The Role Of SRF–microRNA–143/145–TPM4 Axis In The HPMCs Phenotype Transition Induced By High Glucose

IntroductionThe peritoneum can cause changes of structure and function under long term stimulation of peritoneal dialysate of high glucose and leading to the failure of ultrafiltration. The patients have to drop out from the peritoneal dialysis therapy, and turning to therest of renal replacement therapy. Our team previous research have showed that serum response factor increased in the stimulation of high glucose of HPMCs and can promote the phenotypic transition. However, what is the target molecule in the upstream and downstream and how to regulate the phenotypic transition? To date, it is not clear.micro RNA is a kind of small non-coding RNA, which is bound the target m RN A in the 3?UTR, eventually decreased the expression of target protein。Previously the scientist think micro RNA is no useful,only a ?rubbish? in the process of gene transcription. But,professor Lee RC at Harvard first reported that micro RNA have the role of gene expression regulation in the lever of post-transcription since 1993 years, more and moreresearch reported that micro RNA have an important role in the embryonic development. tissue repair. cell proliferation and apoptosis.According to other literature s,the expression of micro RNA-143/145 is lower in a variety of tumor tissue compared to normal tissue and have a certain relationship in invasion and metastasis. However,micro RNA-143/145 increased of expression in HPMCs under the condition of high glucose culture. Thuswise,it is whether take part in the phenotypic transition of HPMCs? It is unknown up to now.The tropomyosin family is made up of TPM1 、TPM2、 TPM3 and TPM4, is characterized that bound to actin filament,regulating the motility and phenotypic transition of cell. We observed that TPM4 protein expression is obvious lower in HPMCs under the condition of high glucose culture. This hint that TPM4 have a role in the course of phenotypic transition of HPMCs.We found that TPM4 is one of the target gene of micro RNA-143/145 via bioinformation prediction and a negative correlation was found in the expression between the micro RNA-143/145 and TPM4.In that way,it is whether related to phenotypic transition under the condition of high glucose culture?whether they can bound,how to bound?These questions will be solved in this project.Objective1) To investigate the expression and function of microRNA-143/145 and TPM4 in HPMCs under the condition of high glucose culture.2) To explore the relationship between micro RNA-143/145 and TPM4.3) To explore the relationship between SRF and microRNA-143/145.Method1) We make use of D-type glucose stimulation HPMCs and tested the expression of micro RNA-143/145 and TPM4 via western blot and q RT-PCR.2) We use micro RNA-143/145 conduct transient transfection,build overexpression and function inhibition cell line and observe the role of those in the phenotypic transition of HPMCs.3) HPMCs transfection by TPM4 Lentivirus vector,build overexpression and silence cell line,observation the role of those in the phenotypic transition of HPMCs.4) Prediction the target gene of micro RNA-143/145 by combined multiple database and validation by dual-luciferase reporter gene.5) HPMCs transfection by SRF Lentivirus vector, build overexpression and silence cell line,observation the expression of micro RNA-143/145.Result1) The expression of micro RNA-143/145 is increased under the condition of high glucose stimulation and have the function of promote the phenotypic transition of HPMCs Using 50mmol/L D-type glucose stimulation HPMCs, tested the expression of micro RNA-143/145 via q RT-PCR, the result show that micro RNA-143/145 expression is increased with time-dependent. Then,we demonstrated that the micro RNA-143/145 have a role of promote the phenotypic transition of HPMCs via mimic and inhibitor transfection.2) The expression of TPM4 is decreased under the condition of high glucose stimulation and have the function of inhibit the phenotypic transition of HPMCsThe result show that TPM4 expression is obvious reduced in the level of protein under the condition of high glucose stimulation. But its m RN A level does not change. Then,we build the overexpression and silence cell model via TPM4 lentivirus vector transfection respectively,we demonstrated that TPM4 have the function of inhibit the phenotypic transition of HPMCs.3) The expression of micro RNA-143/145 and TPM4 are negative correlation under the condition of high glucose stimulation and proved that they can bind.The above result 1 and result 2,we found that the expression omicro RNA-143/145 and TPM4 in HPMCs are negative correlation under high glucose stimulation via western blot and q RT-PCR.Combined three target gene database, including Targetscan. Micro Cosm and mi Rbase predicted the target ge ne of mi RN A-143/145,found that TPM4 is one of the target gene and validation it can bound with each other in the TPM4 3?UTR via dual-luciferase reporter gene.4) SRF can positive regulation the expression of mi RNA-143/145First, we build the overexpression and silence cell model by mean of Lenovirus vector transfection,then tested the expression of micro RNA-143/145 via q RT-PCR. The result show that SRF can regulate the expression of micro RNA-143/145 and validation that SRF- micro RNA-143/145-TPM4 signal axis exist in HPMCs.ConclusionWe demonstrated that micro RNA-143/145 expression was higher under the condition of high glucose stimulation,regulated by SRF in upstream and inhibit the expression of TPM4 protein in downstream, impacting the phenotypic transition in HPMCs through a series of research in this subject. Therefore, SRF- micro RN A-143/145-TPM4 signal axis is exist in HPMCs under the condition of high glucose stimulation, this will provide a new understand for the pathogenesis of phenotypic transition induced by high glucose and have a new target for therapy of peritoneal fibrosis.