MiR-185 targets RhoA and Cdc42 expression and inhibits the proliferation potential of human colorectal cells

Department of Medical Oncology, West China Hospital, Sichuan University, Chengdu, China.
Cancer letters (Impact Factor: 5.62). 02/2011; 301(2):151-60. DOI: 10.1016/j.canlet.2010.11.009
Source: PubMed


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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    • "roliferation of human CRC cells ( Liu et al . , 2011a ) . Its target genes RHOA and CDC42 are well known for their role in regulating G1 / S cell cycle progression and survival . Loss of RHOA or CDC42 expression arrests G1 / S progression in a variety of mammalian cell types , but the mechanisms are likely cell - type dependent and not universal ( Liu et al . , 2011a ) . Li et al . ( 2013c ) reported miR - 330 as another negative regulator of CDC42 in CRC ."
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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 · 4.04 Impact Factor
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    • "In addition, miR-137 was shown to target cdc42 expression, inducing cell cycle g1 arrest and inhibiting invasion in CRC cells [39]. Also miR-185 was shown to target cdc42 and RhoA expression thus inhibiting the proliferation potential of CRC cells [40] while miR-135 affects the Wnt signalling pathway by downregulating the tumour suppressor gene Adenomatous Polyposis Coli (APC) [41]. Another potential tumour-suppressive miRNA in CRC development is miR-143, which might regulate DNA methylation by targeting DNA methyltransferase 3A (DNMT3A) [42]. "
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    ABSTRACT: Recently, involvement of the chemokine/receptor system CCL20/CCR6 in colorectal cancer (CRC) progression was shown. Here, we analyzed the functional interaction of miRNA-518-5p (miR-518a-5p) with CCR6 and its impact on CCR6 expression in CRC cells. MiR-518a-5p was identified by computer software to potentially interact with CCR6. Hence, functional implications of miR-518a-5p with the 3[prime]UTR of CCR6 were analyzed using the Dual Luciferase Reporter assay system. Confirmation of the predicted target site for miR-518a-5p was achieved by site-directed mutagenesis of the seed sequence in the 3[prime]UTR of CCR6 and subsequent application of the mutated seed sequence in a luciferase assay with miR-518a-5p mimics. Accordingly, two CRC cell lines (Caco-2 and HT-29) were transfected with miR-518a-5p miRNA mimics and gene and protein expression of CCR6 was monitored using qRT PCR and immunocytochemistry, respectively. Addition of miR-518a-5p led to significant down-regulation of luciferase activity (P < 0.05), which was significantly reversed in a reporter test system containing the mutated seed sequences in the 3[prime]UTR of CCR6. Following transfection of CRC cell lines with miR-518a-5p mimics and subsequent monitoring of CCR6 expression showed significant down-regulation of CCR6 mRNA and CCR6 protein expression in both CRC cell lines under investigation (P < 0.05). We have shown that miR-518a-5p functionally interacts with CCR6 and that transfection of CRC cells with miR-518a-5p leads to significant CCR6 down-regulation. Consequently, CCR6 expression is regulated by miR-518a-5p in CRC cells indicating that regulation of CCR6 expression by miR-518a-5p might be a regulatory mechanism involved in CRC pathogenesis.
    Journal of Translational Medicine 02/2014; 12(1):48. DOI:10.1186/1479-5876-12-48 · 3.93 Impact Factor
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    • "Further support for the involvement of MIR185 in schizophrenia is provided by findings that two of its validated targets (RhoA, Cdc42; Liu et al., 2011) are associated with altered expression levels in schizophrenia (Hill et al., 2006; Ide and Lewis, 2010). Cdc42 (cell division cycle 42) is a member of the RhoGTPase family (Hill et al., 2006) and promotes dendritic spine formation (Irie and Yamaguchi, 2002; Tada and Sheng, 2006; Wegner et al., 2008) by regulating the polymerization of the actin cytoskeleton into filopodia (Nobes and Hall, 1995). "
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    ABSTRACT: The 22q11.2 deletion is the strongest known genetic risk factor for schizophrenia. Research has implicated microRNA-mediated dysregulation in 22q11.2 deletion syndrome (22q11.2DS) schizophrenia-risk. Primary candidate genes are DGCR8 (DiGeorge syndrome critical region gene 8), which encodes a component of the microprocessor complex essential for microRNA biogenesis, and MIR185, which encodes microRNA 185. Mouse models of 22q11.2DS have demonstrated alterations in brain microRNA biogenesis, and that DGCR8 haploinsufficiency may contribute to these alterations, e.g., via down-regulation of a specific microRNA subset. miR-185 was the top-scoring down-regulated microRNA in both the prefrontal cortex and the hippocampus, brain areas which are the key foci of schizophrenia research. This reduction in miR-185 expression contributed to dendritic and spine development deficits in hippocampal neurons. In addition, miR-185 has two validated targets (RhoA, Cdc42), both of which have been associated with altered expression levels in schizophrenia. These combined data support the involvement of miR-185 and its down-stream pathways in schizophrenia. This review summarizes evidence implicating microRNA-mediated dysregulation in schizophrenia in both 22q11.2DS-related and idiopathic cases.
    Frontiers in Molecular Neuroscience 12/2013; 6:47. DOI:10.3389/fnmol.2013.00047 · 4.08 Impact Factor
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