MicroRNA-31 activates the RAS pathway and functions as an oncogenic MicroRNA in human colorectal cancer by repressing RAS p21 GTPase activating protein 1 (RASA1)

Abstract

MicroRNAs (miRNAs) are known to play a vital role in colorectal cancer. We found a widespread disruption in miRNA expression during colorectal tumorigenesis using microarray and quantitative RT-PCR analysis; of the 161 miRNAs altered in colorectal cancer compared with normal adjacent tissue samples, miR-31 was the most significantly dysregulated. We identified candidate targets of miR-31 using bioinformatics approaches and validated RAS p21 GTPase activating protein 1 (RASA1) as a direct target. First, we found an inverse correlation between miR-31 and RASA1 protein levels in vivo. Second, in vitro evidence demonstrated that RASA1 expression was significantly decreased by treatment with pre-miR-31-LV, whereas anti-miR-31-LV treatment increased RASA1 protein levels. Third, a luciferase reporter assay confirmed that miR-31 directly recognizes a specific location within the 3'-untranslated region of RASA1 transcripts. Furthermore, the biological consequences of miR-31 targeting RASA1 were examined by the cell proliferation assay in vitro and by the immunodeficient mouse xenograft tumor model in vivo. Taken together, our results demonstrate for the first time that miR-31 plays a significant role in activating the RAS signaling pathway through the inhibition of RASA1 translation, thereby improving colorectal cancer cell growth and stimulating tumorigenesis.

FIGURE 1.

Cluster analysis of aberrant miRNA expression in colorectal cancer according to a microarray scan and qRT-PCR validation. a, dendrogram generated by cluster analysis showing the separation of CRC from NAT samples based on miRNA profiling. b, the miRNA expression ratios (CRC relative to NAT) from the qRT-PCR data versus the microarray data. p < 0.05 versus controls.

FIGURE 2.

Hybridization of the 3′ UTR of RASA1 and miR-31 and in vivo inverse correlation between the miR-31 expression and RASA1 protein level. a, schematic depicting the conserved binding sites for miR-31. The seed-recognizing site is marked in red; all nucleotides in this region were completely conserved among several species. Hypothesized duplexes formed by the interaction of the binding sites of the 3′ UTR of RASA1 (top) and miR-31 (bottom) are illustrated, and the predicted free energy of each hybrid was indicated. The short solid lines between the two chains represent hydrogen bonds between adenine (A)-thymine (T) pairs or guanine (G)-cytosine (C) pairs, whereas open lines represent G-U pairings. b, relative miR-31 expression in CRC and NAT samples. **, p < 0.05 versus controls. c, relative RASA1 protein expression in paired CRC and NAT samples.

FIGURE 3.

In vitro identification of RASA1 as a target of miR-31. a, relative miR-31 level after infection with pre-miR-31-LV or anti-miR-31-LV in HT-29 cells. One representative experiment of three is shown, with the average values of triplicate wells. b, relative RASA1 protein expression after infection with pre-miR-31-LV or anti-miR-31-LV in HT-29 cells. One representative experiment of three is shown. c, relative RASA1 mRNA levels after infection with pre-miR-31-LV or anti-miR-31-LV in HT-29 cells. One representative experiment of three is shown. d, relative luciferase activity after transfection with pre-miR-31 or anti-miR-31 in Caco-2 cells. One representative experiment of three is shown. **, p < 0.05 versus controls.

FIGURE 4.

The biological effect of miR-31 in the RAS-MAPK pathway in HT-29 cells. a, the growth-promoting function of miR-31. One representative experiment of three is shown. b, PCNA and KI-67 levels assessed by qRT-PCR after overexpression or knockdown of miR-31. One representative experiment of three is shown. c, the relative level of GTP-bound Ras (RAS-GTP) detected by Western blotting after overexpression or knockdown of miR-31. One representative experiment of three is shown. d, phospho-ERK1/2 and total ERK1/2 levels assessed by Western blotting analysis after overexpression or knockdown of miR-31. One representative experiment of three is shown. **, p < 0.05 versus controls.

FIGURE 5.

The biological role of miR-31 in tumorigenesis by targeting the RAS pathway. a, the volume of xenograft tumors in nude mouse derived from subcutaneous implantation of HT-29 cells (n = 7 per group). b, the relative tumor weight at 21 days post-implantation. c, photograph of nude mice bearing xenograft tumors at 21 days post-implantation. d, photograph of the excised tumors at 21 days post-implantation. **, p < 0.05 versus controls.

FIGURE 6.

Schematic of the entire signaling pathway described, including the interaction between miR-31 and RASA1, along with a brief description of the portion of the RAS-MAPK pathway mentioned in the article.