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A functional assay for microRNA target identification and validation.

ABSTRACT: MicroRNAs (miRNA) are a class of small RNA molecules that regulate numerous critical cellular processes and bind to partially complementary sequences resulting in down-regulation of their target genes. Due to the incomplete homology of the miRNA to its target site identification of miRNA target genes is difficult and currently based on computational algorithms predicting large numbers of potential targets for a given miRNA. To enable the identification of biologically relevant miRNA targets, we describe a novel functional assay based on a 3'-UTR-enriched library and a positive/negative selection strategy. As proof of principle we have used mir-130a and its validated target MAFB to test this strategy. Identification of MAFB and five additional targets and their subsequent confirmation as mir-130a targets by western blot analysis and knockdown experiments validates this strategy for the functional identification of miRNA targets.

SUBMITTER: Gaken J

PROVIDER: S-EPMC3378903 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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A functional assay for microRNA target identification and validation.

Gäken Joop J Mohamedali Azim M AM Jiang Jie J Malik Farooq F Stangl Doris D Smith Alexander E AE Chronis Constantinos C Kulasekararaj Austin G AG Thomas N Shaun B NS Farzaneh Farzin F Tavassoli Mahvash M Mufti Ghulam J GJ

Nucleic acids research 20120209 10

MicroRNAs (miRNA) are a class of small RNA molecules that regulate numerous critical cellular processes and bind to partially complementary sequences resulting in down-regulation of their target genes. Due to the incomplete homology of the miRNA to its target site identification of miRNA target genes is difficult and currently based on computational algorithms predicting large numbers of potential targets for a given miRNA. To enable the identification of biologically relevant miRNA targets, we ...[more]

PMID: 22323518

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Project description:African Americans (AA) exhibit higher rates of prostate cancer incidence and mortality compared with European American (EA) men. In addition to socioeconomic influences, biologic factors are believed to play a critical role in prostate cancer disparities. We investigated whether population-specific and -enriched miRNA-mRNA interactions might contribute to prostate cancer disparities.Integrative genomics was used, combining miRNA and mRNA profiling, miRNA target prediction, pathway analysis, and functional validation, to map miRNA-mRNA interactions associated with prostate cancer disparities.We identified 22 AA-specific and 18 EA-specific miRNAs in prostate cancer versus patient-matched normal prostate, and 10 "AA-enriched/-depleted" miRNAs in AA prostate cancer versus EA prostate cancer comparisons. Many of these population-specific/-enriched miRNAs could be paired with target mRNAs that exhibited an inverse pattern of differential expression. Pathway analysis revealed EGFR (or ERBB) signaling as a critical pathway significantly regulated by AA-specific/-enriched mRNAs and miRNA-mRNA pairings. Novel miRNA-mRNA pairings were validated by qRT-PCR, Western blot, and/or IHC analyses in prostate cancer specimens. Loss/gain of function assays performed in population-specific prostate cancer cell lines confirmed miR-133a/MCL1, miR-513c/STAT1, miR-96/FOXO3A, miR-145/ITPR2, and miR-34a/PPP2R2A as critical miRNA-mRNA pairings driving oncogenesis. Manipulating the balance of these pairings resulted in decreased proliferation and invasion, and enhanced sensitization to docetaxel-induced cytotoxicity in AA prostate cancer cells.Our data suggest that AA-specific/-enriched miRNA-mRNA pairings may play a critical role in the activation of oncogenic pathways in AA prostate cancer. Our findings also suggest that miR-133a/MCL1, miR-513c/STAT1, and miR-96/FOXO3A may have clinical significance in the development of novel strategies for treating aggressive prostate cancer.

Project description:BackgroundThough the roles of microRNAs (miRNAs) in renal diseases have been extensively investigated, a thorough screening and comparison of miRNAs among different types of chronic kidney disease (CKD) has never been performed.MethodsThe intrarenal miRNAs were profiled from fresh kidney tissues of patients with biopsy-proven minimal change disease (MCD), focal segmental glomerular sclerosis (FSGS) and diabetic nephropathy (DN) by using microarray. Commonly dysregulated miRNAs were validated by real-time PCR using paraffin-embedded renal tissues from all three types of CKD patients as well as mouse unilateral ureteral obstruction (UUO) model. Two novel miRNAs were selected and annotations of their target genes were performed using GO and KEGG pathway enrichment analysis. Biological functions of three two candidate miRNAs were explored in TGF-?1-induced cell model using human kidney proximal tubular cells (HK-2).ResultsThe kidney biopsy samples of three disease types represent different levels of damage and fibrosis, which were the mildest in MCD, moderate in FSGS, and the most severe in DN. 116 miRNAs were identified to be commonly dysregulated, including 40 up-regulated and 76 down-regulated in CKD tissues as compared with healthy donor kidney biopsy tissues. Two novel miRNAs, hsa-miR-3607-3p and hsa-miR-4709-3p, were verified as consistently differentially expressed among all three types of patient samples as well as in mouse model. In vitro, hsa-miR-3607-3p was repressed while hsa-miR-4709-3p was induced by TGF-?1 treatment. Inhibition of hsa-miR-3607-3p or overexpression of hsa-miR-4709-3p promoted TGF-?1-induced migration and F-actin assembling in HK-2 cells, which are characteristics of epithelial-mesenchymal transition (EMT). Further study identified that ITGB8 and CALM3 were the bona fide target genes of hsa-miR-3607-3p and hsa-miR-4709-3p respectively.ConclusionsThe present identify a unique miRNAs profile that probably relates to the common fibrosis process of CKD. Results of our study suggest that hsa-miR-3607-3p and hsa-miR-4709-3p may represent as promising therapeutic targets against kidney fibrosis.

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A functional assay for microRNA target identification and validation.

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A functional assay for microRNA target identification and validation.

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