微小RNA-27a在子宫颈癌中的表达变化及作用机制

Expression and mechanism of microRNA-27a in cervical cancer

目的探讨微小RNA(miR)-27a靶向调控F框/WD-40域蛋白7(FBXW7)对子宫颈癌细胞的增殖、凋亡及侵袭的影响。方法30例宫颈癌组织和癌旁组织、30例正常宫颈组织新鲜标本用于实验。Real-time PCR检测宫颈癌、癌旁组织、正常宫颈组织以及宫颈癌细胞(SiHa、Caski、He La、HCC94)、子宫颈鳞状上皮永生化细胞H8中miR-27a的表达。应用脂质体转染法将miR-27a抑制剂(inhibitor)及其阴性对照转染至SiHa细胞,CCK-8法、流式细胞术、Transwell法分别检测miR-27a对SiHa细胞增殖活性、细胞周期、凋亡率和侵袭能力的影响。生物学信息法预测miR-27a的靶向基因,双荧光素酶报告基因实验结合Western bloting验证miR-27a对FBXW7的靶向调控作用。结果与癌旁组织和正常宫颈组织相比,宫颈癌组织中miR-27a高表达(P<0.05);与子宫颈鳞状上皮永生化细胞H8相比,宫颈癌细胞SiHa、Caski、He La、HCC94中miR-27a高表达(P<0.05)。抑制SiHa细胞中miR-27a的表达,能够明显降低细胞的增殖活性(P<0.05),提高G0/G1期细胞比例(P<0.05),降低S期和G2/M期细胞比例(P<0.05),提高细胞凋亡率(P<0.05),抑制细胞的侵袭能力(P<0.05)。生物学信息法预测FBXW7可能是miR-27a的靶向调控基因;双荧光素酶报告基因实验显示,miR-27a可以与FBXW7基因的3’UTR区特异性结合(P<0.05),并负调控FBXW7蛋白的表达(P<0.05)。结论MiR-27a在宫颈癌的发生发展中起着癌基因的作用,抑制miR-27a表达能够明显抑制宫颈癌细胞的恶性生物学行为,其机制可能与靶向调控FBXW7的表达有关。

Objective To investigate the effects of microRNA(miR)-27a on proliferation,apoptosis and invasion of cervical cancer cells by targeting F-box and WD repeat domain containing protein 7(FBXW7)expression.Methods Thirty cases of cervical cancer and paracancerous tissues and 30 cases of normal tissues were used in the experiment.The expression of miR-27a in cervical cancer,paracancerous tissue,normal cervical tissue,cervical cancer cells(SiHa,Caski,He La,HCC94)and cervical squamous epithelial immortalized cell H8 were detected by Real-time PCR.MiR-27a inhibitor and its negative control were transfected into SiHa cells by liposome transfection.CCK-8 assay,flow cytometry and Transwell assay were used to detect the effects of miR-27a on the proliferation,cell cycle,apoptotic rate and invasive ability of SiHa cells.Bioinformatics was used to predict the targeting gene of miR-27a.Double luciferase reporter gene assay combined with Western blotting was used to verify the targeting regulation of miR-27a on FBXW7.Results Compared with the normal cervical tissues and the adjacent tissues,the expression of miR-27a was higher in cervical cancer tissues(P<0.05);Compared with the cervical squamous epithelial immortalized cells H8,the expression of miR-27a in cervical cancer cells SiHa,Caski,He La and HCC94 was higher(P<0.05).Inhibiting the expression of miR-27a in SiHa cells could significantly reduce the proliferation activity of cells(P<0.05),increase the proportion of G0/G1 cells(P<0.05),decrease the proportion of G2/M cells(P<0.05),increase the apoptosis rate(P<0.05),and inhibit the invasive ability of cells(P<0.05).Bioinformatics predicted that FBXW7 might be a target regulatory gene of miR-27a.Double luciferase reporter gene assay showed that miR-27a could specifically bind to the 3’UTR region of FBXW7(P<0.05),and negatively regulate the expression of FBXW7 protein(P<0.05).Conclusion MiR-27a plays an oncogene role in the occurrence and development of cervical cancer.Inhibiting the expression of miR-27a can significantly inhibit the malignant biological behavior of cervical cancer cells,and its mechanism may be related to targeted regulation of FBXW7 expression.