【作者】 马鹏飞；

【导师】 陈俊强；

【作者基本信息】 广西医科大学 ， 胃肠腺体外科， 2012， 硕士

【摘要】 目的探讨siRNA干扰CLIC1基因表达后对胃癌细胞生物学行为的影响。方法利用RT-PCR检测CLIC1基因在胃癌细胞株SGC-7901和MGC-803中的表达；化学合成四对CLIC1siRNA,分别标记为CLIC1siRNA1、CLIC1siRNA2、CLIC1siRNA3和CLIC1siRNA4,利用脂质体Lipofectamine2000瞬时转染胃癌细胞SGC-7901和MGC-803。采用标记有荧光的阴性对照siRNA在荧光显微镜下检测转染效率；转染24h、48h、72h后,通过实时定量PCR和Western blot分别检测CLIC1mRNA和蛋白的表达,筛选出干扰效果最好的siRNA片段；用抑制率最高的siRNA及无义阴性对照再次转染胃癌细胞SGC-7901和MGC-803,设立脂质体对照和空白对照。MTT法检测CLIC1抑制前后细胞增殖情况；转染48h后收集细胞,分别进行AnnexinV-FITC和PI双染,观察细胞各组凋亡变化情况；P工单染,观察各组细胞周期变化情况；转染48h后应用Transwell小室及Matrigel胶,通过计数细胞数目分析细胞侵袭迁移能力的变化。结果1.胃癌细胞株SGC-7901和MGC-803均存在CLIC1基因的表达。2.按照脂质体说明书推荐的比例转染胃癌细胞,转染效率约为80%。3.用实时荧光定量PCR法和Western blot方法分别检测转染24h、48h、72h后CLIC1基因mRNA和蛋白的表达情况。结果显示,转染24h、48h、72h后SGC-7901细胞CLIC1基因mRNA相对表达量在CLIC1siRNA片段转染组中与各对照组中比较差异显著(P<0.05)。在MGGC-803中,转染24h后,只有CLIC1siRNA1、CLIC1siRNA3、CLIC1siRNA4组跟各对照组比较差异显著(P<0.05),siRNA2组跟各对照组比较无明显差异(P>0.05)；转染48h、72h后CLIC1siRNA组基因mRNA跟各对照组比较有显著差异(P<0.05)。两株细胞各个时间点中,空白对照组、脂质体对照组和阴性对照组之间比较差异均不显著(P>0.05)。四个片段中CLIC1siRNA3的抑制作用最好,转染24h、48h、72h后的抑制率在SGC-7901中分别为98.35%、94.15%、95.38%。而在MGC-803中分别为78.33%、86.81%、84.65%。蛋白表达水平的检测结果显示,SGC-7901细胞中,转染24h后只有CLIC1siRNA3、CLIC1siRNA4组跟各对照组比较差异显著(P<0.05),CLIC1siRNAK CLIC1 siRNA2组跟各对照组比较无明显差异(P>0.05)；转染48h、72h后CLIC1siRNA组与各对照组比较,CLIC1蛋白表达则显著下调(P<0.05)。不同片段之间比较,24h、48h、72h时CLIC1siRNA3抑制率最高,分别达到70.00%、76.39%和87.90%,通过对不同时间点抑制率的观察发现,CLIC1siRNA4在24h的抑制效果最好为55.51%,而CLIC1siRNA1、CLIC1siRNA2和CLIC1siRNA3组则在72h时抑制效果最好,分别为46.29%、50.39%和87.90%。在MGC-803细胞中,转染48h后,只有CLIC1siRNA1、CLIC1siRNA3和CLIC1siRNA4组与各对照组细胞比较,CLIC1蛋白的表达显著下调(P<0.05),而CLIC1siRNA2组与各对照组之间比较无明显差异性(P>0.05)。转染24h、72h后,CLIC1siRNA组与各对照组细胞比较,CLIC1蛋白的表达则显著下调(P<0.05)。其中CLIC1siRNA1、CLIC1siRNA2, CLIC1siRNA4在24h的抑制作用最好,分别达到85.57%、51.09%和88.57%。CLIC1siRNA3则在48h的抑制率最好为81.97%。在两株细胞三个时间点中,空白对照组、脂质体对照组、阴性对照组之间比较均无显著差异(P>0.05)。转染CLIC1siRNA后可有效下调胃癌细胞CLIC1蛋白的表达。结果显示,CLIC1siRNA可特异性、高效地下调CLIC1基因的表达；不同的CLIC1siRNA片段对CLIC1基因表达的抑制作用不同；而相同的片段在不同时间点对CLIC1基因表达的抑制作用也不同,总体上CLIC1siRNA对mRNA和蛋白的抑制率在48h最好。从抑制率跟稳定性来看,CLIC1siRNA3片段的效果最好。我们选择CLIC1siRNA3开展后面的实验。5.MTT检测结果：用CLIC1siRNA3转染胃癌细胞SGC-7901和MGC-803。两株细胞转染24h、48h和72h后转染组与空白对照组、脂质体对照组相比较差异均显著(P<0.05)。空白对照组、脂质体对照组之间比较均无明显差异性(P>0.05)。抑制CLIC1表达后,胃癌细胞增殖能力明显增强。SGC-7901中各时间点的增殖率分别为25.60%、23.30%、21.52%；MGC-803中的增殖率分别为15.22%、38.11%、21.43%。6.转染48h后胃癌细胞SGC-7901和MGC-803凋亡情况：空白对照组、脂质体对照组、CLIC1siRNA3组中细胞凋亡率在SGC-7901为23.92%±2.47%、25.56%±3.37%、9.03%±1.20%；在MGC-803为19.59%±3.31%、20.52%±2.88%、9.27%±2.19%。转染组的凋亡细胞明显减少,与各对照组比较差异显著(P<0.05)。空白对照组、脂质体对照组、阴性对照组之间比较均无显著差异性(P>0.05)。7.转染48h后细胞周期分布：空白对照组、脂质体对照组、CLIC1siRNA3组G2/M期细胞比例在SGC-7901分别为6.03%±0.70%、6.84%±0.84%、22.08%±2.41%,在MGC-803分别为8.39%±1.69%、9.16%±1.26%、19.14%±2.18%。siRNA3组G2/M期细胞明显增多(P<0.05),同时,G0/G1期、S期比例相应的减少。空白对照组、脂质体对照组之间比较无明显差异性(P>0.05)。8.侵袭实验显示,转染48h后,空白对照组、脂质体对照组和CLIC1siRNA3组侵袭细胞个数在SGC-7901中分别为65.67±6.03、67.67±3.51、30.00±5.00,在MGC-803细胞中分别为63.00±2.65、64.33±4.51、37.33±4.93。