Effect And Possible Mechanism Of MiR-221 On EMT Of Peritoneal Mesothelial Cells Induced By High Glucose

PrefacePeritoneal dialysis(PD)is one of the effective alternatives for patients with end-stage renal disease(ESRD).Peritoneal fibrosis is the main reason for patients to withdraw from peritoneal dialysis due to various factors such as the exposure of peritoneal and non-physiological high glucose dialysis fluid and the occurrence of inflammatory reaction.Myofibroblasts formed by epithelial-mesenchymal transdifferentiation(EMT)are the primary cells involved in peritoneal fibrosis.EMT is characterized by the loss of epithelial cells marks as well as the accumulation of extracellular matrix.The characteristics mainly include: the loss of epithelial cell adhesion connection such as the decrease of E-cadherin express,reorganization of the cytoskeleton,disintegration of basement membrane,cell migration enhancement and invasion ability enhancement,etc.MicroRNA(miRNA)is a functional,non-coding,small molecule RNA existing in the genome of plants and animals.It has the characteristics of conservative,gene cluster and specific expression.MiRNA plays a very important role in cell life,and it is involved in almost all biological processes.Previous studies have shown that miRNA can be involved in regulating the EMT process of various tissue cells,including the heart,liver,lungs,kidneys,and so on.The latest research shows that miRNA29 c,miRNA193a etc can be mediated high glucose to induce human peritoneal mesothelial cell EMT.MiR-221,which belongs to miR-221/222 gene family,is significantly related to tumor and fibrosis disease.MiR-221 promotes the occurrence of breast cancer and it is significantly increased in thyroid papillary carcinoma and prostate cancer.Studies have shown that its family members may participate in the process of tumor migration and invasion and tissue fibrosis through the regulation of EMT process of tumor cells and other organizations to decrease or reverse the degree of EMT and mitigate fibrosis effectively.It provides new ideas for alleviating EMT and peritoneal fibrosis to explore the influence and mechanism of miR-221 on EMT of peritoneal mesothelial cells induced by glucose.Materials and methods1.Laboratory animals: male rats of clean grade SD,weighing 180-220 g,8 weeks old.2.Human peritoneal mesothelial cells line HMrSV5.3.Intraperitoneal injection of 4.25% standard peritoneal fluid,and establishing a rat model of peritoneal dialysis group with a period of 28 days.Put the rats to death to extract the peritoneal membrane,and extract the histone and mRNA.Detect the expression of E-cadherin、vimentin,PIK3R1 and miR-221 by using Western Blot and Real Time PCR.4.Targetscan software predicts the target gene of miR-221.Luciferase reporter gene identified the target gene of miR-221.5.Use high glucose(2.5%)to stimulate HMrSV5.after 24 h and 48 h,detect the expression of E-cadherin、vimentin,PIK3R1 and mi R-221 by using Western-blot and Realtime PCR.6.Transfect HMrSV5 cells by using MiR-221 inhibitor and set a control group.Detect the expression of E-cadherin、vimentin,PIK3R1 after high-sugar treatment.Use trsnswell to analyze cell migration ability.7.Transfect HMrSV5 cells by using PIK3R1 and set a control group.Detect E-cadherin,Vimentin after high-sugar treatment.Use trsnswell to analyze cell migration ability.The experimental results1.After 28 days of peritoneal dialysis in rats,the expression of E-cadherin in rats was reduced and vimentin expression was increased.The expression of PIK3R1 was decreased and miR-221 expression was increased.2.After 24 h and 48 h since stimulating HMrSV5 by using high glucose(2.5%),the expression of E-cadherin in HMrSV5 cells was decreased and vimentin expression was increased.The PIK3R1 expression was decreased and miR-221 expression was increased.3.Targetscan software was used to analyze the presence of miR-221 binding sites in the PIK3R1 gene 3’UTR region.And further luciferase report gene analysis clearly identified that the PIK3R1 gene 3’UTR zone 522-528 was the binding site of miR-221.4.Transfect HMrSV5 cells by using MiR-221 inhibitor and set a control group.After using high-sugar treatment,compared with that of the control group,the expression of E-cadherin and PIK3R1 in HMrSV5 cells transfected with miR-221 inhibitor was increased and the expression of vimentin was decreased.Cell migration ability was reduced.5.Transfect HMrSV5 cells by using PIK3R1 and set a control group.After using high-sugar treatment,the expression of PIK3R1 and E-cadherin in the HMrSV5 cells transfected with PIK3R1 was increased and the expression of Vimentin was decreased.Cell migration ability was reduced.Conclusion1.During the EMT change in the peritoneum and HMrSV5 cells of rats,the expression of mi R-221 was increased,which was positively correlated with EMT.2.PIK3R1 is one of the main target genes of miR-221.3.During the EMT change in the peritoneum and HMrSV5 cells of rats,the expression of PIK3R1 was decreased,which was negatively correlated with EMT.4.The down-regulation of miR-221 expression can inhibit the EMT of peritoneal mesothelial cells induced by high glucose,which increases the expression of PIK3R1 and decreases the cell migration ability.5.The overexpression of PIK3R1 can inhibit the EMT of peritoneal mesothelial cells induced by high glucose and reduce the cell migration ability.To sum up,miR-221 may participate in peritoneal mesothelial cells EMT by regulating PIK3R1.