转移性肝癌细胞抵抗失巢凋亡的分子机制及相关分子研究
摘要
肝癌是一种在世界范围内发病率和死亡率都非常高的恶性肿瘤,由于进展快,转移、复发率高而使其病人治疗后5年生存率较低,如果能够阐明肝癌转移发生的分子机制并从分子水平对其转移进行早期干预,将会极大地改善肝癌病人的治疗效果。
为了能够到达异位形成转移癌,肿瘤细胞必须完成一系列有序的选择性步骤,其中包括脱离原位癌,侵袭进入脉管系统,在脉管系统中存活,然后侵袭进入异位器官的基质层,长出脉管系统并增殖。转移过程中的肿瘤细胞必须克服在这一过程中所要面临的种种压力才能够最终到达异位形成转移灶。这些压力包括机体的免疫监视能力、体内的乏氧环境、各种抗肿瘤药物的攻击等。另外,肿瘤转移过程中还必须有多种效应分子的参与,其中很大一部分的mRNA和蛋白表达水平在低氧诱导下上调。乏氧被认为是肿瘤细胞在生存过程中必须要克服的一个因素,同时,有越来越多的证据显示乏氧能够促进肿瘤发展。
本课题在成功建立了转移性肝癌细胞模型的基础上,对其有效性以及转移能力进行了验证,并从转移性肝癌细胞对各种刺激(包括外源性TRAIL、AKT/ERK通路抑制剂)的反应性来探讨其抵抗失巢凋亡的分子机制,然后进一步筛选并论证了抵抗失巢凋亡和抵抗低氧刺激的相关分子,为临床上判断肝癌预后以及靶向性治疗奠定坚实的理论基础和实验基础。
目的:
1.建立一种能够模拟转移过程中的肝癌细胞在脉管系统中存活状态的转移性肝癌细胞模型,并研究其聚集稳定性和转移能力。
2.通过研究转移性肝癌细胞对TRAIL诱导凋亡的反应以及对抗肿瘤药物AKT/ERK通路抑制剂的反应来研究其抵抗失巢凋亡的分子机制。
3.探讨转移性肝癌细胞对乏氧刺激的反应。
4.筛选抵抗失巢凋亡和抵抗乏氧刺激相关的分子,以用于抗肿瘤转移的早期诊断、预防和治疗。
方法
1.转移性肝癌细胞模型的建立以及其聚集率和转移能力分析
1.1.转移性肝癌细胞模型的建立
BEL7402和SMMC7721肝癌细胞系以PRMI1640加10%胎牛血清为培养液,在37℃、5%CO_2条件下培养至对数生长期,用0.25%的胰蛋白酶消化成单细胞悬液,以RPMI1640调整好浓度后,加入Poly-HEMA培养板中继续培养24 h,得到悬浮生长的转移性肝癌细胞模型。
1.2.转移性肝癌细胞模型建立条件探索
细胞培养至对数生长期并制备成单细胞悬液后,96孔板中接种不同细胞数量0.5×10~4,1×10~4,1.5×10~4,3×10~4,3×10~4,前四个组细胞接种前Poly-HEMA培养板用无菌PBS洗3遍,第五组不洗涤,每组设六孔重复,继续培养24 h,用CCK-8和trypan blue染色检测细胞活性和死亡率。
1.3.转移性肝癌细胞的聚集率
在铺有Poly-HEMA的96孔板中培养悬浮生长的转移性肝癌细胞,24小时后计数孔中单个细胞数,然后依据如下公式计算聚集率:聚集率=1-培养24h后孔中单个细胞数╱接种细胞数×100%。
1.4.Transwell检测转移性肝癌细胞的侵袭力和运动力
取对数生长期肝癌细胞的单细胞悬液,调整细胞浓度为6×10~5/mL,以500μL/孔接种于预铺有Poly-HEMA和没有预铺Poly-HEMA的24孔板各孔中,铺Poly-HEMA的为悬浮生长组,没有铺Poly-HEMA的为贴壁生长组,培养24小时后收细胞制成单细胞悬液,以含1%FBS的RPMI1640调整细胞浓度,1.5×10~5/孔加入到预先铺有Matrigel基质胶的Transwell小室上室中,其下室中预先加有500μL含10%FBS的RPMI1640,于37℃培养20小时后,穿过小室膜至膜下的细胞经固定、染色后计数,最后将小室膜切下铺于载玻片上,树脂封片后镜下照相。转移性肝癌细胞的运动力实验与侵袭力实验不同之处在于Transwell小室不需要预先铺Matrigel基质胶,并且接种细胞后培养6小时进行固定、染色、计数及封片。
2.转移性肝癌细胞对TRAIL诱导凋亡的反应及其机制探讨
2.1.转移性肝癌细胞对TRAIL诱导凋亡的反应
1).CCK-8、trypan blue染色检测TRAIL诱导的凋亡效应
贴壁生长组和悬浮生长组各分为7组,分别加入终浓度为0ng/mL、5ng/mL、10ng/mL、20ng/mL、50ng/mL、100ng/mL、200ng/mL的TRAIL蛋白,继续培养24 h后,用CCK-8试剂盒和trypanblue染色检测TRAIL诱导转移性肝癌细胞的凋亡效应。
贴壁生长组和悬浮生长组各分为7组,加入终浓度20ng/mL的TRAIL蛋白,继续培养0 h,2 h,5 h,8 h,12 h,24 h,48 h后,用CCK-8试剂盒和trypan blue染色检测TRAIL诱导转移性肝癌细胞的凋亡效应。
2).TUNEL、Caspase-3活性检测分析TRAIL诱导的凋亡效应
贴壁生长组和悬浮生长组细胞培养20 h左右时,实验组加入20ng/mL TRAIL,空白对照组加入等量的PBS。继续培养24 h后,消化、收集细胞(包括上清中漂浮的细胞),洗涤,严格按照TUNEL试剂盒说明书操作,分析细胞的凋亡率;继续培养3 h,6 h,9 h,12 h和24 h后,收集细胞,用Caspase-3活性检测试剂盒测定不同时间点各组的Caspase-3活化程度。
2.2.转移性肝癌细胞抵抗TRAIL诱导凋亡的机制探讨
1).转移性肝癌细胞中TRAIL及其受体的表达情况
Trizol一步法提取贴壁生长和悬浮生长细胞中的总RNA,表达谱芯片分析TRAIL及其受体mRNA的表达情况。并用RT-PCR检测转移性肝癌细胞中TRAIL及其受体在RNA水平的变化。用ELISA法检测培养上清中sTRAIL蛋白的分泌水平,FCM检测转移性肝癌细胞表面膜结合型TRAIL及其受体在蛋白水平的表达情况。
2).转移性肝癌细胞中Caspase-8,-9,-3的活化情况
Western blot检测转移性肝癌细胞及贴壁生长肝癌细胞中Caspase级联反应中Caspase-8,-9,-3前体的表达情况,计算其活化水平。
3.转移性肝癌细胞对AKT/ERK通路抑制剂的反应
贴壁生长组和悬浮生长组细胞培养20 h左右,分别加入PBS、1μM Wortmannin、50μM PD98059以及1μM Wortmannin和50μMPD98059,继续培养24 h后,用CCK-8试剂盒检测细胞生存状态;用trypan blue染色分析细胞死亡率;收集细胞抽提蛋白后,用Western blot方法检测AKT/ERK通路蛋白的活化水平。
4.转移性肝癌细胞对hypoxia的反应
取对数生长期单细胞悬液,接种为悬浮生长组和贴壁生长组各4组,其中两组分别在普通CO_2培养箱和乏氧培养箱中培养24 h后,进行CCK-8试剂盒检测和trypan blue染色。另两组先常规培养24 h后再置于乏氧条件下培养24 h,然后进行CCK-8试剂盒检测和trypan blue染色。
CCK-8检测抑制率计算按如下公式:抑制率=A常氧-A乏氧╱A常氧。trypanblue染色按照如下公式计算死亡率:细胞死亡率=死细胞数╱死细胞数+活细胞数。
5.抵抗失巢凋亡和乏氧刺激的相关分子筛选及论证
5.1.转移性肝癌细胞表达谱芯片结果分析
对贴壁和悬浮生长肝癌细胞做了表达谱芯片研究,利用Cluster软件对其差异表达基因进行聚类分析,找到参与抵抗失巢凋亡同时与乏氧刺激密切相关的差异表达基因。
5.2.MicroRNA芯片结果分析
对贴壁生长细胞、悬浮生长细胞以及乏氧状态下生长的细胞做了microRNA芯片研究,在http://www.targetscan.org/网站对其结果进行分析,查询其中上调和下调的microRNA参与调控的靶基因,将其与表达谱芯片中找到的差异基因进行比对,探寻在抵抗失巢凋亡和抵抗乏氧过程中起关键作用的基因的调控microRNA。
5.3.反义封闭聚集成团细胞的方法学探讨
通过四组方案反义封闭悬浮生长细胞,分别是反义寡核苷酸直接转染贴壁生长和悬浮生长细胞;脂质体介导反义寡核苷酸转染贴壁生长和悬浮生长细胞;脂质体介导反义寡核苷酸无血清条件下转染贴壁生长细胞6h后分组为贴壁生长组和悬浮生长组以及脂质体介导反义寡核苷酸有血清条件下转染贴壁生长细胞6h后分组为贴壁生长组和悬浮生长组。比较不同方案封闭效率及实验稳定性。
5.4.反义封闭相关基因后细胞生物学活性检测
挑选其中的ANGPTL4,CA9,NDRG1,TRAIL和TrkB,设计反义寡核苷酸序列,对悬浮生长组和贴壁生长组进行反义封闭,继续孵育24 h后,用RT-PCR检测封闭效率,用CCK-8试剂盒和trypan blue染色检测细胞生物学活性。
6.统计学分析
结果用mean±SD表示,各组之间差别的统计学意义用SPSS10软件中的T检验或者one-way ANOVA进行统计,P＜0.05被认为有统计学意义。
结果
1.转移性肝癌细胞模型的建立及其聚集稳定性和转移能力分析
1.1.转移性肝癌细胞模型
常规培养的BEL7402细胞和SMMC7721细胞在培养板内伸展开,长成单层细胞,而悬浮生长的细胞培养24 h后,细胞变圆并且聚集成团。悬浮生长24 h聚集率稳定,均大于99.7%。在制作转移性肝癌细胞模型过程中,预铺有Poly-HEMA的培养板如果不洗涤,则细胞死亡率大于90%;96孔板的细胞密度在3×10~4时,细胞死亡率小于5%,低于3×10~4的各个密度与该密度相比,细胞死亡率明显差别(p＜0.05)。
1.2.转移性肝癌细胞的运动力和侵袭力
贴壁生长细胞的运动力和悬浮生长细胞的运动力差别没有统计学意义。悬浮生长组与贴壁生长组具有侵袭力的细胞数分别为19473±1104和9110±706,差别有统计学意义(p＜0.05)。
