The Mechanisms Of MiR-199a/214Cluster Expedites Epithelial-mesenchymal Transition In Human Peritoneal Mesothelial Cells

In the current end stage renal disease is a common and serious disease, it is showedperitoneal dialysis (PD) have lower mortality rates than hemodialysis in lots of survey, Butthis advantage tends to disappear with prolonged dialysis due to peritoneal fibrosis. It isshowed transdifferentiation of mesothelial cells may trigger a large number ofmyofibroblasts lead to fibrosis in present studies. Epithelial-to-mesenchymal transition（EMT）in human peritoneal mesothelial cells (HPMCs) is a key aspect of peritonealfibrosis. EMT in peritoneal mesothelial cells is a complex and dynamic process thatinvolve several factors, It was confirmed that epithelial cell marker molecule expressiongradually reduced or disappeared, while the peritoneal mesothelial cell marker moleculeexpression gradually increases in the process of the EMT. EMT in peritoneal is an earlypathological process of peritoneal fibrosis, and became an early intervention and animportant target. Studies have shown that, disorders of miRNA lead to various diseasessuch as cancer, cardiovascular diseases and fibrosis. miRNA play a role in EMT-relatedfibrosis of organs, including heart, liver, lung and kidney, and are therapeutic targets and diagnostic markers. However, the role of miRNA in peritoneal mesothelial cells EMT andperitoneal fibrosis is unclear. Our study will investigate the mechanisms of miR-199a/214clusters in EMT of HPMCs.【Objectives】1. Expression of miR-199a/214clusters in EMT of HPMCs.2. Up or down-regulated miR-199a/214clusters, expression of markers in EMT ofHPMCs.3. CDH1/CLDN2is the target of miR-199a/214cluster involved in the process of EMT.【Methods】1. HPMCs were cultured48h induced by HG, Fluorescent immunocytochemistry todetect E-cadherin and Claudin2expression. PCR results of miR-199a/214in HPMCsdifferent time induced by HG, while Western blot analysis of epithelial cell markermolecule E-cadherin and Claudin2expression.2. PCR results of miR-199a/214in HPMCs of different duration of dialysis, whileWestern blot analysis of epithelial cell marker molecule E-cadherin and Claudin2expression.3. Rats were infused with4.25%glucose dialysis solution, in situ hybridizationmiR-199a and miR-214expression in peritoneal fibrosis.4. After up-regulated miR-199a/214clusters， HPMCs morphological changes In vitro;Immunofluorescence to detect E-cadherin, Claudin2, ɑ-SMA expression; Western blotanalysis of E-cadherin and Claudin2expression in vitro and ex vivo.5. After down-regulated miR-199a/214clusters， HPMCs morphological changes Invitro; Immunofluorescence to detect E-cadherin, Claudin2, ɑ-SMA expression; Westernblot analysis of E-cadherin and Claudin2expression in vitro and ex vivo.6. Dual-luciferase reporter detect whether the gene CDH1and CLDN2are the target ofmiR-199a/214clusters.【Results】1. The expression of E-cadherin and Claudin2was decreased by immunofluorescencecytochemical staining after HPMCs were cultured48h in HG. HPMCs cultured induced by HG, expression of E-cadherin and Claudin2was decreased by western blotwith PD time extension, while PCR detect miR-199a/214cluster increased gradually.2. The expression of E-cadherin and Claudin2was decreased by western blot with timeextension in HPMCs of different duration of dialysis, while PCR detect miR-199a/214cluster increased gradually.3. In situ hybridization miR-199a and miR-214expression were significantly enhancedin peritoneal fibrosis, while E-cadherin and Claudin2protein levels decreased.4. After up-regulated miR-199a/214clusters，Control group HPMCs are cobblestone-like,mimic group HPMCs are fusiform or fibrous-like. Expression of E-cadherin andClaudin2was decreased and xpression of ɑ-SMA expressionwas increased byimmunofluorescence cytochemical staining. Expression of E-cadherin and Claudin2was decreased in vitro and ex vivo by western blot（P<0.05）.5. After down-regulated miR-199a/214clusters，HG group HPMCs are mixed withcobblestone-like and fusiform, inhibitor group HPMCs are cobblestone-like.Expression of E-cadherin and Claudin2was increased and expression of ɑ-SMA wasdecreased by immunofluorescence cytochemical staining; Expression of E-cadherinand Claudin2was increased in vitro and ex vivo by western blot.6. Dual-luciferase reporter to detect confirmed CDH1and CLDN2are the target ofmiR-199a/214clusters.【Conclusion】MiR-199a/214cluster suppress CDH1and CLDN2post-transcriptional level, reducedE-cadherin and Claudin2protein expression, Promote peritoneal EMT and peritonealfibrosis.