| ObjectiveTo investigate the effect of inhibiting Mcl-1 gene expression on the biological behavior of gastric cancer cell lines SGC7901 and MGC-803 by using a small interference RNA (siRNA) strategy.MethodsMcl-1 expression was evaluated in Human gastric cancer cell lines SGC7901 and MGC-803 by RT-PCR. And then four segments of siRNAs targeting Mcl-1 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 Mcl-1 specific siRNA and was transfected transiently into human gastric cancer SGC7901 and MGC-803 cells. After transfected 24h,48h,72h, quantitative real-time PCR was applied to detect the mRNA expression of Mcl-1 and Western blot was applied to detect the protein expression of Mcl-1 in SGC7901 and MGC-803 cells. Then MTT method was adapted to investigate the proliferation of SGC7901 and MGC-803 cells after transfection of Mcl-1 siRNA. After 48h transfection of Mcl-1 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, negative control group and siRNA 1 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 SGC7901 and MGC-803 expressed Mcl-1 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 Mcl-1 mRNA and protein expression levels in SGC7901 and MGC-803 cells. When SGC7901 and MGC-803 cells were transfected 24h and 48h later, compared with normal group, liposome group and negative control group cells. The Mcl-1 mRNA levels were decreased in different degree in Mcl-1siRNA1, Mcl-1siRNA2, Mcl-1siRNA3 and Mcl-1siRNA4 transfected cells, to different extent(P<0.05). But when SGC7901 cells were transfected 72h later, there were no significant differences among normal group, liposome group, negative control group and Mcl-1 siRNA4 group(P>0.05). When MGC-803 cells were transfected 72h later, there were no significant differences among normal group, liposome group, negative control group, Mcl-1siRNA2 and Mcl-1siRNA4 group(P>0.05). There were no significant differences among normal group, liposome group and negative control group cells at all the time in both SGC7901 and MGC-803 cell lines (P >0.05).The most effective segment was Mcl-1siRNA1. when SGC7901 cells were transfected 24h,48h and 72h later, inhibition rate of the Mcl-1siRNA1 group were 79.4%,73.8% and 66.1%, respectively, and when MGC-803 cells were transfected 24h,48h and 72h later, inhibition rate was 51.9%,67.6% and 63.1%, respectively. When SGC7901 cells were transfected 24h,48h and 72h later, compared to normal group, liposome group and negative control group cells.The Mcl-1 protein levels were decreased in Mcl-1siRNA1, Mcl-1siRNA2, Mcl-1siRNA3, and Mcl-1siRNA4 transfected cells to different extent (P<0.05). Transfected 24h and 48h later, the most effective segment was Mcl-1siRNA2. The inhibition rate was 99.7% and 82.2%, respectively. Transfected 72h later, the most effective segment was Mcl-1siRNA3. The inhibition rate was 57.3%. The highest inhibition rate of Mcl-1 siRNA2 and Mcl-1siRNA3 were 79.3% and 64.0% at 48h. The highest inhibition rate of Mcl-1siRNA1 was 99.7% at 24h. The highest inhibition rate of Mcl-1siRNA4 was 55.8% at 24h.When MGC-803 cells were transfected 24h later, only the Mcl-1 protein levels of the Mcl-1siRNA1 group was 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, Mcl-1siRNA2 group, Mcl-1siRNA3 group and Mcl-1siRNA4 group cells (P> 0.05). Transfected 48h later, the Mcl-1 protein levels of the Mcl-1siRNA1 group and Mcl-1siRNA2 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, Mcl-1siRNA3 group and Mcl-1siRNA4 group cells (P>0.05). Transfected 72h later, the Mcl-1 protein levels were decreased in different degree in Mcl-1siRNAl, Mcl-1siRNA2, Mcl-1siRNA3, and Mcl-1siRNA4 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). The highest inhibitory rate was 96.1% in Mcl-1 siRNAl at 48h. Therefore, the Mcl-1siRNA1 was chosen for the subsequent experiments in vitro.5. The proliferation was inhibited notably when SGC7901 and MGC-803 cells were transfected with Mcl-1siRNA1(P<0.05). The inhibitory rates were 21.4%,39.9%,31.3%, respectively, in SGC7901 cells and 8.9%,21.6%,18.8%, respectively, in MGC-803 cells, after transfected 24h,48h,72h. The highest inhibitory rate was 39.9% in SGC7901 and 21.6% in MGC-803 cells at 48h. There were no significant differences among normal group, liposome group and negative control group cells (P>0.05).6. After transfected 48h, the apoptotic rate of the Mcl-1siRNA1 group obviously increased in both SGC7901 cells (26.66%±1.10%) and MGC-803 cells (19.61%±1.66%), signifieantly higher than control groups(P<0.05).7. After transfected 48h, The S phase proportion of the Mcl-1 siRNAl group obviously increased in both SGC7901 cells (52.93%±1.11%) and MGC-803 cells (57.17%±1.72%), signifieantly higher than control groups(P< 0.05), while there were accordingly decrease among the G0/G1 and G2/M phases proportion(P<0.05).8. After transfected 48h, the number of the Mcl-1siRNA1 group cells invading obviously decreased in both SGC7901 cells (24.00±3.51) and MGC-803 cells (42.00±4.00), signifieantly lower than control groups(P<0.05). The inhibitory rate was 51.25% in SGC7901 and 47.05% in MGC-803 cells.9. After transfected 48h, the number of the Mcl-1siRNA1 group cells migrating obviously decreased in both SGC7901 cells (34.00±4.00) and MGC-803 cells (76.33±3.51), signifieantly lower than control groups(P<0.05). The inhibitory rate was 43.80% in SGC7901 and 29.35% in MGC-803 cells.ConclusionMcl-1 gene obviously expresses in gastric cancer cell lines SGC7901 and MGC-803. Mcl-1siRNA can efficiently inhibit Mcl-1 gene expression in gastric cancer cells. Inhibiting Mcl-1 gene expression effectively suppresses gastric cancer cell proliferation and enhances gastric cancer cell apoptosis in vitro. Inhibiting Mcl-1 gene expression results in S phase arrested in cell cycle in gastric cancer cell. Inhibiting Mcl-1 gene expression suppresses gastric cancer cell invation ability and migration ability in vitro. Our data suggests that the specific down-regulation of Mcl-1 by RNAi is a promising gene therapy approach for the treatment of gastric carcinoma. |
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