miR-519d靶向CDKN1A/p21调控宫颈癌细胞增殖的初步研究  

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

作　　者：刘洁[1] 曾烨[2] 周珏宇 陈志超 张汉荣 赖姝彧 吴小丽[1] 谭令梅[1] 王雪飞[1] LIU Jie;ZENG Ye;ZHOU Jueyu;CHEN Zhichao;ZHANG Hanrong;LAI Shuyu;WU Xiaoli;TAN Lingmei;WANG Xuefei(Department of Obstetrics and Gynecology,Nanfang Hospital,Southern Medical University,Guangzhou 510515,China;Department of Stomatology,Nanfang Hospital,Southern Medical University,Guangzhou 510515,China;Department of Biochemistry and Molecular Biology,School of Basic Medical Sciences,Southern Medical University,Guangzhou 510515,China)

机构地区：[1]南方医科大学南方医院妇产科,广东广州510515 [2]南方医科大学南方医院口腔科,广东广州510515 [3]南方医科大学基础医学院生物化学与分子生物学教研室,广东广州510515

出　　处：《中国现代医生》2018年第21期25-29,共5页China Modern Doctor

基　　金：高等学校博士学科点专项科研基金(20124433120001);广东省高等学校优秀青年教师培养计划(2014);广东省自然科学基金(2014A030313293)

摘　　要：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。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 abilitywas detected by MTT assay, and cell cycle distribution was de- tected by flow eytometry. 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 in- hibitor can effectively down-regulate the expression of miR-519d in HeLa and SiHa ceils of cervical cancer. The re- sults of MrlT 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 iden- tified 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 prolif- eration of cervical cancer ceils by targeting p21.

关 键 词：微小RZqA 宫颈癌 P21 细胞周期 

分 类 号：R737.33[医药卫生—肿瘤]