CLIC1siRNA3组与各对照组比较有显著差异(P<0.05)。空白对照组、脂质体对照组之间比较均无显著差异(P>0.05)。抑制CLIC1表达后对SGC-7901细胞侵袭能力的抑制率为54.31%,对MGC-803细胞侵袭能力的抑制率为40.74%。9.迁移实验显示,转染48h后,空白对照组、脂质体对照组和CLIC1siRNA3组迁移细胞个数在SGC-7901分别为78.33±5.51、76.00±4.58、52.00±5.29,在MGC-803细胞中分别为76.33±7.02、76.00±3.61、54.00±4.58。CLIC1siRNA3组与各对照组比较差异显著(P<0.05)。空白对照组、脂质体对照组之间比较均无显著差异(P>0.05)。抑制CLIC1表达后对SGC-7901细胞迁移能力的抑制率为33.62%,对MGC-803细胞迁移能力的抑制率为29.26%。结论1.胃癌细胞株SGC-7901和MGC-803中存在CLIC1基因的表达,可以用于siRNA抑制CLIC1基因的表达,进行CLIC1基因在胃癌细胞生物学行为中的作用的研究。2.脂质体介导的CLIC1siRNA转染胃癌细胞株SGC-7901和MGC-803,可特异性、高效地下调CLIC1基因的表达。不同的CLIC1siRNA片段对CLIC1基因表达的抑制作用不同；而相同的片段在不同时间点对CLIC1基因表达的抑制作用也不同。总体上讲,siRNA对CLIC1的表达抑制作用48h最强。说明,RNAi是一种抑制胃癌细胞CLIC1基因表达的有效的方法。3.抑制CLIC1表达,导致体外培养的胃癌细胞的增殖明显增强、凋亡减少、细胞周期阻滞在G2/M期。4.抑制CLIC1表达,胃癌细胞侵袭迁移能力受抑。表明,通过RNAi抑制CLIC1是胃癌基因治疗的一个潜在有效的法。为完善胃癌的治疗方法、改善胃癌患者的预后,提供了新的思路。更多还原

【Abstract】 ObjectiveTo investigate the effect of inhibiting CLIC1 gene expression on the biological behavior of gastric cancer cell lines SGC-7901 and MGC-803 by using a small interference RNA (siRNA) strategy.MethodsCLIC1 expression was evaluated in Human gastric cancer cell lines SGC-7901 and MGC-803 by RT-PCR. And then four segments of siRNAs targeting CLIC1 mRNA were designed by bioinformatics technology, and the no-sense control segment was also designed. The transfected efficiency was detected by fluorescence microscope. Lipofectamine 2000 was used to package the CLIC1 specific siRNA and was transfected transiently into human gastric cancer SGC-7901 and MGC-803 cells. After transfected 24h,48h,72h, quantitative real-time PCR was applied to detect the mRNA expression of CLIC1 and Western blot was applied to detect the protein expression of CLIC1 in SGC-7901 and MGC-803 cells. Then MTT method was adapted to investigate the proliferation of SGC-7901 and MGC-803 cells after transfection of CLIC1 siRNA. After 48h transfection of CLIC1 siRNA, the cells were stained by AnnexinV-FIFC and PI, and the flow cytometry was used to examine the apoptosis cells in normal group, liposome group and siRNA3 group. Cell cycle was detected by FCM in different groups. Polycarbonate membrane transwell chamber and Matrigel were used for the detection of the changes of invasion and migration of these two cell lines.Results1. The gastric cancer cell lines SGC-7901 and MGC-803 expressed CLIC1 obviously.2. The transfection rate was approximate 80%.3. Quantitative real-time PCR and Western blot were performed to examine the effect of siRNA transfection on CLIC1 mRNA and protein expression levels in SGC-7901 and MGC-803 cells. When SGC-7901 cells were transfected 24h, 48h and 72h later, compared with normal group, liposome group and negative control group cells. The CLIC1 mRNA levels were decreased in different degree in CLIC1 siRNA 1, CLIC1 siRNA2, CLIC1 siRNA3 and