2.转移性肝癌细胞对TRAIL诱导凋亡的反应及其机制探讨
2.1.转移性肝癌细胞抵抗TRAIL诱导的凋亡效应具有时间依赖性和剂量依赖性
转移性肝癌细胞能够抵抗TRAIL诱导的凋亡效应,并且这种效应具有时间依赖性和剂量依赖性。其中,转移性肝癌细胞在20ng/mL TRAIL处理24 h后的凋亡率是20%,而对照组仅为5%。我们根据时间依赖性和剂量依赖性曲线确定后续实验的剂量为20ng/mL,处理时间为24 h。
2.2.转移性肝癌细胞抵抗TRAIL诱导的细胞凋亡
转移性肝癌细胞经过TRAIL处理后,其TUNEL法检测到的细胞凋亡率为23.1%,明显低于对照组细胞的43.7%。两组细胞的Caspase-3活化水平均在TRAIL作用9 h时达到高峰,但是转移性肝癌细胞的Caspase-3活化水平在各时间点均低于对照组细胞,以9 h时差别最明显。
2.3.转移性肝癌细胞中TRAIL mRNA水平的表达明显高于贴壁生长细胞
基因芯片检测发现在抵抗失巢凋亡的悬浮生长细胞中TRAILmRNA的表达水平比贴壁生长细胞上调了3.9倍。RT-PCR检测发现转移性肝癌细胞中TRAIL mRNA的表达明显高于贴壁生长细胞。
2.4.转移性肝癌细胞中TRAIL蛋白水平的表达存在不一致现象
ELISA法检测发现转移性肝癌细胞培养上清中分泌的sTRAIL蛋白水平明显高于贴壁生长细胞,而FCM检测发现膜结合型TRAIL在转移性肝癌细胞表面表达低于正常生长的贴壁细胞。
2.5.转移性肝癌细胞中TRAIL受体mRNA水平和蛋白水平的表达均明显低于贴壁生长细胞
基因表达谱芯片和RT-PCR检测发现在抵抗失巢凋亡的悬浮生长BEL7402和SMMC7721肝癌细胞中TRAIL受体DR4、DR5、DcR1和DcR2在mRNA水平的表达比贴壁生长细胞低。FCM检测膜结合型TRAIL受体的表达发现转移性肝癌细胞表面的DR4、DR5、DcR1和DcR2表达均低于贴壁生长细胞。
2.6.转移性肝癌细胞在TRAIL作用后Caspase级联反应各分子的活化水平明显低于贴壁生长细胞
Western blot检测发现,经20ng/mL TRAIL处理后,贴壁生长肝癌细胞提取物中Caspase-8,-9,-3前体活化明显,而转移性肝癌细胞提取物中各前体的活化受抑制。
3.转移性肝癌细胞对AKT/ERK通路抑制剂的反应
3.1转移性肝癌细胞抵抗AKT/ERK通路抑制剂的能力更强
显示贴壁生长的肝癌细胞在单独应用AKT/ERK通路抑制剂时,其生物学活性受抑制程度高于悬浮生长细胞,而细胞死亡率也高于悬浮生长细胞。而当联合应用两条通路抑制剂时,转移性肝癌细胞生物学活性较单独应用通路抑制剂显著降低,其降低的幅度明显高于贴壁生长细胞。
3.2在转移性肝癌细胞AKT通路受到抑制时ERK蛋白能够被代偿性激活
Western blot结果显示当AKT通路受抑制时,贴壁生长组细胞的ERK蛋白的相对磷酸化水平为0.67±0.006,而悬浮生长组为0.93±0.055,差别有统计学意义。
4.转移性肝癌细胞对hypoxia的反应
4.1单个悬浮生长细胞乏氧培养24小时细胞生长状态
在肝癌细胞还没有获得抵抗失巢凋亡能力时将其置于乏氧条件下,则CCK-8和trypan blue染色结果提示失去附着的转移性肝癌细胞和正常附着生长的细胞间细胞活性及细胞死亡率差别不大。
4.2聚集成团的悬浮生长细胞乏氧培养24小时的生长状态
将已经获得抵抗失巢凋亡能力的转移性肝癌细胞和贴壁生长的肝癌细胞同时放入低氧环境中,则转移性肝癌细胞的活性明显高于贴壁生长细胞,死亡率明显低于贴壁生长细胞,提示转移性肝癌细胞抵抗低氧的能力明显高于贴壁生长的细胞。
5.抵抗失巢凋亡和低氧刺激的相关分子筛选及论证
5.1.表达谱芯片中参与抵抗失巢凋亡同时耐受低氧的基因
表达谱芯片中在抵抗失巢凋亡过程中起作用同时耐受低氧的基因有许多,其中:ANGPTL4上调了16.29倍,BNIP3上调了7.88倍,CA9上调了10.31倍,IER3上调了7.95倍,IGFBP3上调了31.07倍,NDRG1上调了18.4倍。
5.2.与乏氧和失巢相关的microRNA间存在交叉
microRNA芯片结果显示,乏氧刺激下和失巢刺激下上调的microRNA重叠部分占到两种刺激下上调基因的44.4%,而下调的microRNA重叠部分有18.7%。
5.3.表达谱芯片中差异表达基因与microRNA芯片中差异microRNA调控基因吻合良好
表达谱芯片中差异表达基因与microRNA芯片中差异microRNA调控基因吻合良好,其中表达谱芯片结果中转移性肝癌细胞表达明显增高的BNIP3、CA9、NDRG1相对应的hsa-miR-558、hsa-miR-516a-3p/516b~*、hsa-miR-342、hsa-miR-620等在转移性肝癌细胞中显著下调。
5.4.反义封闭悬浮生长细胞的方法学探讨
PS-asODNs/ANGPTL4直接转染结果不稳定,脂质体介导转染悬浮生长细胞的转染效率低于转染贴壁生长细胞的转染效率,我们对培养至对数生长期的贴壁生长细胞进行反义封闭,6 h后分为贴壁生长组和悬浮生长组继续培养24 h,反义封闭效率基本一致。
5.5.ANGPTL4反义封闭后细胞的生物学活性
RT-PCR结果显示,PS-asODNs/ANGPTL4可显著降低ANGPTL4 mRNA的合成。反义封闭ANGPTL4后,转移性肝癌细胞与正常贴壁生长的肝癌细胞相比活性降低明显,其死亡率也较贴壁生长的肝癌细胞明显增高。
结论
1.转移性肝癌细胞模型的细胞聚集率在24小时稳定达到99.7%左右,进一步验证了模型的有效性。
2.转移性肝癌细胞的侵袭能力明显高于贴壁生长细胞,提示抵抗失巢凋亡的转移性肝癌细胞具有更强的通过第三微环境进而侵入第二微环境的能力。
3.转移性肝癌细胞一方面分泌更多的sTRAIL,另一方面自身能够抵抗TRAIL诱导的凋亡,通过诱导免疫效应细胞发生凋亡来逃逸机体免疫系统的监视,这一过程被称为“肿瘤反击”。转移性肝癌细胞表面的膜结合型DR4,DR5均明显下调,可能是转移性肝癌细胞能够抵抗TRAIL诱导凋亡的机制之一。
4.当AKT或者ERK通路单独受抑制时,转移性肝癌细胞的活性并没有发生多大变化。但是,当两条通路同时受抑制时,产生了一个非常明显的细胞死亡率。说明在这两个信号传导通路之间存在很强的代偿作用。当其中的一条通路受抑制时,另外一条通路的活化被放大来代偿其生存信号的缺失。Western blot结果进一步显示抑制AKT通路后磷酸化ERK蛋白的水平明显提高。
5.转移性肝癌细胞通过抵抗TRAIL诱导的凋亡以及AKT和ERK通路之间存在较强的代偿作用等机制抵抗失巢凋亡。
6.在肝癌细胞还没有获得抵抗失巢凋亡能力时将其置于乏氧条件下,则失去附着和正常附着生长的细胞间活性差别不大,一定程度上都能够耐受低氧刺激,但是,如果将已经获得抵抗失巢凋亡能力的转移性肝癌细胞和贴壁生长的肝癌细胞同时放入低氧环境中,则转移性肝癌细胞抵抗低氧的能力明显高于正常贴壁生长的细胞。说明抵抗失巢凋亡能力的获得有助于转移性肝癌细胞耐受低氧环境。
7.表达谱芯片中差异表达基因与microRNA芯片中差异microRNA调控基因吻合良好,其中表达谱芯片结果中转移性肝癌细胞表达明显增高的NDRG1、CA9、BNIP3相对应的hsa-miR-620、hsa-miR-516a-3p/516b~*、hsa-miR-342、hsa-miR-558等在转移性肝癌细胞中显著下调。
8.利用反义寡核苷酸直接封闭聚集成团的转移性肝癌细胞中的基因比较困难,需要将反义寡核苷酸事先转染入贴壁生长细胞6 h后再分别接种为贴壁生长组和悬浮生长组,以达到最佳的转染效率和转染稳定性。
9.反义封闭ANGPTL4后,转移性肝癌细胞的活性受抑制程度明显高于正常贴壁生长的肝癌细胞,而且其死亡率增高也比正常生长肝癌细胞明显,说明ANGPTL4在转移性肝癌细胞生长过程中发挥重要作用。
创新点及意义:
1.本研究首次报导转移性肝癌细胞通过“肿瘤反击”来逃逸机体的免疫监视功能,并且其侵袭力明显增强。
2.本研究首次报导转移性肝癌细胞的AKT、ERK通路之间存在很强的代偿作用,当抑制AKT通路后磷酸化ERK蛋白的水平明显提高。这种代偿作用可使转移性肝癌细胞抵抗相应抗肿瘤药物的攻击,提示我们可以联合应用这两条通路抑制剂来治疗转移性肝癌。
3.本研究率先提出获得抵抗失巢凋亡能力的转移性肝癌细胞抵抗低氧的能力明显高于贴壁生长的细胞。
4.本研究将表达谱芯片中差异表达基因与microRNA芯片中差异microRNA调控基因相比较,筛选可能参与调控转移性肝癌细胞转移能力的关键分子,临床应用价值更高。
5.本研究首次探讨了反义转染聚集成团细胞的方法,为反义寡核苷酸转染以及其他小分子物质进入聚集成团细胞提供了有价值的参考。
Hepatocarcinoma is a kind of malignant tumor with high morbidity and mortality worldwidely.The 5-year survival rate is very low because of its rapid progress, high recurrence and metastatic rate.If the mechanism of metastasis can be clarified and some early intervention can be put up,it will greatly improve the treatment of hepatocarcinoma.
