Published online Feb 7, 2015. doi: 10.3748/wjg.v21.i5.1488
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
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.
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.