| 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. |
PDF Full Text Request