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| Silence of PLCε Induced by miR-145 Inhibits EMT and Metastasis in Bladder Cancer |
| ZHAO Yan1, HAO Yan-ni1, LIU Nan-jing1, LI Ting1, WU Xiao-hou2, LUO Chun-li1 |
1. Key Laboratory of Clinical Diagnostics Founded by Ministry of Education, College of Laboratory, Chongqing Medical University, Chongqing 400016, China;
2. Department of Urinary Surgery, The First Affiliated Hospital of Chongqing, Medical University, Chongqing 400016, China |
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Abstract Objective:To study the effect of PLCε regulated by miR-145 on EMT and metastasis in bladder cancer cell T24 and and the potential mechanism. Methods:①Using adenovirus infecting T24 cells,the migratory abilities of T24 cells were observed by wound healing and transwell chamber cell migration assay.The expression of EMT related molecules such as E-cadherin, n-cadherin、Vimentin were detected by RT-PCR and Western blot,at genetic and protein level respectively. In order to explore the molecule mechanism,western blot was used to test the protein level of p-GSK-3β and Snail. ② Using bioinformatic to calculate the possible microRNA regulating PLCε,referenced to the reported microRNA array, miR-145 was chosen for the target microRNA.Using miR-145 mimics transfecting T24,the expression of miR-145 and PLC ε were detected by quantitative Real-time-PCR, meantime, the PLCε protein expression was tested by Western blot. ③ After cells transfected with miR-145 mimics,the protein expression of EMT related molecule as well as p-GSK-3β and Snail were detected by Western blot. Wound healing and transwell chamber cell migration assay were adopted to detect he migratory abilities of T24. Results:① Wound healing and transwell chamber cell migration assay showed that the migratory ability was remarkably decreased in the Ad-shPLCε group,compared with the blank contol group and Ad-HK group(P<0.05).The expressions level of both mRNA and protein of N-cadherin, Vimentin were higher,while E-cadherin was lower in Ad-shPLCε group than that in blank contol group and Ad-HK group(P<0.05). Western blot result showed that the protein level of p-GSK-3β and Snail were significantly lower after treated with Ad-shPLCε(P<0.05). ② qPCR result showed that, transfection miR-145 mimics could restore miR-145 expression of bladder cancer cells T24,significantly higher than transfection negative control(P<0.01),by contrst,the PLCε mRNA expression was lower(P<0.05). Western blot result showed that the PLCε protein expression was markedly down-regulated (P<0.05),which was consistent with qPCR result. ③ After transfection miR-145 mimics, the migratory ability of bladder cancer T24 significantly lower than negative control(P<0.05). Western blot result indicated that,on the one hand, the expression of N-cadherin, Vimentin was lower than negative control, while E-cadherin was higher(P<0.05),on the other hand,overexpression of miR-145 could significantly inhibit the expression of p-GSK-3β and Snail(P<0.05).Conclusion:PLCε induce EMT and metastasis via GSK-3β/Snail pathway,overexpression of miR-145 can downregulate the role of PLCε on bladder cancer.
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Received: 27 September 2016
Published: 25 March 2017
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