The Role Of MiRNA-9 In Renal Fibrosis Induced By Hypoxia

There are more than 500 million people with chronic kidney disease.And more than one million people of them died of cardiovascular complication,which was induced by CKD.CKD becomes another disease which seriously threaten people's health,except cardiovascular diseases,cerebrovascular disease,cancer,and diabetes.CKD has become a public health problem that should be paid special attention.Renal fibrosis is a common feature of CKD and a common pathological process to end-stage renal disease.Therefore,it is imperative to investigate the mechanism of renal fibrosis.In recent years,miRNAs become a hot research.Among them,miRNA-9 plays an important role in EMT of cancer and fibrosis of tissue and organ.Previous studies showed that tissue hypoxia can promote EMT of cells,and enhance invasion,metastasis of cancer.In addition,hypoxia is significant in renal EMT and fibrosis.Wheather miRNA-9 has a certain role in renal fibrosis.E-cadherins is a family of cell surface glycoproteins and encoded by the CDH1 gene.It can regulate calcium-dependent intercellular adhesion,and involved in differentiation and morphogenesis of cells.But there is little research in renal fibrosis which induced by hypoxia.We found that CDH1 is one of downstream target genes through bioinformaticsprediction.Wheather E-cadherin participates in renal fibrosis.What's the relationship between miRNA-9 and E-cadherin.Objective1)To analyse the expression of miRNA-9,E-cadherin and fibrosis related molecules in HK-2 cells under low oxygen stimulation.2)To explore the role of miRNA-9 in EMT of HK-2 cells induced by hypoxia.3)To explore the role of E-cadherin in EMT of HK-2 cells induced by hypoxia.4)To study the relationship between miRNA-9 and E-cadherin.5)To test the expression of miRNA-9 and E-cadherin in UUO model and IgA nephropathy.Method1)The expression of miRNA-9,E-cadherin and fibrosis related molecules were tested by q RT-PCR and Western blot in HK-2 cells which were stimulated by low oxygen for different times.2)The expression of miRNA-9,E-cadherin and fibrosis related molecules were measured by q RT-PCR and Western blot in HK-2 cells which were transfected with miRNA-9 mimic or inhibitor after cultured for 48 h under low oxygen stimulation.3)The expression of E-cadherin and fibrosis related molecules expression were evaluated by q RT-PCR and Western blot in HK-2 cells which were transfected with E-cadherin lentivirus or si RNA after cultured for 48 h with low oxygen.4)Seek downstream targets genes of miRNA-9 and verify it.5)The renal tissue of UUO rat model and kidney biopsy samples of IgA nephropathy were collected.The expression of miRNA-9 was tested by q RT-PCR and E-cadherin by q RT-PCR,Western blot and immunohistochemical.Result1)The expression of miRNA-9 and fibrosis related molecules was gradually increased with the incubation time was prolonged under hypoxic conditions,however,the level of E-cadherin was markedly decreased.2)The expression of miRNA-9 and fibrosis related molecules was upregulated in transfected HK-2 cells after treat with miRNA-9 mimic,and correspondingly the expression of E-cadherin was down-regulated.The level of miRNA-9 and fibrosis related molecules was decreased in HK-2 cells after transfected with miRNA-9 inhibitor,but E-cadherin was increased.It illustrated that the miRNA-9 can promote the fibrosis of HK-2 cells which was induced by hypoxia.3)The expression of E-cadherin was significantly upregulated in transfected HK-2 cells after treat with E-cadherin lentivirus,whereas the expression of fibrosis related molecules was down-regulated relative to control cells.The level of fibrosis related molecules was decreased in HK-2 cells after transfected with E-cadherin si RNA,but E-cadherin was increased compared with si RNA negative control cells.It showed that E-cadherin can reverse fibrosis of HK-2 cells induced by low oxygen.In other words,E-cadherin can protect HK-2 cells against fibrosis induced by hypoxia.4)We found that CDH1 was an downstream targets genes in Target scan datebase.And then we verified that miRNA-9 can bound with CDH1 in CDH1 3'UTR by dual-luciferase reporter gene assay.5)The expression of miRNA-9 was obviously increased in renal tissue of UUO rat model and kidney tissue of Ig A nephropathy,however,the level of E-cadherin was markedly decreased.ConclusionIn this study,we aimed to identify the importance of miRNA-9 and the underlying molecular responsible for hypoxia-induced renal fibrosis.Our research indicated that miRNA-9 and E-cadehrin significantly impact on the progression of hypoxia-induced renal fibrosis.And the expression miRNA-9 was increased in the cells with hypoxic stimulation,UUO model and Ig A nephropathy.Furthermore,miRNA-9 negative regulated the expression of CDH1,and then mediate the renal fibrosis which were stimulated by hypoxia.