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目的:观察微小RNA-21(miR-21)对子宫内膜癌细胞PTEN基因的调控作用,并验证PTEN基因3’-UTR区内的微小RNA反应(MRE)-21片段为miR-21作用的靶向位点,为通过miR-21治疗子宫内膜癌提供依据。方法:在人子宫内膜癌HEC-1A细胞中转染不同浓度的miR-21拮抗剂、无关序列和空白对照,利用qRT-PCR法检测miR-21以及靶基因PTEN的mRNA水平,利用免疫印迹法检测PTEN的蛋白表达水平。再利用双荧光素酶报告基因法对miR-21与PTEN3’-UTR区内的MRE-21片段的靶向关系进行验证。结果:在HEC-1A细胞内,随着miR-21拮抗剂浓度的增加,miR-21的表达量逐步减少。miR-21拮抗剂组与无关序列组比较,miR-21表达量的减少差异有统计学意义(P<0.01);PTEN蛋白表达量的增加差异亦有统计学意义(P<0.01)。p MER-报告载体和miR-21拮抗剂共转染HEC-1A细胞时,与p MIR-空白载体转染组以及p MER-报告载体转染组相比,荧光素酶表达水平皆显著上调(P<0.01)。结论:沉默miR-21能上调子宫内膜癌细胞内PTEN基因的表达,PTEN基因3’-UTR区的MRE-21片段为miR-21作用的靶向位点。
AIM: To investigate the regulatory effect of miR-21 on PTEN gene expression in endometrial carcinoma cells and to verify the role of miR-21 in the microRNA response (MRE) -21 in the 3’-UTR region of PTEN gene Targeting site for the treatment of endometrial cancer by miR-21 provide the basis. Methods: Different concentrations of miR-21 antagonist, unrelated sequence and blank control were transfected into human endometrial carcinoma HEC-1A cells. The mRNA levels of miR-21 and target gene PTEN were detected by qRT-PCR. Method to detect the protein expression level of PTEN. The targeting relationship between miR-21 and MRE-21 fragment in PTEN3’-UTR region was verified by dual luciferase reporter assay. Results: In HEC-1A cells, the expression of miR-21 gradually decreased with the increase of miR-21 antagonist concentration. There was significant difference of miR-21 expression in miR-21 antagonist group compared with unrelated sequence group (P <0.01). The difference of PTEN protein expression was also statistically significant (P <0.01). The luciferase expression levels were significantly up-regulated when p-MER-reporter vector and miR-21 antagonist were cotransfected into HEC-1A cells compared to p MIR-empty vector transfection group and p MER-reporter vector transfection group P <0.01). Conclusion: Silencing miR-21 can up-regulate the expression of PTEN gene in endometrial carcinoma cells. The MRE-21 fragment in the 3’-UTR region of PTEN gene is a target site of miR-21.