Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Feb 7, 2015; 21(5): 1488-1497
Published online Feb 7, 2015. doi: 10.3748/wjg.v21.i5.1488
MiR-21/RASA1 axis affects malignancy of colon cancer cells via RAS pathways
Bo Gong, Wan-Wei Liu, Wen-Jing Nie, Dong-Feng Li, Zi-Jun Xie, Chao Liu, Yan-Hui Liu, Ping Mei, Zi-Jun Li
Bo Gong, Wan-Wei Liu, Zi-Jun Li, Department of Gastroenterology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, Guangdong Province, China
Bo Gong, Department of Gastroenterology, the Third People’s Hospital of Huizhou, Huizhou 516002, Guangdong Province, China
Wen-Jing Nie, Graduate School, Southern Medical University, Guangzhou 510515, Guangdong Province, China
Dong-Feng Li, Research Center of Medical Sciences, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, Guangdong Province, China
Zi-Jun Xie, Department of Gastroenterology, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, Guangdong Province, China
Chao Liu, Yan-Hui Liu, Ping Mei, Department of Pathology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, Guangdong Province, China
Author contributions: Gong B and Liu WW contributed equally to this work and are co-first authors; Li ZJ designed the research; Gong B, Liu WW, Nie WJ, Li DF, Xie ZJ, Liu C, Liu YH and Mei P performed the research; Liu WW, Xie ZJ and Liu YH contributed new reagents/analytic tools; Gong B, Liu WW and Li ZJ analyzed the data; Gong B and Li ZJ wrote the paper.
Supported by National Natural Science Foundation of China, No. 81272770; Grants from Guangdong Natural Science Foundation, No. S2013020012746; and Foundation of Guangdong Provincial Department of Science and Technology, No. 2012A030400018.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Zi-Jun Li, MD, PhD, Professor, Department of Gastroenterology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, Guangdong Province, China. zijunli2005@aliyun.com
Telephone: +86-20-83827812 Fax: +86-20-83826085
Received: July 12, 2014
Peer-review started: July 13, 2014
First decision: August 15, 2014
Revised: August 28, 2014
Accepted: September 30, 2014
Article in press: September 30, 2014
Published online: February 7, 2015
Processing time: 211 Days and 19.6 Hours
Abstract

AIM: To determine how the oncogene miR-21 regulates the RAS signaling pathways and affects colon cancer cell behaviors.

METHODS: RAS p21 GTPase activating protein 1 (RASA1) protein expression in six colon cancer cell lines was assessed by Western blot. Colon cancer RKO cells were chosen for transfection because they are KRAS wild type colon cancer cells whose RASA1 expression is significantly decreased. RKO cells were transfected with vectors overexpressing or down-regulating either miR-21 or RASA1. Furthermore, a luciferase reporter assay was used to determine whether RASA1 is a gene target of miR-21. Then, changes in mRNA and protein levels of RASA1, RAS-GTP, and other components of the RAS signaling pathways were assessed in transfected RKO cells by real-time quantitative reverse transcription-polymerase chain reaction, Western blot and immunoprecipitation. Finally, cell proliferation, apoptosis, invasion, and tumor formation ability were assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye assay, flow cytometry, transwell assay, and animal experiment, respectively.

RESULTS: RASA1 protein levels were significantly decreased in RKO cells compared with the other 5 colon cancer cell lines, and RASA1 was confirmed as a target gene of miR-21. Interestingly, RASA1 mRNA and protein levels in pre-miR-21-LV (up-regulation of miR-21) cells were lower than those in anti-miR-21-LV (down-regulation of miR-21) cells (P < 0.05). In addition, pre-miR-21-LV or siRASA1 (down-regulation of RASA1) cells showed higher cell proliferation, reduced apoptosis, increased expression of RAS-GTP, p-AKT, Raf-1, KRAS, and p-ERK1/2, and higher invasion and tumor formation ability, compared with control, anti-miR-21-LV or pcDNA3.1-RASA1 (up-regulation of RASA1) cells (P < 0.05).

CONCLUSION: RASA1 is a target gene of miR-21, which promotes malignant behaviors of RKO cells through regulation of RASA1 expression.

Keywords: Colon cancer; miR-21; RAS; RASA1; RAS signaling pathways

Core tip: In this work, we confirmed RAS p21 GTPase activating protein 1 (RASA1) as an miR-21 target and assessed the role of the miR-21/RASA1 axis in colon cancer cells. Our data suggested that miR-21 activated the RAS signaling pathways by down-regulating RASA1 expression, and it played an important role in promoting cell proliferation, anti-apoptosis and tumor cell formation.