Cao N J, Hou H H, Li F, Guo S T, Wang Y
Department of Biochemistry and Molecular Biology, Basic Medical College, Shanxi Medical University, Taiyuan 030001, China.
Shanxi Cancer Hospital, Taiyuan 030013, China.
Zhonghua Yi Xue Za Zhi. 2020 Dec 15;100(46):3689-3693. doi: 10.3760/cma.j.cn112137-20200407-01111.
To investigate the effects of miR-191-5p on cell migration, clone formation and proliferation of gastric cancer (GC) cells. The level of miR-191-5p expression was detected by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in 60 paired GC tissues and their adjacent normal tissues. miR-191-5p overexpression was achieved by transfection of construct pcDNA-miR-191-5p into GC cells. The migration, clone formation and proliferation of GC cells were detected by the scratch wound assay, clone formation assay and cell counting kit-8 (CCK-8), respectively. Low expression of miR-191-5p was achieved with miRNA-191-5p inhibitor. The binding sites of cyclin-dependent kinase 6 (CDK6) and miR-191-5p were analyzed using TargetScan software, and the interaction of CDK6 and miR-191-5p was verified using dual-fluorescence reporter gene expression. Western blot (WB) was used to detect the effect of miR-191-5p on the expression of p21 and CDK6 proteins. miR-191-5p decreased in 53 cases (88%) of GC tissues compared to their controls. Furthermore, overexpression of miR-191-5p effectively inhibited the migration, clone formation and proliferation of GC cells (<0.05). Dual-fluorescence reporter confirmed that miR-191-5p bound to 3'UTR of CDK6. WB showed that pcDNA-miR-191-5p inhibited the CDK6 expression but promoted the p21. Down-regulation of miR-191-5p has a correlation with the progression of GC. Overexpression of miR-191-5p can decrease the expression of CDK6 and inhibit the growth of GC cells.
探讨miR-191-5p对胃癌（GC）细胞迁移、克隆形成及增殖的影响。采用实时逆转录聚合酶链反应（RT-PCR）检测60对GC组织及其癌旁正常组织中miR-191-5p的表达水平。通过将构建体pcDNA-miR-191-5p转染至GC细胞实现miR-191-5p过表达。分别采用划痕试验、克隆形成试验和细胞计数试剂盒-8（CCK-8）检测GC细胞的迁移、克隆形成及增殖情况。使用miRNA-191-5p抑制剂实现miR-191-5p的低表达。利用TargetScan软件分析细胞周期蛋白依赖性激酶6（CDK6）与miR-191-5p的结合位点，并通过双荧光报告基因表达验证CDK6与miR-191-5p的相互作用。采用蛋白质免疫印迹法（WB）检测miR-191-5p对p21和CDK6蛋白表达的影响。与对照相比，53例（88%）GC组织中miR-191-5p表达降低。此外，miR-191-5p过表达有效抑制了GC细胞的迁移、克隆形成及增殖（<0.05）。双荧光报告证实miR-191-5p与CDK6的3'UTR结合。WB显示pcDNA-miR-191-5p抑制CDK6表达但促进p21表达。miR-191-5p下调与GC进展相关。miR-191-5p过表达可降低CDK6表达并抑制GC细胞生长。
