抑制结直肠癌SW480细胞的增殖和迁移。

Inhibits Proliferation and Migration of Colorectal Cancer SW480 Cells.

作者信息

Long Zhi H, Bai Zhi G, Song Jian N, Zheng Zhi, Li Jun, Zhang Jun, Cai Jun, Yao Hong W, Wang Jin, Yang Ying C, Yin Jie, Zhang Zhong T

机构信息

Department of General Surgery, Beijing Friendship Hospital, Capital Medical University, Beijing Key Laboratory of Cancer Invasion and Metastasis Research & National Clinical Research Center for Digestive Diseases, Xi-Cheng District, Beijing, P.R. China.

School of Rehabilitation, Capital Medical University, Department of General Surgery, Beijing Bo'ai Hospital, China Rehabilitation Research Center, Beijing, P.R. China.

出版信息

Anticancer Res. 2017 Aug;37(8):4345-4352. doi: 10.21873/anticanres.11828.

DOI:10.21873/anticanres.11828

PMID:28739727

Abstract

BACKGROUND

This study was designed to determine the molecular function of miR-141 and the underlying mechanisms in colorectal cancer (CRC).

MATERIALS AND METHODS

SW480 cells in which miR-141 was up- or down-regulated were established. Reverse transcription, quantitative polymerase chain reaction and Western blotting were used to examine the microRNA and protein expression. Cell-cycle progression was analyzed by flow cytometry. Proliferation marker Ki-67 was evaluated by immunofluorescence. Transwell assay was conducted to determine the migration rates of cells. Subcutaneous xenograft models were used to examine the effect of miR-141 on tumorigenicity. Human mitogen-activated protein kinase (MAPK) and receptor tyrosine kinase (RTK) pathway phosphorylation array assays were used to interrogate MAPK and RTK pathway activation.

RESULTS

miR-141 directly targeted zinc finger E-box-binding homeobox 1/2 (ZEB1/2). We first determined the expression levels of ZEB1 and ZEB2 in miR-141-expressing cells and miR-141-knockdown cells and found that inhibition of miR-141 significantly increased the expression of ZEB2. In vitro study revealed that miR-141 overexpression inhibited the expression of Ki-67. Furthermore, overexpression of miR-141 led to a significant reduction in the proliferation of SW480 cells via induction of cell-cycle arrest at the G stage. In contrast, inhibition of miR-141 markedly promoted the proliferation of SW480 cells by promoting cell-cycle progression. Moreover, overexpression of miR-141 significantly inhibited SW480 cell migration in vitro. In addition, overexpression of miR-141 significantly reduced tumor size and weight, and inhibited the growth of SW480 cell-derived tumor in nude mice. Notably, overexpression of miR-141 also suppressed the liver metastasis of SW480 cells in nude mice. Using RTK and MAPK arrays, we found increased phosphorylation of hepatocyte growth factor receptor (HGFR/c-MET) following inhibition of miR-141, but phosphorylation of P53, AKT, ERK1/2, P38 and mTOR, etc., in SW480 cells was not affected by miR-141.

CONCLUSION

Our results suggest that miR-141 functions as a tumor suppressor through ZEB2 and HGFR in CRC cells.

摘要

背景

本研究旨在确定miR-141在结直肠癌（CRC）中的分子功能及潜在机制。

材料与方法

构建miR-141上调或下调的SW480细胞。采用逆转录、定量聚合酶链反应和蛋白质印迹法检测微小RNA和蛋白质表达。通过流式细胞术分析细胞周期进程。通过免疫荧光评估增殖标志物Ki-67。进行Transwell实验以确定细胞的迁移率。使用皮下异种移植模型检测miR-141对肿瘤发生的影响。使用人类丝裂原活化蛋白激酶（MAPK）和受体酪氨酸激酶（RTK）通路磷酸化阵列分析来探究MAPK和RTK通路的激活情况。

结果

miR-141直接靶向锌指E盒结合同源框1/2（ZEB1/2）。我们首先测定了miR-141表达细胞和miR-141敲低细胞中ZEB1和ZEB2的表达水平，发现抑制miR-141可显著增加ZEB2的表达。体外研究表明，miR-141过表达抑制Ki-67的表达。此外，miR-141过表达通过诱导细胞周期停滞在G期导致SW480细胞增殖显著减少。相反，抑制miR-141通过促进细胞周期进程显著促进SW480细胞增殖。此外，miR-141过表达在体外显著抑制SW480细胞迁移。另外，miR-141过表达显著减小肿瘤大小和重量，并抑制裸鼠中SW480细胞来源肿瘤的生长。值得注意的是，miR-141过表达还抑制了裸鼠中SW480细胞的肝转移。使用RTK和MAPK阵列，我们发现在抑制miR-141后肝细胞生长因子受体（HGFR/c-MET）的磷酸化增加，但SW480细胞中P53、AKT、ERK1/2、P38和mTOR等的磷酸化不受miR-141影响。