To be able to arrival a second site to form a metastatic carcinoma,tumor cells have to complete a series of selective and orderly steps,including department from the carcinoma in situ,invasion into the vascular system,survival in vascular system,invasion of the matrix into the ectopic organ,spreading out of vascular system and proliferating.They have to overcome pressures before they finally forming metastases,including the immune surveillance capability,the hypoxic environment,a variety of attacks,such as antineoplastic agents.In addition,a variety of effector molecules involved in the process of tumor metastasis,many of them are upregulated at mRNA and protein expression levels under hypoxia. Hypoxia is considered a factor that tumor cells have to overcome.At the same time,there is increasing evidence indicating that hypoxia can promote tumor development.
We have successfully constructed the metastatic hepatoma cell model and we further validate its validity and metastatic capacity,as well as discussing the mechnism of enhanced metastatic capacity by forcing the metastatic hepatoma cells to various stimuli(including the exogenous TRAIL as well as the AKT/ERK pathway inhibitors),and further filter the key moleculars which can resist anoikis and hypoxia at the same time.
Objectives:
1.To construct the metastatic hepatoma cell model and explore its aggregated rate and metastatic capacity.
2.To demonstrate molecular mechanism of anoikis-resistance by study response of metastatic hepatoma cells to TRAIL-induced apoptosis to AKT/ERK pathway inhibitors.
3.To explore the response of metastatic hepatoma cells to hypoxic stimuli.
4.To screen the key molecules for anoikis-resistance and hypoxia-resistance in metastatic hepatoma cells.
Methods:
1.Metastatic hepatoma cell model and its aggregated rate and metastatic ability
1.1.Metastatic model of hepatocellular carcinoma cells BEL7402 and SMMC7721 cell lines were cultured in RPMI1640 with 10%FCS at 37℃5%CO_2 conditions to cultivate logarithmic phase,then digested by 0.25% trypsin into single cell suspension,adjusted the concentration,and cultured in Poly-HEMA culture plates for 24 h,that is the metastatic hepatoma cell model which analog metastatic hepatoma cells in vascular system.
1.2.Conditions of metastatic hepatoma cell model
Cells were cultured to logarithmic phase and prepared into single cell suspension, then seeded into the 96-well plates in different cells numbers as 0.5×10~4,1×10~4,1.5×10~4,3×10~4,3×10~4.The Poly-HEMA plates of the first four groups were washed 3 times using sterile PBS before seeding cells,the fifth group was not washed,each tin duplicated and cultured for another 24 h.Cell activity and mortality were detected by using CCK-8 and trypan blue staining.
1.3.Aggregated rate of metastatic hepatoma cells
Hepatoma cells were seeded into plates without or with poly-HEMA coating as attached or detached experimental groups and cultured for 24h.Single, non-aggregated cells were counted.The percentage of aggregated cells was calculated as(1-Ne/Nc)×100%,where Ne is the number of single cells after incubation for 24 h and Nc is the number of single cells before incubation.
1.4.Transwell detection of metastatic hepatoma cells' invasion and migration About 1.5×10~5 separating detached or attached hepatoma cells were seeded to the top chamber(diluted with 200ul RPMI 1640 in the presence of 1%FBS) of the Polycarbonate Membrane Transwell Inserts pre-coated with diluted Matrigel~(TM) Basement Membrane Matrix.The bottom chamber was added 0.5ml RPMI 1640 with 10%FBS in advance.After 20 h of incubation at 37℃,cells on the lower part were fixed,stained and scored in 10 randomly chosen microscopic fields to decide the invasion of anoikis-resistant cells.For migration studies,cells were seeded to the upper compartment of the uncoated inserts and cultured for 6h.
2.Response of metastatic hepatoma cells to TRAIL-induced apoptosis and its mechanisms
2.1.Response of metastatic hepatoma cells to TRAIL-induced apoptosis
1).TRAIL induced apoptosis detected by CCK-8 and Trypan blue assay
Attached and suspended groups were divided into 7 groups respectively,add 0ng/mL,5ng/mL,10ng/mL,20ng/mL,50ng/mL,100ng/mL,200 ng/mL of TRAIL protein.After 24h cell culture,CCK-8 and trypan blue staining were used to detect TRAIL-induced apoptosis.Attached and suspended groups were divided into 7 groups respectively,add 20ng/mL of TRAIL protein and cultured for 0 h,2 h,5 h,8 h,12 h,24 h and 48 h.Then,CCK-8 and trypan blue staining were used to detect TRAIL-induced apoptosis.
2).TRAIL induced apoptosis detected by TUNEL and Caspase3 activation assay
Hepatoma cells were seeded into 24-well plates as attached or detached groups for 20h,and treated with 20ng/ml recombinant sTRAIL for another 24h.Apoptotic cells were labeled with TUNEL by using an In Situ Cell Death Detection Kit.The degree of apoptosis was determined by flow cytometry.Caspase-3 activities of hepatoma cells in 6-well plates treated with sTRAIL for 3 h,6 h,9 h,12 h and 24 h were detected by Caspase-3/CPP32 Colorimetric Assay Kit.
2.2.Mechanism of metastatic hepatoma cells resist TRAIL-induced apoptosis
1).Expression of TRAIL and its receptors in metastatic hepatoma cells
Total RNA was extracted by trizol one-step extraction from attached and detached cells.The mRNA expression of TRAIL and its receptors were analysesed by profiling microarray.RT-PCR was used to detect the mRNA expression of TRAIL and its receptors of metastatic hepatoma cells.ELISA assay was used to indicate the secretion of sTRAIL protein in culture supernatant,FCM was used to detect the surface-bound TRAIL and its receptors on metastatic hepatoma cells.
2).Caspase-8,-9 and -3 activation of metastatic hepatoma cells determined by Western blot
We further detected the Caspase-8,-9 and -3 activation after 20ng/ml of sTRAIL treatment by using Western blot.
3.Responses of metastatic hepatoma cells to PI-3K/AKT or ERK pathway inhibitors
Cells in either adherent or suspended culture conditions were treated with 1μM of Wortmannin or 50μM of PD98059 for 24 h.cell viability was measured by CCK-8 kit as well as trypan blue exclusion assay.Phosphorylation level of protein was detected by western blot.
4.Response of metastatic hepatoma cells to hypoxie stress
Attached and detached cells in 96-well plates were cultured at 37℃with 2%O_2 for 24 h as hypoxia treated group or cultured at 37℃with 20%O_2 as nomoxia control.Another two groups of conventional culture and then placed under hypoxic conditions for another 24 hours.Cell viability and proliferation status were measured by using the CCK-8 assay or trypan blue staining.
Inhibition rate calculated by the following formula InhibitRate=Anomoxia-Ahypoxia/Anomoxia.Mortality rates in accordance with the following formula:DeadRate=DeadCells/DeadCells+LiveCells.
5.Screening and demonstrating associated genes in anoikis-resistance and hypoxia-resistance
5.1.Analysesing the microarray expression profiling of attached and detached hepatoma cells
Expression profiling of attached and detached hepatoma cells were analysesed by gene chip.Using cluster software to define the functional annotation clustering of different expressed genes and find genes participated in anoikis-resistance and hypoxia-resistance.
5.2.Analyses the different microRNAs in attached,detached and hypoxia hepatoma cells
MicroRNA chip was used to find different microRNAs in attached,detached and hypoxia hepatoma cells.The results were analyzed at http://www.targetscan.org/ website,target genes of up-regulated and down-regulated microRNA were screened and compared with the results of gene chip.Genes participated in anoikis-resistance and hypoxic stimulus were selected.
5.3.Block efficiency of different antisense protocol
We used four protocol to block aggregated cells by antisense oligonucleotide.①Transfecting attached and detached hepatoma cells directly by antisense oligonucleotide.②Transfecting attached and detached hepatoma cells by liposome-mediated antisense oligonucleotide.③Transfecting attached hepatoma cells by liposome-mediated antisense oligonucleotide without FBS,and then divided the attached cells into attached group and detached group.④Transfecting attached hepatoma cells by liposome-mediated antisense oligonucleotide with FBS,and then divided the attached cells into attached group and detached group.The efficiency of different programs and experimental stability was compared.
5.4.Biological activity of cells blocked by antisense oligonucleotides of associated genes
We designed antisense oligonucleotide sequence of ANGPTL4,CA9,NDRG1, TRAIL and TrkB,and blocked the attached and detached hepatoma cells for 24h, then detected block efficiency by Real-Time PCR and RT-PCR as well as determined the biological activity by using CCK-8 or trypan blue staining assay.
