微小RNA-7-5p靶向调节锌指蛋白核转录因子4基因抑制口腔鳞状细胞癌细胞增殖和诱导凋亡的体外研究

Inhibition of proliferation and induction of apoptosis in oral squamous cell carcinoma cells by miR-7-5p targeting KLF4 gene

目的探讨微小RNA(miR)-7-5p对口腔鳞状细胞癌细胞增殖、凋亡及锌指蛋白核转录因子4(KLF4)基因表达调控的影响。方法研究起止时间为2018年3-10月。实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测miR-7-5p在人正常口腔黏膜成纤维细胞hOMF及口腔鳞状细胞癌细胞SCC25、Cal127和Tca8113中的表达量。将miR-7-5p模拟物(miR-7-5p mimics)及阴性对照序列(mimics Control)分别转染至SCC25细胞,分为三组:Control组(未转染的SCC25细胞)、NC组(转染mimics Con-trol的SCC25细胞)、miR-7-5p组(转染miR-7-5p mimics的SCC25细胞)。以qRT-PCR检测miR-7-5p在SCC25细胞中的转染效果;MTT法检测miR-7-5p对SCC25细胞增殖的影响;流式细胞术检测miR-7-5p对SCC25细胞凋亡的影响;通过双荧光素酶报告基因实验、qRT-PCR和蛋白质印迹法(Western blotting)检测miR-7-5p对KLF4的靶向关系。结果miR-7-5p在3种口腔鳞状细胞癌细胞SCC25、Cal127和Tca8113中的表达量[(0.21±0.02)、(0.43±0.04)、(0.48±0.05)]明显低于人正常口腔黏膜成纤维细胞hOMF(1.00±0.09)(P<0.05);转染miR-7-5p mimics能够上调SCC25细胞中miR-7-5p的表达(P<0.05);miR-7-5p过表达能够明显抑制SCC25细胞增殖能力[Control组、NC组72 h吸光度(1.43±0.13)、(1.46±0.15)比miR-7-5p组(0.81±0.09)](P<0.05),并诱导细胞凋亡[Control组、NC组凋亡率(9.48±2.65)%、(10.21±3.02)%比miR-7-5p组(24.41±8.15)%](P<0.05);双荧光素酶报告基因实验结果表明miR-7-5p过表达可抑制KLF4-Wt报告基因载体细胞相对荧光活性;qRT-PCR和Western blotting进一步证实了miR-7-5p能够靶向调节KLF4 mRNA和蛋白表达。结论miR-7-5p直接靶向调控KLF4基因的表达来抑制口腔鳞状细胞癌SCC25细胞增殖,诱导细胞凋亡。

Objective To investigate the effect of microRNA(miR)-7-5p on proliferation,apoptosis and regulation of zinc finger pro-tein nuclear factor 4(KLF4)gene expression in oral squamous cell carcinoma cells.Methods The study started from March 2018 and ended in October 2018.Real-time fluorescence quantitative reverse transcription polymerase chain reaction(qRT-PCR)was used to de-tect the expressions of miR-7-5p in human normal oral mucosal fibroblast hOMF and oral squamous cell carcinoma cells SCC25,Cal127 and Tca8113;miR-7-5p mimics and mimics Control were transfected into SCC25 cells,respectively.The cells were assigned in-to the following three groups:Control group(untransfected SCC25 cells),NC group(SCC25 cells transfected with mimics Control)and miR-7-5p group(SCC25 cells transfected with miR-7-5p mimics).The transcriptional effect of miR-7-5p in SCC25 cells was detected by qRT-PCR.The proliferation of SCC25 cells by miR-7-5p was detected by MTT assay.The effect of miR-7-5p on apoptosis of SCC25 cells was detected by flow cytometry.The targeting relationship of miR-7-5p to KLF4 was detected by double luciferase reporter gene assay,qRT-PCR and Western blotting.Results The expressions of miR-7-5p in three oral squamous cell carcinoma cells SCC25,Cal127 and Tca8113 were significantly lower[(0.21±0.02),(0.43±0.04),(0.48±0.05)]than that in normal oral mucosa fibroblast hOMF(1.00±0.09)(P<0.05).Transfection of miR-7-5p mimics significantly up-regulated the expression of miR-7-5p in SCC25 cells(P<0.05).Overexpression of miR-7-5p significantly inhibited the proliferation[(72 h absorbance:Control group(1.43±0.13),NC group(1.46±0.15)vs.miR-7-5p group(0.81±0.09)]of SCC25 cells and induced apoptosis[apoptosis rate:Control group(9.48±2.65)%,NC group(10.21±3.02)%vs.miR-7-5p group(24.41±8.15)%](P<0.05).Dual luciferase reporter gene assay results showed that miR-7-5p overex-pression significantly inhibited the relative fluorescence activity of KLF4-Wt reporter vector cells.Results of qRT-PCR and Western blotting further confirmed that miR-7-5p can target KLF4 mRNA and protein expression.Conclusion MiR-7-5p directly targets the expression of KLF4 gene to inhibit the proliferation of oral squamous cell carcinoma SCC25 cells and induce apoptosis.