CLIC1 siRNA4 transfected cells, to different extent(P< 0.05). when MGC-803 cells were transfected 24h later, there were no significant differences among normal group, liposome group, negative control group and CLIC1 siRNA2 group(P>0.05). But When MGC-803 cells were transfected 48h and 72h later, compared with normal group, liposome group and negative control group cells. The CLIC1 mRNA levels were decreased in different degree in CLIC1 siRNA 1, CLIC1siRNA2, CLIC1siRNA3 and CLIC1siRNA4 transfected cells, to different extent(P<0.05). There were no significant differences among normal group, liposome group and negative control group cells at all the time in both SGC-7901 and MGC-803 cell lines (P>0.05). The most effective segment was CLIC1siRNA3. when SGC-7901 cells were transfected 24h,48h and 72h later, inhibition rate of the CLIC1siRNA3 group were 98.35%、94.15% and 95.38%, respectively, and when MGC-803 cells were transfected 24h,48h and 72h later, inhibition rate was 78.33%、86.81% and 84.65%, respectively. When SGC-7901 cells were transfected 24h later, compared to normal group, liposome group and negative control group cells, the CLIC1 protein levels were decreased in CLIC1 siRNA3 and CLIC1siRNA4 transfected cells to different extent (P<0.05),and they were no significant differences in CLIC1siRNA1and CLIC1siRNA2 transfected cells(P>0.05). Transfected 48h and 72h later, compared with normal group, liposome group and negative control group cells. The CLIC1 protein levels were decreased in different degree in CLIC1siRNA1, CLIC1siRNA2, CLIC1siRNA3 and CLIC1siRNA4 transfected cells, to different extent(P< 0.05). The comparison between different segments showed that the most effective segment was CLIC1siRNA3, transfected 24h,48h and 72h later, the inhibition rate were 70.00%,76.39% and 87.90%, respectively.The most effective segment was CLIC1siRNA3, transfected 24h,48h and 72h later, the inhibition rate were 70.00%,76.39 and 87.90. When MGC-803 cells were transfected 48h later, only the CLIC1 protein levels of the CLIC1siRNA1, CLIC1siRNA3, CLIC1siRNA4 group were decreased compared to normal group, liposome group and negative control group cells (P<0.05). There were no significant differences among normal group, liposome group, negative control group, CLIC1siRNA2 group cells (P>0.05). Transfected 24h and 72h later, the CLIC1 protein levels of the CLIC1siRNAs group were decreased compared to normal group, liposome group and negative control group cells (P <0.05). There were no significant differences among normal group, liposome group, negative control group, (P>0.05). The highest inhibitory rate of CLIC1siRNAl,CLIC1siRNA2 and CLIC1siRNA4 were 85.57%,51.09% and 88.57%, respectively, after transfected 24h later. The highest inhibitory rate of CLIC1siRNA3 was 81.97% after