXCR4-shRNA1was transfected into gastric cancer cell SGC7901, via MTT method to draw growth curve, the results show that:the blank group and the control group cells grew rapidly, the difference between that two groups had no significant (P>0.05), the degree of cell proliferation decreased significantly after the experimental group was transfected with adenovirus vector, and compared to the former two groups, there was significant difference between them(P<0.05).
6. Cell invasion experiment results showed that:in the CXCL12Chemotaxis24h, the transmembrane cell number in blank group, negative control group and the experimental group were264.7±15.7,270.5±16.9,113.8±9.2, compared shRNA-CXCR4adenovirus vectors to blank group and the control group, the transmembrane cell number significantly decreased, difference between them was significant (P<0.05). And the transmembrane cell number in blank group and control group didn't obviously decrease, no significant difference between that two groups(P>0.05).
7. Three groups of gastric cancer cell abdominal cavity metastasis tumor model were successfully built in nude mice, CXCR4-shRNA group was compared with SGC7901group and empty virus group, ascites produced time was delayed and less capacity; abdominal organs metastasis tumors'number, weight, diameter were lower than the latter two, the tumor inhibition rate reached to68.1%; nude mice's survival time and survival rate was prolonged, life extension rate reached to40.4%. SGC7901group compared with empty virus group, the results had no significant difference (P>0.05). Western Blo was used to detect CXCR4.VEGF-C, MMP-2protein expression in three groups of abdominal metastasis tissue,the results show that CXCR4-shRNA group compared with SGC7901group, empty virus group, CXCR4,VEGF-C, MMP-2protein expression were low with significant difference (P<0.01), SGC7901group was contrasted to empty viral group, the protein expression of CXCR4,VEGF-C, MMP-2had no significant difference (P>0.05).
Conclusion
1. Study found that compared with normal gastric tissues, CXCR4/CXCL12in gastric cancer tissues presented high expression level, alone with occurrence、progression and metastasis of gastric cancer, CXCR4expression gradually increased.it prompt that CXCR4high positive expression level can promote gastric career to occur、reproduct、grow and transfer.gastric cancer cells with CXCR4/CXCL12excessive expression were more aggressive and metastatic, the biological axis may become an important molecular markers to cooperate with gastric cancer progression, invasion, metastasis and indicate poor prognosis.gastric carcinoma cell strain SGC7901was capable of high expression of CXCR4, which can be used as further cell and molecular research subjects.
2. CXCR4-shRNA adenovirus expression vector was constructed successfully and the highest scilence efficiency group was screened out.
3. CXCR4-shRNA recombinant adenovirus vector was transfected into gastric cancer cell SGC7901successfully and it demonstrated that recombinant adenovirus vector CXCR4-shRNA can inhibit CXCR4gene mRNA and protein expression in gastric cancer cell strain SGC7901.
4. Recombinant adenovirus vector CXCR4-shRNA can significantly inhibit the proliferation of gastric cancer cell SGC7901.
5. Recombinant adenovirus vector CXCR4-shRNA can reduce invasion force of gastric cancer cell SGC7901.
6. CXCR4-shRNA adenovirus vector can effectively inhibit orthotopic transplantation tumor nude mice's ascites production, inhibit tumor growth, reduce tumor volume and weight, inhibit gastric cancer cell from transfering to the various organs of the abdominal cavity;at the same time,can significantly prolong nude mice's survival time, reduce mortality. CXCR4-shRNA adenovirus vector downregulated the expression of CXCR4, then VEGF-C, MMP-2protein expression in gastric carcinoma tissue decreased corresponding, the growth and metastasis of gastric cancer tissue was inhibited.VEGF-C, MMP-2expression downregulation may be one of the mechanism of CXCR4-shRNA adenovirus vector inhibits the growth and metastasis of gastric carcinoma.
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