miR-185 targets RhoA and Cdc42 expression and inhibits the proliferation potential of human colorectal cells
ABSTRACT Increasing evidence in the past few years has shown that miRNAs could serve functionally as "oncogenes" or "tumor suppressor genes" and regulate multiple cellular processes relevant to carcinogenesis and cancer progression. Both RhoA and Cdc42, two members of the Rho GTPase family, are found to be upregulated in several types of human tumors including colorectal cancer, and have been implicated in cancer initiation and progression. In the present studies, we found that miR-185 expression greatly inhibited the proliferation potential of Hela cells. An examination of the predicted targets of miR-185 revealed RhoA and Cdc42 among the putative targets that are crucial for cell proliferation. A genomic sequence analysis indicated that nt 1844-1852 of the RhoA 3'UTR and nt 1382-1396 of the cdc42 3'UTR encode for miR-185 target matching sequences and they are highly conserved across different species. Using a luciferase-reporter assay, we show that miR-185 expression significantly suppressed the RhoA and Cdc42 3'UTR activities, and the inhibitory effect was lost when the putative target sites for miR-185 were mutated. Consistent with these results, ectopic expression of miR-185 reduced protein levels of RhoA and Cdc42 in cells, indicating miR-185 functionally regulates RhoA and Cdc42 abundance. Similar to the effects of knocking down RhoA and/or Cdc42 expression, miR-185 effectively inhibited proliferation, induced G1 cell cycle arrest and apoptosis, and blocked invasion of colorectal cancer cells. Thus, miR-185 is a negative regulator of RhoA and Cdc42 and their cellular activities, and could inhibit proliferation and invasion of colorectal cancer cells.
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ABSTRACT: MicroRNAs (miRNA/miR) play an important role in gene regulatory networks through targeting mRNAs. They are involved in diverse biological processes such as cell proliferation, differentiation, angiogenesis, and apoptosis. Due to their pivotal effects on multiple genes and pathways, dysregulated miRNAs have been reported to be associated with different diseases, including colorectal cancer (CRC). Recent evidence indicates that aberrant miRNA expression is tightly linked with the initiation and progression of CRC. To elucidate the influence of miRNA regulation in CRC, it is critical to identify dysregulated miRNAs, their target mRNA genes and their involvement in gene regulatory and signaling networks. Various experimental and computational studies have been conducted to decipher the function of miRNAs involved in CRC. Experimental studies that are used for this purpose can be classified into two categories: direct/individual and indirect/high-throughput gene expression studies. Here we review miRNA target identification studies related to CRC with an emphasis on experimental data based on Luciferase reporter assays. Recent advances in determining the function of miRNAs and the signaling pathways they are involved in have also been summarized. The review helps bioinformaticians and biologists to find extensive information about downstream targets of dysregulated miRNAs, and their pro-/anti-CRC effects. © 2015 Wiley Periodicals, Inc.Genes Chromosomes and Cancer 03/2015; 54(3). DOI:10.1002/gcc.22231
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ABSTRACT: It has become increasingly clear that microRNAs play an important role in many human diseases including cancer. Here, we show that expression of miR-21 in HEK293 and several colorectal cancer cells was found inversely correlated with ras homolog gene family, member B (RhoB) expression. miR-21 expression significantly suppressed RhoB 3' UTR luciferase-reporter activity, but the inhibitory effect was lost when the putative target sites were mutated. Exogenous miR-21 over-expression mimicked the effect of RhoB knockdown in promoting proliferation and invasion and inhibiting apoptosis, whereas anti-miR-21 or RhoB expression yielded opposite effects, in colorectal cancer cells. These results suggest that miR-21 is a regulator of RhoB expression and RhoB could be a useful target in exploring the potential therapeutic benefits of miR-21 mediated tumor cell behaviors in colorectal cancer.FEBS letters 08/2011; 585(19):2998-3005. DOI:10.1016/j.febslet.2011.08.014
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ABSTRACT: MicroRNA (miRNA) is an endogenous non-coding RNA species that either inhibits RNA translation or promotes degradation of target mRNAs. miRNAs often regulate cellular signaling by targeting multiple genes within the pathways. In the present study, using Gene Set Analysis, a useful bioinformatics tool to identify miRNAs with multiple target genes in the same pathways, we identified miR-185 as a key candidate regulator of cardiac hypertrophy. Using a mouse model, we found that miR-185 was significantly down-regulated in myocardial cells during cardiac hypertrophy induced by transverse aortic constriction. To confirm that miR-185 is an anti-hypertrophic miRNA, genetic manipulation studies such as overexpression and knock-down of miR-185 in neonatal rat ventricular myocytes were conducted. The results showed that up-regulation of miR-185 led to anti-hypertrophic effects, while down-regulation led to pro-hypertrophic effects, suggesting that miR-185 has an anti-hypertrophic role in the heart. Our study further identified Camk2d, Ncx1, and Nfatc3 as direct targets of miR-185. The activity of Nuclear Factor of Activated T-cell (NFAT) and calcium/calmodulin-dependent protein kinase II delta (CaMKIIδ) was negatively regulated by miR-185 as assessed by NFAT-luciferase activity and western blotting. The expression of phospho-phospholamban (Thr-17), a marker of CaMKIIδ activity, was also significantly reduced by miR-185. In conclusion, miR-185 effectively blocked cardiac hypertrophy signaling through multiple targets, rendering it a potential drug target for diseases such as heart failure.PLoS ONE 03/2015; 10(3):e0122509. DOI:10.1371/journal.pone.0122509