6.Statistical analysis
Results were expressed as mean±SD.The statistical significance of differences between the groups was determined by Student's paired T tests or one-way ANOVA using SPSS10 statistical software.P＜0.05 was considered statistically significant.。
Results
1.Aggregated rate and metastatic ability of metastatic hepatoma cell model
1.1.Metastatic hepatoma cell model
Conventional cultured BEL7402 and SMMC7721 cells spread out as monolayers, while the suspension cells became round and self-aggregated into big cell clusters after cultured for 24 h.The aggregation rate stability was very well.It was always more than 99.7%.Poly-HEMA coated plates must be washed before using.The best cell density of 96 well plates is 3×10~4 per well.
1.2.metastatic hepatoma cells' invasion and migration
Mobility analysis revealed no significant difference between the attached and detached cells.However,the amount of invasive cells in the anoikis-resistant detached cell group was much greater than that of attached cells(19473±1104 vs. 9110±706 cells,p＜0.05).
2.Response of metastatic hepatoma cells to TRAIL-induced apoptosis and its mechanisms
2.1.Metastatic hepatoma cells resist TRAIL-induced apoptosis in a dose-dependent and time-dependent manner
Metastatic hepatoma cells resist TRAIL-induced apoptosis in a dose-dependent and time-dependent manner.Specifically,the apoptosis rate in the attached hepatoma cells after treatment with 20ng/ml of sTRAIL for 24 h was 20%while that in the detached cells was 5%,a value similar to that of control hepatoma cells without sTRAIL treatment.We used the dose of 20ng/mL and processing time for 24 h in the following experiments according to time-dependent and dose-dependent curve.
2.2.Metastatic hepatoma cells were more resistant to TRAIL-induced apoptosis
After treated with TRAIL,metastatic hepatoma cells' apoptotic rate was significantly lower than the control group cells detected by TUNEL method. Caspase-3 activated level arrived the peak at 9 h after treated with TRAIL. Caspase-3 activated level of metastatic hepatoma cells were lower than control group cells at different time points and the difference at 9 h was most obviously.
2.3.Expression of TRAIL mRNA was increased significantly in metastatic hepatoma cells
All assays of detecting TRAIL mRNA,including microarray analysis and semi-quantitative RT-PCR indicated that expression of TRAIL mRNA was increased significantly in Synoikis-like suspended BEL7402 cells compared with that of attached counterpart.
2.4.A discrepancy of TRAIL protein expression in metastatic hepatoma cells There is a discrepancy at the protein level of TRAIL expression.sTRAIL secreted in the supernatant was increased significantly in the detached hepatoma cells,while membrane binding TRAIL(mTRAIL) was down-regulated in the detached cells compared with that of attached cells.
2.5.TRAIL receptors of metastatic hepatoma cells were decreased significantly on mRNA level and protein level
Gene chip and semi-quantitative RT-PCR indicated that expression of DR4,DR5, DcR1,and DcR2 mRNA of detached synoikis-like hepatoma cells was decreased significantly compared with that of attached control groups.There is also a substantial decreased expression of TRAIL membrane binding receptors at the protein level on the detached cells as compared with that of attached cells detected by FCM.
2.6.Loss of anchorage blocked TRAIL-induced caspase cascade activation TRAIL induced significant activation of procaspase-8,procaspase-9 and procaspase-3.However,this activation of caspase cascade was effectively blocked upon loss of cell anchorage.
3.Response of metastatic hepatoma cells to AKT/ERK pathway inhibitors
3.1 Metastatic hepatoma cells were less compromised to AKT or ERK pathway inhibition
Growth of attached hepatoma cells were greatly inhibited under the effect of AKT or ERK inhibition,however,the detached hepatoma cells were much more resistant to protein kinase inhibition when the inhibitor was used separately. Surprisingly,when both of the AKT and ERK pathways were blocked together, the viability of detached cells decreased dramatically to a degree even greater than that of attached groups.
3.2 A strong compensated activation mechanism exit in the metastatic hepatoma cells
It showed that the ERK phosphorylation level was dramatically increased when AKT pathway inhibitor was used,which indicated a strong compensated activation mechanism in the metastatic hepatoma cells.
4.Response of metastatic hepatoma cells to hypoxia
There was no difference of cell viability between detached and attached hepatoma cells when placed under hypoxic conditions before they obtained the ability of anoikis-resistance detected by CCK-8 and trypan blue assay.They were all able to tolerant the hypoxia stimuli to some extent.
If expand the anoikis-resistant hepatoma cells to hypoxia,its ability to resist hypoxic environment is much higher than the attached hepatoma cells detected by CCK-8 and trypan blue assay.
5.Screening and demonstrating associated genes in anoikis-resistance and hypoxia-resistance
5.1.Genes involved in resistance to anoikis and hypoxia in gene chip
There were a large number of genes that involved in resistance to anoikis and regulated by hypoxia in gene chip.Such as IER3(up-regulated for 7.95 times), CA9(up-regulated for 10.31 times),BNIP3(up-regulated for 7.88 times), NDRG1(up-regulated for 18.4 times),ANGPTL4(up-regulated for 16.29 times), IGFBP3(up-regulated for 31.07 times).
5.2.Crosstalk between microRNAs regulate anoikis and hypoxia
There was a large proportion of crosstalk between microRNAs which regulate anoikis and hypoxia.For example,the crosstalking proportion of upregulated microRNA is more than 44.4%,and that of downregulated is about 18.7%.
5.3.There was a well consistent between the gene chip and microRNA chip There was a well consistent between the differentially expressed genes of gene chip and target genes of different microRNAs in microRNA chip.For example, NDRG1,CA9 and BNIP3 were increased in metastatic hepatoma cells,and their possible regulate microRNAs hsa-miR-620,hsa-miR-516a-3p/516b*, hsa-miR-342,hsa-miR-558 were down-regulated significantly.
5.4.Methods of PS-asODNs/ANGPTL4 block
The results of PS-asODNs/ANGPTL4 direct transfection were unstable. Transfection efficiency of liposome-mediated transfection of detached cells was lower than that of attached cells.We cultured attached cells to logarithmic phase and transfected them with liposome-mediation for 6 h,then divided them into attached and suspension groups to continue to culture for another 24 h,the efficiency of antisense had no difference.
5.5.Biology activity of cells whose ANGPTL4 had been blocked
Real-Time PCR and RT-PCR results showed that,PS-asODNs/ANGPTL4 can significantly reduce the mRNA synthesis of ANGPTL4(p＜0.05).When ANGPTL4 was blocked,the viability of metastatic hepatoma cells was much more inhibited than that of the attached hepatoma cells.The mortality rate was also higher than the normal cultured hepatoma cells.
Conclusions:
1.Cell aggregated rate of metastatic hepatoma cell model at 24 hours reached 99.7%steadily,which further verify the validity of the metastatic hepatoma cell model.
2.Metastatic hepatoma cells had significantly higher invasiveness than the adherent cells.These results suggest that anoikis-resistant metastatic hepatoma cells are more invasive to go through the third microenvironment and arrive the second microenvironment,that is to say they are more aggressive.
3.Metastatic hepatoma cells secret more sTRAIL on the one hand and it can resist TRAIL-induced apoptosis on the other hand,which can induce apoptosis of immune effector cells in order to escape from immune system surveillance,the process is called "tumor counterattack".Metastatic hepatoma cells membrane-binding DR4,DR5 were significantly reduced. Maybe it is one of the mechanisms of metastatic hepatoma cells resist TRAIL-induced apoptosis.
4.When AKT or ERK pathway was inhibited separately,it did not take much effect on the viability of the anoikis-resistant cells.However,when both of these two protein kinase pathway were blocked,it caused a dramatically cell death.This indicates that there is a strong interplay between these two pro-survival pathways.When one of these ways was inhibited,the signal of the other pathway will be over-activated and compensate for loss of this survival signal.Interestingly,our western blot data showed that inhibition of AKT pathway dramatically increased the phosphorylation level of ERK pathway.
5.Metastatic hepatoma cells may be resist anoikis by resisting TRAIL-induced apoptosis and interplay of AKT and ERK pathway.
6.There was no difference of cell viability between detached and attached hepatoma cells when placed under hypoxic conditions before they obtained the ability of anoikis-resistance.They were all able to tolerant the hypoxia stimuli to some extent.But,if expand the anoikis-resistant hepatoma cells to hypoxia,its ability to resist hypoxic environment is much higher than the attached hepatoma cells.That is to say,acquisition of anoikis-resistance contributes to hypoxia tolerance of metastatic hepatoma cells.
7.There was a well consistent between the differentially expressed genes of gene chip and target genes of different microRNAs in microRNA chip.For example,NDRG1,CA9 and BNIP3 were increased in metastatic hepatoma cells,and their possible regulate microRNAs hsa-miR-620, hsa-miR-516a-3p/516b*,hsa-miR-342,hsa-miR-558 were down-regulated significantly.
8.It is difficult to block genes of metastatic hepatoma cells by transfecting oligonucleotide directly.It is better to transfect antisense oligonucleotide into attached hepatoma cells and block for 6 h,followed by dividing them into attached and detached cell groups.
9.When ANGPTL4 was blocked,the viability of metastatic hepatoma cells was much more inhibited than that of the attached hepatoma cells.The mortality rate was also higher than the normal cultured hepatoma cells.These indicate that ANGPTL4 may play an important role in the growth of metastatic hepatoma cells.
Innovations and significances:
1.This is the first report that metastatic hepatoma cells can escape from immunological surveillance by "tumor counterattack" and have increased invasiveness.
2.It is the first time to report this compensated activation in detached hepatoma cells when one of the major survival pathway was blocked.This vigorous compensated activation may offer anchorage-deprived hepatoma cells more survival opportunities and metastatic potential when using relative therapeutic agents.Thus,combination of kinase inhibitors is likely to yield a substantial advance in successfully producing downstream phenotypic response in anoikis-resistant hepatoma cells.
3.In this study,we first put forward that the ability of resistance to hypoxia of metastatic hepatoma cells was significantly higher than that of normal adherent cells.
4.In this study,we combined the gene chip and microRNA chip to screen key molculars that may be involved in regulation of metastatic hepatocellular carcinoma cells.It will have higher clinical application value.
