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目的:预测并鉴定miRNA-486-5p在人胃癌细胞SGC7901中的靶基因及其表达。方法:采用生物信息学技术预测miRNA-486-5p的作用靶点,构建miRNA-486-5p过表达质粒(GV214-miR)并转染入SGC7901细胞(SGC7901-miR)中,以空质粒转染SGC7901细胞(SGC7901-miR-NC)为阴性对照,以SGC7901细胞为空白对照。Real-time PCR检测转染细胞中miRNA-486-5p及其靶基因神经纤毛蛋白2(neuropilin-2,NRP2)mRNA的表达,Western blotting检测NRP2的表达,双荧光素酶实验验证miRNA-486-5p对NRP2基因的调控机制。结果:经生物信息学预测,选择与胃癌生物学行为密切相关的NRP2作为miRNA-486-5p的靶基因。与空白组相比,GV214-miR转染后的SGC7901细胞miRNA-486-5p表达显著上调[(8.21±1.18)vs(1.02+0.26),P<0.01],NRP2 mRNA表达无明显变化(P>0.05),而NRP2蛋白表达则明显下调[(0.36±0.06)vs(0.76±0.05),P<0.05],双荧光素酶实验证实miRNA-486-5p可与NRP2 mRNA 3'-UTR直接结合,从而发挥对NRP2转录后翻译的抑制作用。结论:miRNA-486-5p在胃癌细胞SGC7901中可直接作用于NRP2 mRNA 3'UTR,从而抑制其表达。
Objective: To predict and identify miRNA-486-5p target gene and its expression in human gastric cancer cell line SGC7901. Methods: The target of miRNA-486-5p was predicted by bioinformatics technique. The miRNA-486-5p overexpression plasmid (GV214-miR) was constructed and transfected into SGC7901 cells (SGC7901-miR) SGC7901 cells (SGC7901-miR-NC) as a negative control to SGC7901 cells as a blank control. The expression of miRNA-486-5p and its target gene neuropilin-2 (NRP2) mRNA in transfected cells was detected by Real-time PCR. The expression of NRP2 was detected by Western blotting. The miRNA-486- 5p on NRP2 gene regulation mechanism. Results: According to bioinformatics prediction, NRP2, which is closely related to the biological behavior of gastric cancer, was selected as the target gene of miRNA-486-5p. Compared with the blank group, the expression of miRNA-486-5p in SGC7901 cells after GV214-miR transfection was significantly up-regulated [(8.21 ± 1.18) vs (1.02 + 0.26), P <0.01] 0.05), while NRP2 protein expression was significantly down-regulated [(0.36 ± 0.06) vs (0.76 ± 0.05), P <0.05]. Dual luciferase assay confirmed that miRNA-486-5p directly binds to 3'-UTR of NRP2 mRNA, Thereby exerting an inhibitory effect on NRP2 post-translational translation. Conclusion: miRNA-486-5p can directly act on the 3'UTR of NRP2 mRNA in gastric cancer cell line SGC7901 to inhibit its expression.