MiR-21/RASA1 axis affects malignancy of colon cancer cells via RAS pathways.

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.

• Authors: Bo Gong, Wan-Wei Liu, Wen-Jing Nie, Dong-Feng Li, Zi-Jun Xie, Chao Liu, Yan-Hui Liu, Ping Mei, Zi-Jun Li
• Journal: World journal of gastroenterology (World J Gastroenterol) Vol. 21 Issue 5 Pg. 1488-97 (Feb 07 2015) ISSN: 2219-2840 [Electronic] United States
• PMID: 25663768 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• KRAS protein, human
• MIRN21 microRNA, human
• MicroRNAs
• Proto-Oncogene Proteins
• RASA1 protein, human
• RNA, Messenger
• p120 GTPase Activating Protein
• Proto-Oncogene Proteins p21(ras)
• ras Proteins

Topics

• Animals
• Apoptosis
• Caco-2 Cells
• Cell Proliferation
• Colonic Neoplasms (genetics, metabolism, pathology)
• Gene Expression Regulation, Neoplastic
• HCT116 Cells
• HT29 Cells
• Humans
• Male
• Mice, Inbred BALB C
• Mice, Nude
• MicroRNAs (genetics, metabolism)
• Proto-Oncogene Proteins (genetics, metabolism)
• Proto-Oncogene Proteins p21(ras)
• RNA, Messenger (metabolism)
• Signal Transduction
• Time Factors
• Transfection
• Tumor Burden
• p120 GTPase Activating Protein (genetics, metabolism)
• ras Proteins (genetics, metabolism)