5.This study explored firstly the methods of PS-asODNs transfecting aggregated cells,which will provide a useful reference for PS-asODNs as well as other small molecules to transfer into the aggregated cells.
为了能够到达异位形成转移癌,肿瘤细胞必须完成一系列有序的选择性步骤,其中包括脱离原位癌,侵袭进入脉管系统,在脉管系统中存活,然后侵袭进入异位器官的基质层,长出脉管系统并增殖。转移过程中的肿瘤细胞必须克服在这一过程中所要面临的种种压力才能够最终到达异位形成转移灶。这些压力包括机体的免疫监视能力、体内的乏氧环境、各种抗肿瘤药物的攻击等。另外,肿瘤转移过程中还必须有多种效应分子的参与,其中很大一部分的mRNA和蛋白表达水平在低氧诱导下上调。乏氧被认为是肿瘤细胞在生存过程中必须要克服的一个因素,同时,有越来越多的证据显示乏氧能够促进肿瘤发展。
本课题在成功建立了转移性肝癌细胞模型的基础上,对其有效性以及转移能力进行了验证,并从转移性肝癌细胞对各种刺激(包括外源性TRAIL、AKT/ERK通路抑制剂)的反应性来探讨其抵抗失巢凋亡的分子机制,然后进一步筛选并论证了抵抗失巢凋亡和抵抗低氧刺激的相关分子,为临床上判断肝癌预后以及靶向性治疗奠定坚实的理论基础和实验基础。
目的:
1.建立一种能够模拟转移过程中的肝癌细胞在脉管系统中存活状态的转移性肝癌细胞模型,并研究其聚集稳定性和转移能力。
2.通过研究转移性肝癌细胞对TRAIL诱导凋亡的反应以及对抗肿瘤药物AKT/ERK通路抑制剂的反应来研究其抵抗失巢凋亡的分子机制。
3.探讨转移性肝癌细胞对乏氧刺激的反应。
4.筛选抵抗失巢凋亡和抵抗乏氧刺激相关的分子,以用于抗肿瘤转移的早期诊断、预防和治疗。
方法
1.转移性肝癌细胞模型的建立以及其聚集率和转移能力分析
1.1.转移性肝癌细胞模型的建立
BEL7402和SMMC7721肝癌细胞系以PRMI1640加10%胎牛血清为培养液,在37℃、5%CO_2条件下培养至对数生长期,用0.25%的胰蛋白酶消化成单细胞悬液,以RPMI1640调整好浓度后,加入Poly-HEMA培养板中继续培养24 h,得到悬浮生长的转移性肝癌细胞模型。
1.2.转移性肝癌细胞模型建立条件探索
细胞培养至对数生长期并制备成单细胞悬液后,96孔板中接种不同细胞数量0.5×10~4,1×10~4,1.5×10~4,3×10~4,3×10~4,前四个组细胞接种前Poly-HEMA培养板用无菌PBS洗3遍,第五组不洗涤,每组设六孔重复,继续培养24 h,用CCK-8和trypan blue染色检测细胞活性和死亡率。
1.3.转移性肝癌细胞的聚集率
在铺有Poly-HEMA的96孔板中培养悬浮生长的转移性肝癌细胞,24小时后计数孔中单个细胞数,然后依据如下公式计算聚集率:聚集率=1-培养24h后孔中单个细胞数╱接种细胞数×100%。
1.4.Transwell检测转移性肝癌细胞的侵袭力和运动力
取对数生长期肝癌细胞的单细胞悬液,调整细胞浓度为6×10~5/mL,以500μL/孔接种于预铺有Poly-HEMA和没有预铺Poly-HEMA的24孔板各孔中,铺Poly-HEMA的为悬浮生长组,没有铺Poly-HEMA的为贴壁生长组,培养24小时后收细胞制成单细胞悬液,以含1%FBS的RPMI1640调整细胞浓度,1.5×10~5/孔加入到预先铺有Matrigel基质胶的Transwell小室上室中,其下室中预先加有500μL含10%FBS的RPMI1640,于37℃培养20小时后,穿过小室膜至膜下的细胞经固定、染色后计数,最后将小室膜切下铺于载玻片上,树脂封片后镜下照相。转移性肝癌细胞的运动力实验与侵袭力实验不同之处在于Transwell小室不需要预先铺Matrigel基质胶,并且接种细胞后培养6小时进行固定、染色、计数及封片。
2.转移性肝癌细胞对TRAIL诱导凋亡的反应及其机制探讨
2.1.转移性肝癌细胞对TRAIL诱导凋亡的反应
1).CCK-8、trypan blue染色检测TRAIL诱导的凋亡效应
贴壁生长组和悬浮生长组各分为7组,分别加入终浓度为0ng/mL、5ng/mL、10ng/mL、20ng/mL、50ng/mL、100ng/mL、200ng/mL的TRAIL蛋白,继续培养24 h后,用CCK-8试剂盒和trypanblue染色检测TRAIL诱导转移性肝癌细胞的凋亡效应。
贴壁生长组和悬浮生长组各分为7组,加入终浓度20ng/mL的TRAIL蛋白,继续培养0 h,2 h,5 h,8 h,12 h,24 h,48 h后,用CCK-8试剂盒和trypan blue染色检测TRAIL诱导转移性肝癌细胞的凋亡效应。
2).TUNEL、Caspase-3活性检测分析TRAIL诱导的凋亡效应
贴壁生长组和悬浮生长组细胞培养20 h左右时,实验组加入20ng/mL TRAIL,空白对照组加入等量的PBS。继续培养24 h后,消化、收集细胞(包括上清中漂浮的细胞),洗涤,严格按照TUNEL试剂盒说明书操作,分析细胞的凋亡率;继续培养3 h,6 h,9 h,12 h和24 h后,收集细胞,用Caspase-3活性检测试剂盒测定不同时间点各组的Caspase-3活化程度。
2.2.转移性肝癌细胞抵抗TRAIL诱导凋亡的机制探讨
1).转移性肝癌细胞中TRAIL及其受体的表达情况
Trizol一步法提取贴壁生长和悬浮生长细胞中的总RNA,表达谱芯片分析TRAIL及其受体mRNA的表达情况。并用RT-PCR检测转移性肝癌细胞中TRAIL及其受体在RNA水平的变化。用ELISA法检测培养上清中sTRAIL蛋白的分泌水平,FCM检测转移性肝癌细胞表面膜结合型TRAIL及其受体在蛋白水平的表达情况。
2).转移性肝癌细胞中Caspase-8,-9,-3的活化情况
Western blot检测转移性肝癌细胞及贴壁生长肝癌细胞中Caspase级联反应中Caspase-8,-9,-3前体的表达情况,计算其活化水平。
3.转移性肝癌细胞对AKT/ERK通路抑制剂的反应
贴壁生长组和悬浮生长组细胞培养20 h左右,分别加入PBS、1μM Wortmannin、50μM PD98059以及1μM Wortmannin和50μMPD98059,继续培养24 h后,用CCK-8试剂盒检测细胞生存状态;用trypan blue染色分析细胞死亡率;收集细胞抽提蛋白后,用Western blot方法检测AKT/ERK通路蛋白的活化水平。
4.转移性肝癌细胞对hypoxia的反应
取对数生长期单细胞悬液,接种为悬浮生长组和贴壁生长组各4组,其中两组分别在普通CO_2培养箱和乏氧培养箱中培养24 h后,进行CCK-8试剂盒检测和trypan blue染色。另两组先常规培养24 h后再置于乏氧条件下培养24 h,然后进行CCK-8试剂盒检测和trypan blue染色。
CCK-8检测抑制率计算按如下公式:抑制率=A常氧-A乏氧╱A常氧。trypanblue染色按照如下公式计算死亡率:细胞死亡率=死细胞数╱死细胞数+活细胞数。
5.抵抗失巢凋亡和乏氧刺激的相关分子筛选及论证
5.1.转移性肝癌细胞表达谱芯片结果分析
对贴壁和悬浮生长肝癌细胞做了表达谱芯片研究,利用Cluster软件对其差异表达基因进行聚类分析,找到参与抵抗失巢凋亡同时与乏氧刺激密切相关的差异表达基因。
5.2.MicroRNA芯片结果分析
对贴壁生长细胞、悬浮生长细胞以及乏氧状态下生长的细胞做了microRNA芯片研究,在http://www.targetscan.org/网站对其结果进行分析,查询其中上调和下调的microRNA参与调控的靶基因,将其与表达谱芯片中找到的差异基因进行比对,探寻在抵抗失巢凋亡和抵抗乏氧过程中起关键作用的基因的调控microRNA。
5.3.反义封闭聚集成团细胞的方法学探讨
通过四组方案反义封闭悬浮生长细胞,分别是反义寡核苷酸直接转染贴壁生长和悬浮生长细胞;脂质体介导反义寡核苷酸转染贴壁生长和悬浮生长细胞;脂质体介导反义寡核苷酸无血清条件下转染贴壁生长细胞6h后分组为贴壁生长组和悬浮生长组以及脂质体介导反义寡核苷酸有血清条件下转染贴壁生长细胞6h后分组为贴壁生长组和悬浮生长组。比较不同方案封闭效率及实验稳定性。
5.4.反义封闭相关基因后细胞生物学活性检测
挑选其中的ANGPTL4,CA9,NDRG1,TRAIL和TrkB,设计反义寡核苷酸序列,对悬浮生长组和贴壁生长组进行反义封闭,继续孵育24 h后,用RT-PCR检测封闭效率,用CCK-8试剂盒和trypan blue染色检测细胞生物学活性。
6.统计学分析
结果用mean±SD表示,各组之间差别的统计学意义用SPSS10软件中的T检验或者one-way ANOVA进行统计,P＜0.05被认为有统计学意义。
结果
1.转移性肝癌细胞模型的建立及其聚集稳定性和转移能力分析
1.1.转移性肝癌细胞模型
常规培养的BEL7402细胞和SMMC7721细胞在培养板内伸展开,长成单层细胞,而悬浮生长的细胞培养24 h后,细胞变圆并且聚集成团。悬浮生长24 h聚集率稳定,均大于99.7%。在制作转移性肝癌细胞模型过程中,预铺有Poly-HEMA的培养板如果不洗涤,则细胞死亡率大于90%;96孔板的细胞密度在3×10~4时,细胞死亡率小于5%,低于3×10~4的各个密度与该密度相比,细胞死亡率明显差别(p＜0.05)。
1.2.转移性肝癌细胞的运动力和侵袭力
