miR-663通过靶向TGFB1对肺癌细胞A549增殖的调控

目的利用双荧光蛋白报告基因分析系统,验证miR-663的直接靶基因TGFB1,探讨miR-663促进肺癌细胞A549增殖的可能机制。方法实时定量RT-PCR检测10对肺癌组织和正常肺组织中miR-663的表达水平;利用细胞计数和集落形成实验来验证细胞转染miR-663 ASO后的A549细胞增殖。选取表达绿色荧光蛋白的质粒pcDNA3/EGFP,将TGFB1 3′UTR的一段特异性序列插入该质粒中,并与miR-663及表达红色荧光蛋白质pDsRed2-N1共同转染肺癌细胞系A549,转染后细胞提取的蛋白样品,荧光分光光度计进行定性和定量检测。结果 miR-663在肺癌组织中的表达高于在正常肺组织中的表达;miR-663表达明显促进了细胞A549的增殖;共转miR-663和pcDNA3/EGFP-TGFB13′UTR质粒后,绿色荧光蛋白的表达量明显低于pcDNA3和pcDNA3/EGFP-TGFB13′UTR共转组。结论 miR-663可能通过靶定靶基因TGFB1,促进了肺癌细胞A549的增殖。

Regulation of Lung Cancer Proliferation by miR-663 through Targeting TGFB1

Objective To identify the miR-663 targeted gene TGFB1 using a dual fluorescent protein reporter assay system and to reveal the possible mechanism of miR-663 to promote the proliferation of A549 lung cancer cells.Methods The expression of miR-663 in lung cancer in 10 pairs of lung cancer tissues and in their adjacent normal tissues was measured using microRNA specific qRT-PCR.Than the effects of miR-663 on the proliferation of A549 cells transfected by miR-663 LNA(locked nucleic acid) was analyzed by cell growth curve and colony formation assay.A sequence of TGFB1 3′UTR(untranslated region) was inserted into the plasmid which expressed green fluorescent protein(pcDNA3/EGFP).This plasmid(pcDNA3/EGFP-TGFB1 3′UTR) and miR-663 and the plasmid expressed red fluorescent protein(pDsRed2-N1) were cotransfected into A549 cells.The cells and the extracted protein had been detected under fluorescence microscope and the fluorescence spectrophotometer respectively.Results MiR-663 was highly expressed in lung cancer tissues and A549 cells.Decreased level of miR-663 could significantly inhibit the proliferation of lung cancer cells.After miR-663 and the plasmid of pcDNA3/EGFP-TGFB1 3′UTR being cotransfected,the intensity of green fluorescent protein was significantly lower than that in the group of cotransfected pcDNA3/EGFP-TGFB1 3′UTR with pcDNA3.Conclusion This study demonstrated that miR-663 contributes to A549 cell proliferation through direct regulating of the expression of TGFB1 directly.