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微小RNA-27a通过Wnt/β-连环蛋白信号通路靶向分泌型卷曲相关蛋白1促进结肠癌细胞的增殖和侵袭

MicroRNA-27a Promotes the Proliferation and Invasiveness of Colon Cancer Cells by Targeting SFRP1 through the Wnt/β-Catenin Signaling Pathway.

作者信息

Ba Sang, Xuan Yi, Long Zi-Wen, Chen Hai-Yong, Zheng Shu-Sen

机构信息

Department of Surgery, Shigatse People's Hospital, Shigatse, China.

Department of Gastric Cancer and Sugery, Fudan University Shanghai Cancer Center, Shanghai, China.

出版信息

Cell Physiol Biochem. 2017;42(5):1920-1933. doi: 10.1159/000479610. Epub 2017 Aug 3.

Abstract

OBJECTIVE

This study aims to explore the effects of microRNA-27a (miR-27a) on the proliferation and invasiveness of colon cancer cells through the Secreted Frizzled-related protein 1 (SFRP1) and the Wnt/β-catenin signaling pathway.

METHODS

Colon cancer tissues and adjacent normal tissues from 125 colon cancer patients, together with the HCEpic, HCT-116, HT-29, SW480 and SW620 cell lines, were prepared for this study. The transfected HCT-116 cells were divided into the miR-27a mimics, miR-27a-NC, anti-miR-27a, blank, Lv-SFRP1, Lv-NC, and miR-27a mimics + Lv-SFRP1 groups. RT-qPCR was performed to detect the expressions of miR-27a and SFRP1 mRNA. A dual-luciferase reporter assay was conducted to examine the effect of miR-27a on SFRP1. Western blotting was used to measure the expressions of the SFRP1, β-catenin, GSK-3β, p-β-catenin, p-GSK-3β, c-Myc and cyclin D1 proteins. MTT, soft agar clone formation and Transwell chamber assays were performed to detect cell proliferation and invasion.

RESULTS

Compared with normal tissues and cells, colon cancer tissues and cells demonstrated significantly higher expression of miR-27a, but lower expressions of SFRP1 mRNA and protein. MiR-27a negatively regulated the expression of SFRP1 mRNA. SFRP1 was also found to be a target gene of miR-27a. In the miR-27a mimic group, the proliferation and invasiveness of colon cancer cells were significantly increased, while the expressions of GSK-3 β and p-β-catenin were remarkably down-regulated; in contrast, the expressions of p-GSK-3β, -catenin, c-Myc and cyclin D1 were up-regulated. While the proliferation and invasiveness of colon cancer cells in the anti-miR-27a and Lv-SFRP1 groups were decreased, the expressions of GSK-3β and p-β-catenin were elevated, and the expressions of p-GSK-3β, β-catenin, c-Myc and cyclin D1 were decreased.

CONCLUSION

These findings indicated that miR-27a could promote the proliferation and invasiveness of colon cancer cells by targeting SFRP1 through the Wnt/β-catenin signaling pathway.

摘要

目的

本研究旨在通过分泌型卷曲相关蛋白1(SFRP1)和Wnt/β-连环蛋白信号通路,探讨微小RNA-27a(miR-27a)对结肠癌细胞增殖和侵袭能力的影响。

方法

本研究采用了125例结肠癌患者的癌组织及癌旁正常组织,以及人结肠上皮细胞系(HCEpic)、人结肠癌细胞系HCT-116、HT-29、SW480和SW620。将转染后的HCT-116细胞分为miR-27a模拟物组、miR-27a阴性对照组、抗miR-27a组、空白组、慢病毒载体SFRP1组、慢病毒载体阴性对照组、miR-27a模拟物+慢病毒载体SFRP1组。采用RT-qPCR检测miR-27a和SFRP1 mRNA的表达。通过双荧光素酶报告基因实验检测miR-27a对SFRP1的作用。采用蛋白质免疫印迹法检测SFRP1、β-连环蛋白、糖原合成酶激酶-3β(GSK-3β)、磷酸化β-连环蛋白(p-β-catenin)、磷酸化GSK-3β(p-GSK-3β)、原癌基因c-Myc和细胞周期蛋白D1的表达。采用MTT法、软琼脂克隆形成实验和Transwell小室实验检测细胞的增殖和侵袭能力。

结果

与正常组织和细胞相比,结肠癌组织和细胞中miR-27a的表达显著升高,而SFRP1 mRNA和蛋白的表达降低。miR-27a对SFRP1 mRNA的表达具有负向调控作用,SFRP1是miR-27a的靶基因。在miR-27a模拟物组中,结肠癌细胞的增殖和侵袭能力显著增强,同时GSK-3β和p-β-catenin的表达显著下调;相反,p-GSK-3β、β-连环蛋白、c-Myc和细胞周期蛋白D1的表达上调。在抗miR-27a组和慢病毒载体SFRP1组中,结肠癌细胞的增殖和侵袭能力降低,GSK-3β和p-β-catenin的表达升高,p-GSK-3β、β-连环蛋白、c-Myc和细胞周期蛋白D1的表达降低。

结论

这些研究结果表明,miR-27a可通过Wnt/β-连环蛋白信号通路靶向SFRP1,从而促进结肠癌细胞的增殖和侵袭。

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