贴壁生长细胞的运动力和悬浮生长细胞的运动力差别没有统计学意义。悬浮生长组与贴壁生长组具有侵袭力的细胞数分别为19473±1104和9110±706,差别有统计学意义(p＜0.05)。
2.转移性肝癌细胞对TRAIL诱导凋亡的反应及其机制探讨
2.1.转移性肝癌细胞抵抗TRAIL诱导的凋亡效应具有时间依赖性和剂量依赖性
转移性肝癌细胞能够抵抗TRAIL诱导的凋亡效应,并且这种效应具有时间依赖性和剂量依赖性。其中,转移性肝癌细胞在20ng/mL TRAIL处理24 h后的凋亡率是20%,而对照组仅为5%。我们根据时间依赖性和剂量依赖性曲线确定后续实验的剂量为20ng/mL,处理时间为24 h。
2.2.转移性肝癌细胞抵抗TRAIL诱导的细胞凋亡
转移性肝癌细胞经过TRAIL处理后,其TUNEL法检测到的细胞凋亡率为23.1%,明显低于对照组细胞的43.7%。两组细胞的Caspase-3活化水平均在TRAIL作用9 h时达到高峰,但是转移性肝癌细胞的Caspase-3活化水平在各时间点均低于对照组细胞,以9 h时差别最明显。
2.3.转移性肝癌细胞中TRAIL mRNA水平的表达明显高于贴壁生长细胞
基因芯片检测发现在抵抗失巢凋亡的悬浮生长细胞中TRAILmRNA的表达水平比贴壁生长细胞上调了3.9倍。RT-PCR检测发现转移性肝癌细胞中TRAIL mRNA的表达明显高于贴壁生长细胞。
2.4.转移性肝癌细胞中TRAIL蛋白水平的表达存在不一致现象
ELISA法检测发现转移性肝癌细胞培养上清中分泌的sTRAIL蛋白水平明显高于贴壁生长细胞,而FCM检测发现膜结合型TRAIL在转移性肝癌细胞表面表达低于正常生长的贴壁细胞。
2.5.转移性肝癌细胞中TRAIL受体mRNA水平和蛋白水平的表达均明显低于贴壁生长细胞
基因表达谱芯片和RT-PCR检测发现在抵抗失巢凋亡的悬浮生长BEL7402和SMMC7721肝癌细胞中TRAIL受体DR4、DR5、DcR1和DcR2在mRNA水平的表达比贴壁生长细胞低。FCM检测膜结合型TRAIL受体的表达发现转移性肝癌细胞表面的DR4、DR5、DcR1和DcR2表达均低于贴壁生长细胞。
2.6.转移性肝癌细胞在TRAIL作用后Caspase级联反应各分子的活化水平明显低于贴壁生长细胞
Western blot检测发现,经20ng/mL TRAIL处理后,贴壁生长肝癌细胞提取物中Caspase-8,-9,-3前体活化明显,而转移性肝癌细胞提取物中各前体的活化受抑制。
3.转移性肝癌细胞对AKT/ERK通路抑制剂的反应
3.1转移性肝癌细胞抵抗AKT/ERK通路抑制剂的能力更强
显示贴壁生长的肝癌细胞在单独应用AKT/ERK通路抑制剂时,其生物学活性受抑制程度高于悬浮生长细胞,而细胞死亡率也高于悬浮生长细胞。而当联合应用两条通路抑制剂时,转移性肝癌细胞生物学活性较单独应用通路抑制剂显著降低,其降低的幅度明显高于贴壁生长细胞。
3.2在转移性肝癌细胞AKT通路受到抑制时ERK蛋白能够被代偿性激活
Western blot结果显示当AKT通路受抑制时,贴壁生长组细胞的ERK蛋白的相对磷酸化水平为0.67±0.006,而悬浮生长组为0.93±0.055,差别有统计学意义。
4.转移性肝癌细胞对hypoxia的反应
4.1单个悬浮生长细胞乏氧培养24小时细胞生长状态
在肝癌细胞还没有获得抵抗失巢凋亡能力时将其置于乏氧条件下,则CCK-8和trypan blue染色结果提示失去附着的转移性肝癌细胞和正常附着生长的细胞间细胞活性及细胞死亡率差别不大。
4.2聚集成团的悬浮生长细胞乏氧培养24小时的生长状态
将已经获得抵抗失巢凋亡能力的转移性肝癌细胞和贴壁生长的肝癌细胞同时放入低氧环境中,则转移性肝癌细胞的活性明显高于贴壁生长细胞,死亡率明显低于贴壁生长细胞,提示转移性肝癌细胞抵抗低氧的能力明显高于贴壁生长的细胞。
5.抵抗失巢凋亡和低氧刺激的相关分子筛选及论证
5.1.表达谱芯片中参与抵抗失巢凋亡同时耐受低氧的基因
表达谱芯片中在抵抗失巢凋亡过程中起作用同时耐受低氧的基因有许多,其中:ANGPTL4上调了16.29倍,BNIP3上调了7.88倍,CA9上调了10.31倍,IER3上调了7.95倍,IGFBP3上调了31.07倍,NDRG1上调了18.4倍。
5.2.与乏氧和失巢相关的microRNA间存在交叉
microRNA芯片结果显示,乏氧刺激下和失巢刺激下上调的microRNA重叠部分占到两种刺激下上调基因的44.4%,而下调的microRNA重叠部分有18.7%。
5.3.表达谱芯片中差异表达基因与microRNA芯片中差异microRNA调控基因吻合良好
表达谱芯片中差异表达基因与microRNA芯片中差异microRNA调控基因吻合良好,其中表达谱芯片结果中转移性肝癌细胞表达明显增高的BNIP3、CA9、NDRG1相对应的hsa-miR-558、hsa-miR-516a-3p/516b~*、hsa-miR-342、hsa-miR-620等在转移性肝癌细胞中显著下调。
5.4.反义封闭悬浮生长细胞的方法学探讨
PS-asODNs/ANGPTL4直接转染结果不稳定,脂质体介导转染悬浮生长细胞的转染效率低于转染贴壁生长细胞的转染效率,我们对培养至对数生长期的贴壁生长细胞进行反义封闭,6 h后分为贴壁生长组和悬浮生长组继续培养24 h,反义封闭效率基本一致。
5.5.ANGPTL4反义封闭后细胞的生物学活性
RT-PCR结果显示,PS-asODNs/ANGPTL4可显著降低ANGPTL4 mRNA的合成。反义封闭ANGPTL4后,转移性肝癌细胞与正常贴壁生长的肝癌细胞相比活性降低明显,其死亡率也较贴壁生长的肝癌细胞明显增高。
结论
1.转移性肝癌细胞模型的细胞聚集率在24小时稳定达到99.7%左右,进一步验证了模型的有效性。
2.转移性肝癌细胞的侵袭能力明显高于贴壁生长细胞,提示抵抗失巢凋亡的转移性肝癌细胞具有更强的通过第三微环境进而侵入第二微环境的能力。
3.转移性肝癌细胞一方面分泌更多的sTRAIL,另一方面自身能够抵抗TRAIL诱导的凋亡,通过诱导免疫效应细胞发生凋亡来逃逸机体免疫系统的监视,这一过程被称为“肿瘤反击”。转移性肝癌细胞表面的膜结合型DR4,DR5均明显下调,可能是转移性肝癌细胞能够抵抗TRAIL诱导凋亡的机制之一。
4.当AKT或者ERK通路单独受抑制时,转移性肝癌细胞的活性并没有发生多大变化。但是,当两条通路同时受抑制时,产生了一个非常明显的细胞死亡率。说明在这两个信号传导通路之间存在很强的代偿作用。当其中的一条通路受抑制时,另外一条通路的活化被放大来代偿其生存信号的缺失。Western blot结果进一步显示抑制AKT通路后磷酸化ERK蛋白的水平明显提高。
5.转移性肝癌细胞通过抵抗TRAIL诱导的凋亡以及AKT和ERK通路之间存在较强的代偿作用等机制抵抗失巢凋亡。
6.在肝癌细胞还没有获得抵抗失巢凋亡能力时将其置于乏氧条件下,则失去附着和正常附着生长的细胞间活性差别不大,一定程度上都能够耐受低氧刺激,但是,如果将已经获得抵抗失巢凋亡能力的转移性肝癌细胞和贴壁生长的肝癌细胞同时放入低氧环境中,则转移性肝癌细胞抵抗低氧的能力明显高于正常贴壁生长的细胞。说明抵抗失巢凋亡能力的获得有助于转移性肝癌细胞耐受低氧环境。
7.表达谱芯片中差异表达基因与microRNA芯片中差异microRNA调控基因吻合良好,其中表达谱芯片结果中转移性肝癌细胞表达明显增高的NDRG1、CA9、BNIP3相对应的hsa-miR-620、hsa-miR-516a-3p/516b~*、hsa-miR-342、hsa-miR-558等在转移性肝癌细胞中显著下调。
8.利用反义寡核苷酸直接封闭聚集成团的转移性肝癌细胞中的基因比较困难,需要将反义寡核苷酸事先转染入贴壁生长细胞6 h后再分别接种为贴壁生长组和悬浮生长组,以达到最佳的转染效率和转染稳定性。
9.反义封闭ANGPTL4后,转移性肝癌细胞的活性受抑制程度明显高于正常贴壁生长的肝癌细胞,而且其死亡率增高也比正常生长肝癌细胞明显,说明ANGPTL4在转移性肝癌细胞生长过程中发挥重要作用。
创新点及意义:
1.本研究首次报导转移性肝癌细胞通过“肿瘤反击”来逃逸机体的免疫监视功能,并且其侵袭力明显增强。
2.本研究首次报导转移性肝癌细胞的AKT、ERK通路之间存在很强的代偿作用,当抑制AKT通路后磷酸化ERK蛋白的水平明显提高。这种代偿作用可使转移性肝癌细胞抵抗相应抗肿瘤药物的攻击,提示我们可以联合应用这两条通路抑制剂来治疗转移性肝癌。
3.本研究率先提出获得抵抗失巢凋亡能力的转移性肝癌细胞抵抗低氧的能力明显高于贴壁生长的细胞。
4.本研究将表达谱芯片中差异表达基因与microRNA芯片中差异microRNA调控基因相比较,筛选可能参与调控转移性肝癌细胞转移能力的关键分子,临床应用价值更高。
5.本研究首次探讨了反义转染聚集成团细胞的方法,为反义寡核苷酸转染以及其他小分子物质进入聚集成团细胞提供了有价值的参考。
Hepatocarcinoma is a kind of malignant tumor with high morbidity and mortality worldwidely.The 5-year survival rate is very low because of its rapid progress, high recurrence and metastatic rate.If the mechanism of metastasis can be clarified and some early intervention can be put up,it will greatly improve the treatment of hepatocarcinoma.
