miRNA-486-5p对胃癌细胞SGC7901中NRP2表达的影响  

Effect of miRNA-486-5p on expression of NRP2 in gastric carcinoma SGC7901 cells

作　　者：连海峰[1] 李明[1] 刘成霞[1] 李锟[1] 李丹[2] 

机构地区：[1]滨州医学院附属医院消化内科,山东滨州256603 [2]滨州医学院护理学院内科护理学教研室,山东滨州256603

出　　处：《中国肿瘤生物治疗杂志》2014年第3期298-302,共5页Chinese Journal of Cancer Biotherapy

基　　金：山东省高校科技计划基金资助项目(No.J11LF75);滨州医学院科研启动基金资助项目(No.BY2010KYQD02)~~

摘　　要：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 the target genes of microRNA-486-5p（ miRNA-486-5p） in human gastric cancer SGC7901 cells. Methods： Possible target genes of miRNA-486-5p were predicted by bioinformatics techniques and accordingly miRNA-486-5p over-expressing plasmid（ GV214-miR） against the identified target gene,neuropilin-2（ NRP-2） was constructed. SGC7901 cells were transfected with a control miRNA and an NRP-2-specific miRNA-486-5p. In the transfectants and non-transfected control cells,miRNA-486-5p and NRP-2 mRNA levels and NRP-2 protein levels were analyzed by real-time PCR and Western blotting respectively,and the NRP-2 promoter activity was evaluated by a dual luciferase reporter assay. Results： The expression of miRNA-486-5p in miRNA-486-5p-transfected SGC7901 cells（ SGC7901-miR cells） was significantly up-regulated compared with that in the control group（ 8. 21 ± 1. 18 vs 1. 02 ± 0. 26,P 〈 0. 01）. No significant difference in NRP2 mRNA abundance was observed（ P 〉 0. 05）. However,the NRP2 protein level was significantly reduced in SGC7901-miR cells（ 0. 36 ± 0. 06） as compared with SGC7901 cells transfected with the control plasmid（ 0. 76 ± 0. 05,P 〈 0. 05）. Dual luciferase reporter assay demonstrated that miRNA-486-5p directly targeted the 3 ’-untranslated region（ UTR） of the NRP2 gene,resulting in inhibition of the post-transcriptional translation of NRP2. Conclusion： Sequence-specific miRNA-486-5p may suppress the expression of NRP2 at the protein level in human gastric cancer cells by binding to NRP2 mRNA 3’UTR directly.

关 键 词：miRNA-486-5p 胃癌 生物信息学技术 神经纤毛蛋白2 转染 

分 类 号：R735.2[医药卫生—肿瘤] R730.54[医药卫生—临床医学]