上调微小RNA-613表达对人肾细胞癌细胞生物学行为的影响

Effect of up-regulating the expression of microRNA-613 on biological behavior of human renal cell carcinoma cells

目的观察微小RNA-613(miR-613)对人肾细胞癌细胞生物学行为的影响,并探究其分子机制。方法分别将合成的miR-613模拟物(mimics)和miR-NC通过Lipofectamine 3000转入786-0细胞中,分为观察组和对照组。实时定量聚合酶链反应(qPCR)检测2组细胞中miR-613的表达量验证转染是否成功。噻唑蓝(MTT)法检测786-0细胞增殖,Transwell细胞迁移实验检测786-0细胞迁移。生物信息学预测miR-613的靶基因。双荧光素酶报告实验验证miR-613与靶基因之间的靶向关系及结合位点。qPCR和免疫印迹法(Western blot)检测靶基因的表达水平。结果 qPCR检测结果显示观察组中miR-613的表达量(16. 22±1. 08)显著高于对照组(1. 06±0. 20),差异有统计学意义(P<0. 01)。MTT结果显示观察组细胞在转染后的4、5 d时间点均表现出吸光度值降低(P<0. 05)。Transwell细胞迁移实验结果显示,对照组和观察组细胞从Transwell上室迁移到下室的细胞数分别为(199. 10±22. 74)个和(95. 55±17. 88)个,差异有统计学意义(P <0. 05)。生物信息学预测miR-613的靶基因为皮层肌动蛋白(CTTN)。双荧光素酶报告实验验证miR-613能够靶向调控CTTN基因(P<0.05)。转染后观察组细胞中CTTN mRNA及蛋白的表达量显著低于对照组(P<0.01)。结论上调miR-613的表达可抑制肾细胞癌细胞的增殖和迁移能力,其可能的分子机制是miR-613靶向抑制CTTN基因的表达。

Objective To observe the effect of microRNA-613 （miR-613） on the biological behav- ior of human renal cell carcinoma cells and explore its molecular mechanism. Methods The synthesized miR-613 mimics and miR-NC were respectively transferred into 786-0 cells by Lipofectamine 3000 and di- vided into experimental group and control group. Real time quantitative polymerase chain reaction （qPCR） was used to detect the expression of miR-613 in cells of two groups to verify the transfection success. The proliferation of 786-0 cells was detected by methyl thiazolyl tetrazolium （MTT） assay and the migration of 786-0 cells was detected by Transwell migration assay. Bioinformatics predicts the target gene of miR-613. Dual luciferase reporter assay validates the targeted relationship between miR-613 and target genes, and their binding sites, qPCR and Western blot were used to detect the expression of target genes. Results qPCR test results showed that the expression of miR-613 in the experimental group （ 16. 22 ± 1.08 ） was sig- nificantly higher than that in the control group （1.06±0. 20） ,with statistically significant difference （P 〈 0. 01 ）. The results of MTT showed that the cells in the experimental group showed a decrease in absorbanee value at the d-th point after transfection （ P 〈 0. 05 ）. Transwell cell migration test results showed that the cells in the control group and experimental group migrated from the upper chamber of Transwell to the lower chamber of the cells were respectively （95.55 ± 17.88 ） and （ 199. 10 ± 22. 74）, with statistically signifi- cant difference （P 〈 0. 05 ）. Bioinformatics predicts that the target gene of miR-613 is Cortactin （CTrN）. Dual luciferase reporter assay confirmed that miR-613 can target CTTN gene （P 〈 0. 05 ）. miR-613 can sig- nificantly inhibit the CTTN gene expression in renal cell carcinoma ceils （ P 〈 0. 01 ）. Conclusions Up- regulation of miR-613 expression can inhibit the proliferation and migration of renal cell carcinoma ceils, and its possible molecular mechanism is that miR-613 inhibits the expression of CTFN gene.