To be able to arrival a second site to form a metastatic carcinoma,tumor cells have to complete a series of selective and orderly steps,including department from the carcinoma in situ,invasion into the vascular system,survival in vascular system,invasion of the matrix into the ectopic organ,spreading out of vascular system and proliferating.They have to overcome pressures before they finally forming metastases,including the immune surveillance capability,the hypoxic environment,a variety of attacks,such as antineoplastic agents.In addition,a variety of effector molecules involved in the process of tumor metastasis,many of them are upregulated at mRNA and protein expression levels under hypoxia. Hypoxia is considered a factor that tumor cells have to overcome.At the same time,there is increasing evidence indicating that hypoxia can promote tumor development.
We have successfully constructed the metastatic hepatoma cell model and we further validate its validity and metastatic capacity,as well as discussing the mechnism of enhanced metastatic capacity by forcing the metastatic hepatoma cells to various stimuli(including the exogenous TRAIL as well as the AKT/ERK pathway inhibitors),and further filter the key moleculars which can resist anoikis and hypoxia at the same time.
Objectives:
1.To construct the metastatic hepatoma cell model and explore its aggregated rate and metastatic capacity.
2.To demonstrate molecular mechanism of anoikis-resistance by study response of metastatic hepatoma cells to TRAIL-induced apoptosis to AKT/ERK pathway inhibitors.
3.To explore the response of metastatic hepatoma cells to hypoxic stimuli.
4.To screen the key molecules for anoikis-resistance and hypoxia-resistance in metastatic hepatoma cells.
Methods:
1.Metastatic hepatoma cell model and its aggregated rate and metastatic ability
1.1.Metastatic model of hepatocellular carcinoma cells BEL7402 and SMMC7721 cell lines were cultured in RPMI1640 with 10%FCS at 37℃5%CO_2 conditions to cultivate logarithmic phase,then digested by 0.25% trypsin into single cell suspension,adjusted the concentration,and cultured in Poly-HEMA culture plates for 24 h,that is the metastatic hepatoma cell model which analog metastatic hepatoma cells in vascular system.
1.2.Conditions of metastatic hepatoma cell model
Cells were cultured to logarithmic phase and prepared into single cell suspension, then seeded into the 96-well plates in different cells numbers as 0.5×10~4,1×10~4,1.5×10~4,3×10~4,3×10~4.The Poly-HEMA plates of the first four groups were washed 3 times using sterile PBS before seeding cells,the fifth group was not washed,each tin duplicated and cultured for another 24 h.Cell activity and mortality were detected by using CCK-8 and trypan blue staining.
1.3.Aggregated rate of metastatic hepatoma cells
Hepatoma cells were seeded into plates without or with poly-HEMA coating as attached or detached experimental groups and cultured for 24h.Single, non-aggregated cells were counted.The percentage of aggregated cells was calculated as(1-Ne/Nc)×100%,where Ne is the number of single cells after incubation for 24 h and Nc is the number of single cells before incubation.
1.4.Transwell detection of metastatic hepatoma cells' invasion and migration About 1.5×10~5 separating detached or attached hepatoma cells were seeded to the top chamber(diluted with 200ul RPMI 1640 in the presence of 1%FBS) of the Polycarbonate Membrane Transwell Inserts pre-coated with diluted Matrigel~(TM) Basement Membrane Matrix.The bottom chamber was added 0.5ml RPMI 1640 with 10%FBS in advance.After 20 h of incubation at 37℃,cells on the lower part were fixed,stained and scored in 10 randomly chosen microscopic fields to decide the invasion of anoikis-resistant cells.For migration studies,cells were seeded to the upper compartment of the uncoated inserts and cultured for 6h.
2.Response of metastatic hepatoma cells to TRAIL-induced apoptosis and its mechanisms
2.1.Response of metastatic hepatoma cells to TRAIL-induced apoptosis
1).TRAIL induced apoptosis detected by CCK-8 and Trypan blue assay
Attached and suspended groups were divided into 7 groups respectively,add 0ng/mL,5ng/mL,10ng/mL,20ng/mL,50ng/mL,100ng/mL,200 ng/mL of TRAIL protein.After 24h cell culture,CCK-8 and trypan blue staining were used to detect TRAIL-induced apoptosis.Attached and suspended groups were divided into 7 groups respectively,add 20ng/mL of TRAIL protein and cultured for 0 h,2 h,5 h,8 h,12 h,24 h and 48 h.Then,CCK-8 and trypan blue staining were used to detect TRAIL-induced apoptosis.
2).TRAIL induced apoptosis detected by TUNEL and Caspase3 activation assay
Hepatoma cells were seeded into 24-well plates as attached or detached groups for 20h,and treated with 20ng/ml recombinant sTRAIL for another 24h.Apoptotic cells were labeled with TUNEL by using an In Situ Cell Death Detection Kit.The degree of apoptosis was determined by flow cytometry.Caspase-3 activities of hepatoma cells in 6-well plates treated with sTRAIL for 3 h,6 h,9 h,12 h and 24 h were detected by Caspase-3/CPP32 Colorimetric Assay Kit.
2.2.Mechanism of metastatic hepatoma cells resist TRAIL-induced apoptosis
1).Expression of TRAIL and its receptors in metastatic hepatoma cells
Total RNA was extracted by trizol one-step extraction from attached and detached cells.The mRNA expression of TRAIL and its receptors were analysesed by profiling microarray.RT-PCR was used to detect the mRNA expression of TRAIL and its receptors of metastatic hepatoma cells.ELISA assay was used to indicate the secretion of sTRAIL protein in culture supernatant,FCM was used to detect the surface-bound TRAIL and its receptors on metastatic hepatoma cells.
2).Caspase-8,-9 and -3 activation of metastatic hepatoma cells determined by Western blot
We further detected the Caspase-8,-9 and -3 activation after 20ng/ml of sTRAIL treatment by using Western blot.
3.Responses of metastatic hepatoma cells to PI-3K/AKT or ERK pathway inhibitors
Cells in either adherent or suspended culture conditions were treated with 1μM of Wortmannin or 50μM of PD98059 for 24 h.cell viability was measured by CCK-8 kit as well as trypan blue exclusion assay.Phosphorylation level of protein was detected by western blot.
4.Response of metastatic hepatoma cells to hypoxie stress
Attached and detached cells in 96-well plates were cultured at 37℃with 2%O_2 for 24 h as hypoxia treated group or cultured at 37℃with 20%O_2 as nomoxia control.Another two groups of conventional culture and then placed under hypoxic conditions for another 24 hours.Cell viability and proliferation status were measured by using the CCK-8 assay or trypan blue staining.
Inhibition rate calculated by the following formula InhibitRate=Anomoxia-Ahypoxia/Anomoxia.Mortality rates in accordance with the following formula:DeadRate=DeadCells/DeadCells+LiveCells.
5.Screening and demonstrating associated genes in anoikis-resistance and hypoxia-resistance
5.1.Analysesing the microarray expression profiling of attached and detached hepatoma cells
Expression profiling of attached and detached hepatoma cells were analysesed by gene chip.Using cluster software to define the functional annotation clustering of different expressed genes and find genes participated in anoikis-resistance and hypoxia-resistance.
5.2.Analyses the different microRNAs in attached,detached and hypoxia hepatoma cells
MicroRNA chip was used to find different microRNAs in attached,detached and hypoxia hepatoma cells.The results were analyzed at http://www.targetscan.org/ website,target genes of up-regulated and down-regulated microRNA were screened and compared with the results of gene chip.Genes participated in anoikis-resistance and hypoxic stimulus were selected.
5.3.Block efficiency of different antisense protocol
We used four protocol to block aggregated cells by antisense oligonucleotide.①Transfecting attached and detached hepatoma cells directly by antisense oligonucleotide.②Transfecting attached and detached hepatoma cells by liposome-mediated antisense oligonucleotide.③Transfecting attached hepatoma cells by liposome-mediated antisense oligonucleotide without FBS,and then divided the attached cells into attached group and detached group.④Transfecting attached hepatoma cells by liposome-mediated antisense oligonucleotide with FBS,and then divided the attached cells into attached group and detached group.The efficiency of different programs and experimental stability was compared.
5.4.Biological activity of cells blocked by antisense oligonucleotides of associated genes
We designed antisense oligonucleotide sequence of ANGPTL4,CA9,NDRG1, TRAIL and TrkB,and blocked the attached and detached hepatoma cells for 24h, then detected block efficiency by Real-Time PCR and RT-PCR as well as determined the biological activity by using CCK-8 or trypan blue staining assay.
