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目的研究微小核糖核酸(microRNA-5p,miR)-17-5p通过靶向调节细胞周期蛋白依赖性激酶抑制因子1A(CDKN1A)对鼻咽癌细胞株CNE2增殖的影响。方法将miR-17-5p模拟物(mimics)转染人鼻咽癌细胞系CNE2,使用定量逆转录-聚合酶链反应(qRT-PCR)和Western blot检测miR-17-5p对CDKN1A的影响;生物信息学预测miR-17-5p是否有CDKN1A的结合位点;荧光素酶实验检测miR-17-5p是否可靶向调节CDKN1A;通过Western blot和克隆形成实验验证miR17-5p可否通过调节CDKN1A影响鼻咽癌细胞株CNE2的增殖。结果 Western blot和qRT-PCR实验显示,在蛋白水平和mRNA水平与对照组相比,过表达miR-17-5p可降低CDKN1A的表达(P<0.01);Target scan靶基因预测软件分析发现,在CDKN1A的3’UTR有两处miR-17-5p的结合位点;荧光素酶检测结果证实miR-17-5p可特异性的作用于这两个位点;克隆形成实验显示,Western blot和qRT-PCR实验显示,在蛋白水平和mRNA水平,过表达miR-17-5p可显著提高CNE2细胞的克隆形成率(P<0.05),同时过表达miR-17-5p和CDKN1A,则抵消了miR-17-5p对细胞增殖的影响(P<0.05)。结论 CDKN1A是miR-17-5p的靶基因,miR-17-5p通过靶向性抑制CDKN1A的表达可促进鼻咽癌细胞的增殖。
Objective To study the effect of microRNA-5p (miR) -17-5p on the proliferation of nasopharyngeal carcinoma cell line CNE2 by targeting the expression of CDKN1A. Methods miR-17-5p mimics were transfected into human nasopharyngeal carcinoma cell line CNE2 and the effect of miR-17-5p on CDKN1A was detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. Bioinformatics predicts whether miR-17-5p has a CDKN1A binding site; luciferase assay detects whether miR-17-5p can target CDKN1A; and whether miR17-5p can influence CDKN1A expression by Western blot and clonogenic assay Proliferation of nasopharyngeal carcinoma cell line CNE2. Results Western blot and qRT-PCR showed that overexpression of miR-17-5p reduced the expression of CDKN1A at the protein and mRNA levels (P <0.01) The 3’UTR of CDKN1A had two miR-17-5p binding sites. Luciferase assay confirmed that miR-17-5p specifically interacted with these two sites. Clone formation assay showed that Western blot and qRT -PCR experiments showed that overexpression of miR-17-5p at the protein level and mRNA level significantly increased the clonogenic rate of CNE2 cells (P <0.05), while overexpression of miR-17-5p and CDKN1A abolished the expression of miR- 17-5p on cell proliferation (P <0.05). Conclusion CDKN1A is the target gene of miR-17-5p, and miR-17-5p can promote the proliferation of nasopharyngeal carcinoma cells through the targeted inhibition of CDKN1A expression.