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      miR-200 Expression Regulates Epithelial to Mesenchymal Transition in Bladder Cancer Cells and Reverses Resistance to EGFR Therapy

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          Abstract

          Purpose

          The epithelial to mesenchyme transition (EMT) is a cell development-regulated process in which noncoding RNAs act as crucial modulators. Recent studies have implied that EMT may contribute to resistance to epidermal growth factor receptor (EGFR)-directed therapy. The aims of this study were to determine the potential role of microRNAs (miRNAs) in controlling EMT and the role of EMT in inducing the sensitivity of human bladder cancer cells to the inhibitory effects of the anti-EGFR therapy.

          Experimental Design

          miRNA array screening and real-time reverse transcription-polymerase chain reaction were used to identify and validate the differential expression of miRNAs involved in EMT in 9 bladder cancer cell lines. A list of potential miR-200 direct targets was identified through the TargetScan database. The precursor of miR-200b and c were expressed in UMUC3 and T24 cells using a retrovirus or a lentivirus construct, respectively. Protein expression and signaling pathway modulation as well as intracellular distribution of EGFR and ERRFI-1 were validated through western blot analysis and confocal microscopy, whereas ERRFI-1 direct target of miR-200 members was validated by using the wild-type and mutanty 3′UTR/ERRFI-1/Luciferse reporters.

          Results

          We identified a tight association between the expression of miRNAs of the miR-200 family, epithelial phenotype, and sensitivity to EGFR inhibitors-induced growth inhibition in bladder carcinoma cell lines. Stable expression of miR-200 in mesenchymal UMUC3 cells increased E-cadherin levels, decreased expression of ZEB-1, ZEB-2, ERRFI-1, and cell migration, and increased sensitivity to EGFR blocking agents. The changes in EGFR sensitivity by silencing or forced expression of ERRFI-1 or by miR-200 expression have also been validated in additional cell lines, UMUC5 and T24. Finally, luciferase assays using 3′UTR/ERRFI-1/Luc and miR-200 co-transfections demonstrated that the direct down-regulation of ERRFI-1 was miR-200-dependent since mutations in the two putative miR-200-binding sites have rescued the inhibitory effect.

          Conclusions

          Members of the miR-200 family appear to control the EMT process and sensitivity to EGFR therapy, in bladder cancer cells and that expression of miR-200 is sufficient to restore EGFR dependency, at least in some of the mesenchymal bladder cancer cells. The targets of miR-200 include ERRFI-1, which is a novel regulator of EGFR-independent growth.

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          Author and article information

          Journal
          9502500
          8794
          Clin Cancer Res
          Clin. Cancer Res.
          Clinical cancer research : an official journal of the American Association for Cancer Research
          1078-0432
          2 May 2018
          11 August 2009
          15 August 2009
          08 May 2018
          : 15
          : 16
          : 5060-5072
          Affiliations
          The University of Texas, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030
          Article
          PMC5938624 PMC5938624 5938624 nihpa830321
          10.1158/1078-0432.CCR-08-2245
          5938624
          19671845
          618a7a49-bb58-4ba0-aa02-7bb58dcdf74c
          History
          Categories
          Article

          E-cadherin,miRNA-200,EGFR,ERRFI-1,TGF-α
          E-cadherin, miRNA-200, EGFR, ERRFI-1, TGF-α

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