微小RNA-27a在子宫颈癌中的表达变化及作用机制

doi:10.16098/j.issn.0529-1356.2021.01.012

摘要/Abstract

摘要：

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、HeLa、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、HeLa、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。

关键词: 微小RNA-27a, 子宫颈癌, F框/WD-40域蛋白7, 实时定量聚合酶链反应, 流式细胞术, 免疫印迹法, 人

Abstract:

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, HeLa, 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, HeLa 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.

Key words: Micro RNA-27a, Cervical cancer, F-box and WD repeat domain containing protein 7, Real-time PCR, Flow cytometry, Western blotting, Human

中图分类号:

R737.33

仉红平李峰牛占杰. 微小RNA-27a在子宫颈癌中的表达变化及作用机制[J]. 解剖学报, 2021, 52(1): 78-83.

ZHANG Hong-ping LI Feng NIU Zhan-jie. Expression and mechanism of microRNA-27a in cervical cancer[J]. Acta Anatomica Sinica, 2021, 52(1): 78-83.

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