transfected 48h later. the CLIC1 protein levels were decreased in different degree in CLIC1siRNAl, CLIC1siRNA3, and CLIC1siRNA4 transfected cells compared to normal group, liposome group and negative control group cells (P<0.05). There were no significant differences among normal group, liposome group and negative control group cells at all the time in both cell lines (P>0.05). In general, the most effective segment was CLIC1siRNA3 and the best time at 48h. Therefore, the CLIC1siRNA1 was chosen for the subsequent experiments in vitro.5. The proliferation was enhanced notably when SGC-7901 and MGC-803 cells were transfected with CLIC1siRNA3(P<0.05). The growth rates were 25.60%x 23.30%、21.52%, respectively, in SGC-7901 cells and 25.60%、23.30%,21.52%, respectively, in MGC-803 cells, after transfected 24h,48h,72h. the highest growth rates were 25.60% in SGC-7901 for CLIC1 siRNA3 at 24h and 38.11% in MGC-803 cells for CLIC1 siRNA3 at 48h. There were no significant differences among normal group and liposome group cells (P>0.05).6. After transfected 48h, the apoptotic rate of the CLIC1siRNA3 group obviously decreased in both SGC-7901 cells (9.03%±1.20%) and MGC-803 cells (9.27%±2.19%), signifieantly lower than control groups(P<0.05).7. After transfected 48h, The G2/M phase proportion of the CLIC1 siRNA3 group obviously increased in both SGC-7901 cells (22.08%±2.4%) and MGC-803 cells (19.14%±2.18%), signifieantly higher than control groups(P< 0.05), while there were accordingly decrease among the G0/G1 and S phases proportion(P<0.05). 8. After transfected 48h, the number of the CLIC1siRNA3 group cells invading obviously decreased in both SGC-7901 cells (30.00±5.00) and MGC-803 cells (37.33±4.93), signifieantly lower than control groups(P<0.05). The inhibitory rate was 54.31% in SGC-7901 and 40.74% in MGC-803 cells.9. After transfected 48h, the number of the CLIC1siRNA3 group cells migrating obviously decreased in both SGC-7901 cells (52.00±5.29) and MGC-803 cells (54.00±4.58), signifieantly lower than control groups(P<0.05). The inhibitory rate was 33.62% in SGC-7901 and 29.26% in MGC-803 cells.ConclusionCLIC1 gene obviously expresses in gastric cancer cell lines SGC-7901 and MGC-803. CLIC1siRNA can efficiently inhibit CLIC1 gene expression in gastric cancer cells. Inhibiting CLIC1 gene expression effectively enhanced gastric cancer cell proliferation and decreased gastric cancer cell apoptosis in vitro. Inhibiting CLIC1 gene expression results in G2/M phase arrested in cell cycle in gastric cancer cell. Inhibiting CLIC1 gene expression suppresses gastric cancer cell invation ability and migration ability in vitro. Our data suggests that the specific down-regulation of CLIC1 by RNAi is a promising gene therapy approach for the treatment of gastric carcinoma.更多还原