shRNAs对胃癌细胞系BGC-823胃泌素表达的抑制效应

INHIBITION EFFECTS OF VARIOUS GASTRIN-shRNAs ON GASTRIN EXPRESSION IN GASTRIC CANCER CELL LINE BGC-823

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 inhibition effects of various gastrin-shNAs on gastrin expression in gastric cancer cell line BGC-823. Methods Four nucleotide sequences of shRNA were designed corresponding to various sites of gastrin gene. Four shRNAs were synthesized by in vitro transcription and transfected into gastric cancer cell line BGC-823 at the final concentration of 10nmol/L, 20nmol/L,40nmol/L and 80nmol/L respectively. In situ hybridization and immunohistochemistry techniques were applied to investigate the inhibition of gastrin expression and screen the most effective shRNA. The inhibitory effect on gastrin mRNA of screened shRNA was further identified by RT-PCR. MTT assay was used to determine the inhibitory effect of 4 shRNAs at various final concentrations on the growth of BGC-823 cells. Results The gastrin mRNA and protein exression were suppressed distinctly 24,48, and 72hours after transfection, and exhibited time-and concentration-dependent tendency. The highest suppression efficiency on both mRNA（54.27 ± 0.042） % and protein（41.69 ± 0.038） % level occurred 72 hours later in the cells transfected with shRNAs. The RT-PCR result showed that the inhibitory ratio of shRNA3 on gastrin mRNA of BGC-823 was 48.1%. MTT displayed a proliferative inhibition of the BGC-823 cells after transfection of shRNAs with a concentration-denpendent tendency except the shRNA4 treated cells. Conclusion Four gastrin-shRNAs showed a significant inhibition effect on gastrin expression of gastric cancer cell BGC-823 on mRNA and protein level, shRNAs might be the most effective gastrin-shRNA. Inhibited gastrin expression by shRNAs resulted in a significant decrease of proliferative ability of BGC-823 cells.