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目的研究shRNAs对胃癌细胞系BGC-823胃泌素表达的抑制效应。方法设计4条针对胃泌素基因不同位点的寡核苷酸序列,通过体外转录法合成相应的shRNAs。以10nmol/L、20nmol/L、40nmol/L和80nmol/L的终浓度,将4条shRNAs分别转染胃癌细胞BGC-823。应用原位杂交及免疫细胞化学方法检测胃泌素表达的抑制效果,筛选最有效的shRNA。应用RT-PCR进一步验证其对胃泌素mRNA的抑制效应。应用MTT法检测4条shRNAs在不同浓度下对BGC-823细胞的增殖抑制效应。结果转染后24h、48h及72h,胃泌素表达均被明显地抑制,并呈现浓度及时间依赖的趋势。shRNA3转染后72h,mRNA及蛋白水平表现出最佳的抑制效率,分别为(54.27±0.042)%和(41.69±0.038)%。RT-PCR结果显示,shRNA3对BGC-823细胞胃泌素mRNA的抑制率为48.1%。MTT结果显示,除shRNA4处理组外,其余3组处理细胞均表现出浓度依赖性的增殖抑制趋势。结论4条shRNAs在mRNA及蛋白水平均明显地抑制了胃癌细胞BGC-823胃泌素的表达,shRNA3可能为最有效的胃泌素-shRNA。shRNA对胃泌素表达的抑制,显著降低了BGC-823细胞增殖能力。
Objective To study the inhibitory effect of shRNAs on gastrin expression in gastric cancer cell line BGC-823. Methods Four oligonucleotide sequences targeting different positions of gastrin gene were designed and the corresponding shRNAs were synthesized by in vitro transcription. Four shRNAs were transfected into BGC-823 gastric cancer cells with 10nmol / L, 20nmol / L, 40nmol / L and 80nmol / L respectively. In situ hybridization and immunocytochemistry were used to detect the inhibitory effect of gastrin expression and the most effective shRNA was screened. The inhibitory effect on gastrin mRNA was further verified by RT-PCR. The inhibitory effects of 4 shRNAs on the proliferation of BGC-823 cells were detected by MTT assay. Results The expression of gastrin was significantly inhibited at 24h, 48h and 72h after transfection, which showed a concentration-dependent and time-dependent manner. At 72h after shRNA3 transfection, mRNA and protein levels showed the best inhibitory efficiency (54.27 ± 0.042)% and (41.69 ± 0.038)%, respectively. The result of RT-PCR showed that the inhibitory rate of shRNA3 on gastrin mRNA of BGC-823 cells was 48.1%. The results of MTT showed that except the shRNA4 treatment group, the other three groups of cells showed a concentration-dependent inhibition of proliferation. Conclusion The four shRNAs significantly inhibited the expression of gastrin in BGC-823 gastric cancer cells at both mRNA and protein levels, and shRNA3 may be the most potent gastrin-shRNA. Inhibition of gastrin expression by shRNA significantly reduced the proliferation of BGC-823 cells.