6.Statistical analysis
Results were expressed as mean±SD.The statistical significance of differences between the groups was determined by Student's paired T tests or one-way ANOVA using SPSS10 statistical software.P＜0.05 was considered statistically significant.。
Results
1.Aggregated rate and metastatic ability of metastatic hepatoma cell model
1.1.Metastatic hepatoma cell model
Conventional cultured BEL7402 and SMMC7721 cells spread out as monolayers, while the suspension cells became round and self-aggregated into big cell clusters after cultured for 24 h.The aggregation rate stability was very well.It was always more than 99.7%.Poly-HEMA coated plates must be washed before using.The best cell density of 96 well plates is 3×10~4 per well.
1.2.metastatic hepatoma cells' invasion and migration
Mobility analysis revealed no significant difference between the attached and detached cells.However,the amount of invasive cells in the anoikis-resistant detached cell group was much greater than that of attached cells(19473±1104 vs. 9110±706 cells,p＜0.05).
2.Response of metastatic hepatoma cells to TRAIL-induced apoptosis and its mechanisms
2.1.Metastatic hepatoma cells resist TRAIL-induced apoptosis in a dose-dependent and time-dependent manner
Metastatic hepatoma cells resist TRAIL-induced apoptosis in a dose-dependent and time-dependent manner.Specifically,the apoptosis rate in the attached hepatoma cells after treatment with 20ng/ml of sTRAIL for 24 h was 20%while that in the detached cells was 5%,a value similar to that of control hepatoma cells without sTRAIL treatment.We used the dose of 20ng/mL and processing time for 24 h in the following experiments according to time-dependent and dose-dependent curve.
2.2.Metastatic hepatoma cells were more resistant to TRAIL-induced apoptosis
After treated with TRAIL,metastatic hepatoma cells' apoptotic rate was significantly lower than the control group cells detected by TUNEL method. Caspase-3 activated level arrived the peak at 9 h after treated with TRAIL. Caspase-3 activated level of metastatic hepatoma cells were lower than control group cells at different time points and the difference at 9 h was most obviously.
2.3.Expression of TRAIL mRNA was increased significantly in metastatic hepatoma cells
All assays of detecting TRAIL mRNA,including microarray analysis and semi-quantitative RT-PCR indicated that expression of TRAIL mRNA was increased significantly in Synoikis-like suspended BEL7402 cells compared with that of attached counterpart.
2.4.A discrepancy of TRAIL protein expression in metastatic hepatoma cells There is a discrepancy at the protein level of TRAIL expression.sTRAIL secreted in the supernatant was increased significantly in the detached hepatoma cells,while membrane binding TRAIL(mTRAIL) was down-regulated in the detached cells compared with that of attached cells.
2.5.TRAIL receptors of metastatic hepatoma cells were decreased significantly on mRNA level and protein level
Gene chip and semi-quantitative RT-PCR indicated that expression of DR4,DR5, DcR1,and DcR2 mRNA of detached synoikis-like hepatoma cells was decreased significantly compared with that of attached control groups.There is also a substantial decreased expression of TRAIL membrane binding receptors at the protein level on the detached cells as compared with that of attached cells detected by FCM.
2.6.Loss of anchorage blocked TRAIL-induced caspase cascade activation TRAIL induced significant activation of procaspase-8,procaspase-9 and procaspase-3.However,this activation of caspase cascade was effectively blocked upon loss of cell anchorage.
3.Response of metastatic hepatoma cells to AKT/ERK pathway inhibitors
3.1 Metastatic hepatoma cells were less compromised to AKT or ERK pathway inhibition
Growth of attached hepatoma cells were greatly inhibited under the effect of AKT or ERK inhibition,however,the detached hepatoma cells were much more resistant to protein kinase inhibition when the inhibitor was used separately. Surprisingly,when both of the AKT and ERK pathways were blocked together, the viability of detached cells decreased dramatically to a degree even greater than that of attached groups.
3.2 A strong compensated activation mechanism exit in the metastatic hepatoma cells
It showed that the ERK phosphorylation level was dramatically increased when AKT pathway inhibitor was used,which indicated a strong compensated activation mechanism in the metastatic hepatoma cells.
4.Response of metastatic hepatoma cells to hypoxia
There was no difference of cell viability between detached and attached hepatoma cells when placed under hypoxic conditions before they obtained the ability of anoikis-resistance detected by CCK-8 and trypan blue assay.They were all able to tolerant the hypoxia stimuli to some extent.
If expand the anoikis-resistant hepatoma cells to hypoxia,its ability to resist hypoxic environment is much higher than the attached hepatoma cells detected by CCK-8 and trypan blue assay.
5.Screening and demonstrating associated genes in anoikis-resistance and hypoxia-resistance
5.1.Genes involved in resistance to anoikis and hypoxia in gene chip
There were a large number of genes that involved in resistance to anoikis and regulated by hypoxia in gene chip.Such as IER3(up-regulated for 7.95 times), CA9(up-regulated for 10.31 times),BNIP3(up-regulated for 7.88 times), NDRG1(up-regulated for 18.4 times),ANGPTL4(up-regulated for 16.29 times), IGFBP3(up-regulated for 31.07 times).
5.2.Crosstalk between microRNAs regulate anoikis and hypoxia
There was a large proportion of crosstalk between microRNAs which regulate anoikis and hypoxia.For example,the crosstalking proportion of upregulated microRNA is more than 44.4%,and that of downregulated is about 18.7%.
5.3.There was a well consistent between the gene chip and microRNA chip There was a well consistent between the differentially expressed genes of gene chip and target genes of different microRNAs in microRNA chip.For example, NDRG1,CA9 and BNIP3 were increased in metastatic hepatoma cells,and their possible regulate microRNAs hsa-miR-620,hsa-miR-516a-3p/516b*, hsa-miR-342,hsa-miR-558 were down-regulated significantly.
5.4.Methods of PS-asODNs/ANGPTL4 block
The results of PS-asODNs/ANGPTL4 direct transfection were unstable. Transfection efficiency of liposome-mediated transfection of detached cells was lower than that of attached cells.We cultured attached cells to logarithmic phase and transfected them with liposome-mediation for 6 h,then divided them into attached and suspension groups to continue to culture for another 24 h,the efficiency of antisense had no difference.
5.5.Biology activity of cells whose ANGPTL4 had been blocked
Real-Time PCR and RT-PCR results showed that,PS-asODNs/ANGPTL4 can significantly reduce the mRNA synthesis of ANGPTL4(p＜0.05).When ANGPTL4 was blocked,the viability of metastatic hepatoma cells was much more inhibited than that of the attached hepatoma cells.The mortality rate was also higher than the normal cultured hepatoma cells.
Conclusions:
1.Cell aggregated rate of metastatic hepatoma cell model at 24 hours reached 99.7%steadily,which further verify the validity of the metastatic hepatoma cell model.
2.Metastatic hepatoma cells had significantly higher invasiveness than the adherent cells.These results suggest that anoikis-resistant metastatic hepatoma cells are more invasive to go through the third microenvironment and arrive the second microenvironment,that is to say they are more aggressive.
3.Metastatic hepatoma cells secret more sTRAIL on the one hand and it can resist TRAIL-induced apoptosis on the other hand,which can induce apoptosis of immune effector cells in order to escape from immune system surveillance,the process is called "tumor counterattack".Metastatic hepatoma cells membrane-binding DR4,DR5 were significantly reduced. Maybe it is one of the mechanisms of metastatic hepatoma cells resist TRAIL-induced apoptosis.
4.When AKT or ERK pathway was inhibited separately,it did not take much effect on the viability of the anoikis-resistant cells.However,when both of these two protein kinase pathway were blocked,it caused a dramatically cell death.This indicates that there is a strong interplay between these two pro-survival pathways.When one of these ways was inhibited,the signal of the other pathway will be over-activated and compensate for loss of this survival signal.Interestingly,our western blot data showed that inhibition of AKT pathway dramatically increased the phosphorylation level of ERK pathway.
5.Metastatic hepatoma cells may be resist anoikis by resisting TRAIL-induced apoptosis and interplay of AKT and ERK pathway.
6.There was no difference of cell viability between detached and attached hepatoma cells when placed under hypoxic conditions before they obtained the ability of anoikis-resistance.They were all able to tolerant the hypoxia stimuli to some extent.But,if expand the anoikis-resistant hepatoma cells to hypoxia,its ability to resist hypoxic environment is much higher than the attached hepatoma cells.That is to say,acquisition of anoikis-resistance contributes to hypoxia tolerance of metastatic hepatoma cells.
7.There was a well consistent between the differentially expressed genes of gene chip and target genes of different microRNAs in microRNA chip.For example,NDRG1,CA9 and BNIP3 were increased in metastatic hepatoma cells,and their possible regulate microRNAs hsa-miR-620, hsa-miR-516a-3p/516b*,hsa-miR-342,hsa-miR-558 were down-regulated significantly.
8.It is difficult to block genes of metastatic hepatoma cells by transfecting oligonucleotide directly.It is better to transfect antisense oligonucleotide into attached hepatoma cells and block for 6 h,followed by dividing them into attached and detached cell groups.
9.When ANGPTL4 was blocked,the viability of metastatic hepatoma cells was much more inhibited than that of the attached hepatoma cells.The mortality rate was also higher than the normal cultured hepatoma cells.These indicate that ANGPTL4 may play an important role in the growth of metastatic hepatoma cells.
Innovations and significances:
1.This is the first report that metastatic hepatoma cells can escape from immunological surveillance by "tumor counterattack" and have increased invasiveness.
2.It is the first time to report this compensated activation in detached hepatoma cells when one of the major survival pathway was blocked.This vigorous compensated activation may offer anchorage-deprived hepatoma cells more survival opportunities and metastatic potential when using relative therapeutic agents.Thus,combination of kinase inhibitors is likely to yield a substantial advance in successfully producing downstream phenotypic response in anoikis-resistant hepatoma cells.
3.In this study,we first put forward that the ability of resistance to hypoxia of metastatic hepatoma cells was significantly higher than that of normal adherent cells.
4.In this study,we combined the gene chip and microRNA chip to screen key molculars that may be involved in regulation of metastatic hepatocellular carcinoma cells.It will have higher clinical application value.
5.This study explored firstly the methods of PS-asODNs transfecting aggregated cells,which will provide a useful reference for PS-asODNs as well as other small molecules to transfer into the aggregated cells.
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