miR-519d靶向CDKN1A/p21调控宫颈癌细胞增殖的初步研究

目的探讨miR-519d对人宫颈癌细胞中CDKN1A/p21基因的靶向调控作用以及对细胞增殖、周期的影响。方法通过荧光定量PCR技术检测miR-519d抑制物对其活性的调控作用。在宫颈癌He La和Si Ha细胞中下调miR-519d表达,利用MTT法检测细胞增殖能力,通过流式细胞术检测细胞周期的分布情况。将miR-519d mimic转染He La和Si Ha细胞,用荧光定量PCR、Western Blot分别检测p21 m RNA和蛋白水平的表达。利用双荧光素酶报告基因系统确认miR-519d与p21的靶向关系。结果 miR-519d抑制物能有效下调宫颈癌He La和Si Ha细胞内miR-519d的表达。MTT实验和细胞周期分析结果提示,下调细胞内miR-519d的表达能够显著降低细胞增殖活力,并使细胞周期阻滞于G1期。进一步通过双荧光素酶报告基因系统鉴定miR-519d能够结合p21 m RNA3′UTR有效抑制其表达。q RT-PCR和Western blot检测结果表明,过表达miR-519d能在m RNA和蛋白水平上抑制p21的表达。结论 p21是miR-519d的直接靶基因,miR-519d可能通过靶向p21调控宫颈癌细胞增殖。

Preliminary study on miR-519d targeting CDKN1A/p21 in the regulation of cell proliferation of cervical cancer cells

Objective To investigate the role of miR-519d in the targeted regulation of CDKN1A/p21 gene in human cervical cancer cells and its effect on cell proliferation and cell cycle.Methods The effect of miR-519d inhibitor on its activity was detected by fluorescence quantitative PCR.The miR-519d expressions in cervical cancer cell HeLa and SiHa were down-regulated.The cell proliferation ability was detected by MTT assay,and cell cycle distribution was detected by flow cytometry.MiR-519d mimic was transfected into HeLa and SiHa cells,and the expression of p21 mRNA and protein was detected by fluorescence quantitative PCR and Western Blot,respectively.A dual luciferase reporter gene system was used to confirm the targeting relationship between miR-519d and p21.Results The miR-519d inhibitor can effectively down-regulate the expression of miR-519d in HeLa and SiHa cells of cervical cancer.The results of MTT assay and cell cycle analysis suggested that the down-regulation of miR-519d expression in cells could significantly reduce cell proliferation and arrest cell cycle at G1 phase.Further the dual luciferase reporter system identified that miR-519d was able to in combination with p21 mRNA 3''UTR and effectively inhibit the expression of p21.The results of qRT-PCR and Western blot showed that overexpression of miR-519d inhibited the expression of p21 at mRNA and protein levels.Conclusion p21 is a direct target gene of miR-519d,and miR-519d may regulate the proliferation of cervical cancer cells by targeting p21.