Tumor suppressor activity of miR-451: Identification of CARF as a new target

Abstract

microRNAs (miRs) have recently emerged as small non-coding regulators of gene expression. We performed a loss-of-function screening by recruiting retrovirus mediated arbitrary manipulation of genome coupled with escape of cells from 5-Aza-2'-deoxycytidine (5-Aza-dC)-induced senescence. miRNA pool from cells that emerged from 5-Aza-dC-induced senescence was subjected to miR-microarray analysis with respect to the untreated control. We identified miR-451 as one of the upregulated miRs and characterized its functional relevance to drug resistance, cell growth, tumor suppressor proteins p53 and pRb, and stress response. We report that miR-451 caused growth arrest in cells leading to their resistance to 5-Aza-dC-induced senescence. Decrease in cyclin D1, CDK4 and phosphorylated pRB supported the growth arrest in miR-451 transfected cells. We demonstrate that Collaborator of ARF (CARF) protein is a new target of miR-451 that intermediates its function in tumor suppressor and stress signaling.

Figure 1

miR-451 is downregulated in cancer cells. Real-time PCR analysis of miR-451 in human normal and cancer cells showed its higher level of expression in normal cells. Cancer cells showed 2 to 10 fold less expression.

Figure 2

miR-451 overexpression inhibited cancer cell proliferation in vitro and in vivo. Viability of control (untransfected), GFP and miR-451 overexpressing cells showed less number of viable cells in the latter (A). Growth curve analysis revealed that miR-451 overexpression suppressed U2OS cell growth as compared to the control (untransfected) and GFP-expressing cells (B). Long term survival by colony forming assay showed reduction (60% and 40% in U2OS and Saos-2, respectively) of cell colonies in miR-451 overexpressing derivatives (C). Cell cycle analysis revealed increase in number of cells at G0/G1 phase and decrease in number of cells at G2/M and S phases (D). Tumor growth assays in subcutaneous xenograft of control (untransfected) and miR-451 overexpressing cells showed reduced tumor forming capacity of the miR-451 derivatives as compared to the control cells (E). Body weight of the mice during the course of experiment showed no difference (E).

Figure 3

miR-451 overexpression mediated growth arrest involves upregulation of pRB and p21^WAF1 tumor suppressor pathways. miR-451 overexpressing derivatives that showed growth arrest possessed lower level of expression of pRB, phospho-pRB, E2F-5, CDK4 and cyclin D1, and higher level of expression of p21^WAF1 as compared to the control (untransfected) cells and determined by Western blotting (A) and immunostaining (B). mRNA expression analyses revealed, decrease in the level of pRb, cyclin D1, CDK4 and increase in p21^WAF1 in miR-451 overexpressing cells (C). p53 dependent promoter driven-reporter assays in control (untransfected) and miR-335 transfected cells showed upregulation of p21^WAF1 and no change in p53 (D).

Figure 4

miR-451 overexpressing cells showed downregulation of p53 and upregulation of HDM2. miR-451 overexpressing derivatives possessed lower level of expression of p53 protein as well as mRNA levels (A–C). HDM2 was upregulated in miR-451 overexpressing as compared to the control (untransfected) cells at both mRNA and protein levels (D–F).

Figure 5

miR-451 directly targets CARF, but not p53. Western blotting (A) immunostaining (B) and qRT-PCR (C) analysis for CARF showed decreased level of expression in miR-451 overexpressing as compared to the control (untransfected) cells. Increase in p21^WAF1 and HDM2 occurred in p53−/− (Saos-2) cells (D). 3′UTR reporter assay for CARF, p21^WAF1, p53 in control and miR-451 overexpressing cells showed that miR-451 directly targets CARF (E).

Figure 6

miR-335 and miR-451 co-transfection caused stronger knockdown of CARF resulting in apoptosis of cells. miR-4335 and miR-451 double derivatives showed stronger knockdown of CARF resulting in decrease in pro-caspase-3, pro-PARP-1 and Bcl-2 (A) and apoptosis (B).

Figure 7

miR-451 regulates stress response of cells through CARF signaling. Phenotype of human fibroblasts under normal and a variety of stressed conditions (as shown in the table) (A). Western blotting showing decrease in CARF associated with apoptosis of stressed cells and its recovery in cells treated with a herbal extract (B). miR-451 expression was upregulated in stressed as compared to the untreated control cells, and showed downregulation in recovered cells (C).