The Role Of LncRNA MIAT In High Glucose-Induced Podocyte Injury

Background:Diabetic nephropathy(DN),one of smoldering destructive complications of diabetes mellitus(DM),is becoming the leading cause of end-stage renal disease(ESRD)in developed countries.Proteinuria,which is the most noticeable symptom of DN,has direct relevance to the damage of podocyte,especially podocyte foot process effacement(FPE).However,the concrete mechanism between proteinuria and podocyte injury hasn't been elucidated clearly yet.Myocardial infarction-associated transcript(MIAT),expressed greatly in brain and retinal tissue,is identified as a key gene that responsible for morbidity and mortality of myocardial infarction because of the variant of one single nucleotide polymorphism(SNP)in exon 513.Recently,a research showed the relationship between MIAT and diabetic retinopathy(DR),one of the common complications of DM,that MIAT was up-regulated in STZ-induced DM mice.Moreover,the suppression of MIAT could decrease Rat Retinal Muller cells(rMC-1)apoptosis and increase rMC-1 activity.To date,documentations that lncRNA MIAT is relevant to podocyte injury has,t been accomplished.P38 MAPK is a member of the MAPK family,which involved in the production of inflammatory mediators and regulates cytoskeletal stability.Our preliminary work has showed that p38 MAPK was activated in high glucose environment and participates in the development of DN.Therefore,in this study,the conditional immortalized mouse podocyte line was used as the research object to observe the changes of lncRNA MIAT and p38 MAPK under high glucose-induced conditions,and to explore the role and mechanism of lncRNA MIAT in regulating p38 MAPK in podocyte injury.The theoretical basis and experimental basis for providing therapeutic targets to prevent or delay the progression of DN.Part I To elucidate the effect of lncRNA MIAT on podocyte injury under high glucose-induced conditionsObjective:1.To determine whether the high glucose environment changes the expression and distribution of lncRNA MIAT in podocyte.2.To investigate whether lncRNA MIAT is involved in high glucose-induced podocyte injury.Methods:1.Cell culture:Conditional immortalized mouse podocyte(MPC)line was donated by Professor Peter Mundel.The podocytes proliferated in 33? incubator and coated with type I collagen containing 10%FBS and 10 U/ml y-IFN.When the cells grow to 70-80%,they are transferred to a medium containing no y-IFN at 37? for 10-14 days.After the cells are differentiated and matured,the cells are synchronized and used.2.(1)Real-time PCR was used to detect the expression of lncRNA MIAT in podocytes induced by high glucose(30mmol/L);(2)RNA fluorescence in situ hybridization to detect the distribution of lncRNA MIAT in podocytes induced by high glucose.3.Construction of podocyte-specific lncRNA MIAT shRNA lentiviral vector for further detection(1)Real-time PCR was used to detect the expression of lncRNA MIAT in podocytes;(2)Real-time PCR,western blot were used to detect the expression of synaptopodin,Desmin,FSP-1 and a-SMA;(3)Transwell was used to measure the mobility ability of podocytes.Results:1.After 48h of high glucose culture,lncRNA MIAT was expressed in mouse podocytes,and its transcription RNA was mainly located in the nucleus;high glucose could significantly up-regulate the expression of lncRNA MIAT in podocytes(P<0.05).2.Podocyte-specific lncRNA MIAT shRNA significantly inhibited the expression of lncRNA MIAT(P<0.05);and corrected the expression of protein related to podocyte injury(P<0.05).3.After 48h of high glucose culture,the expression of synaptopodin,one of the podocyte-specific markers,was significantly decreased,while the Desmin,FSP-1 and a-SMA were re-expressed(P<0.05),at the same time,the mobility ability of podocytes increased significantly(P<0.05).Conclusions:High glucose environment leads to increased expression of lncRNA MIAT in podocytes and the specific knockdown of lncRNA MIAT expression can alleviate the degree of podocyte injury induced by high glucose.Part II To verify that lncRNA MIAT mediates podocyte injury by modulating p-p38 MAPK under high glucose-induced conditionsObjective:1.To investigate whether p38 MAPK is associated with podocyte injury.2.To investigate the effect of MIAT on the phosphorylation level of p38 MAPK.Methods:Transfection of lncRNA MIAT shRNA lentiviral vector and p38 MAPK inhibitor(SB203580)were served for further detection:(1)Western blot was used to estimate the phosphorylation level of p38 MAPK in podocytes;(2)Immunofluorescence were used to detect the expression of podocyte-specific marker Desmin,FSP-1,a-SMA and synaptopodin.Results:1.After 48h of high glucose stimulation,the phosphorylation level of p38 MAPK in mouse podocytes increased(P<0.05).2.SB203580,p38 MAPK inhibitor,significantly inhibited the expression of p38 MAPK(P<0.05)and alleviated the changes of podocyte injury-related protein expression levels(P<0.05).3.Transfection of podocyte-specific lncRNA MIAT shRNA lentiviral vector significantly inhibited the phosphorylation of p38 MAPK(P<0.05).Conclusions:1.High glucose environment induced increased phosphorylation of p38 MAPK in podocytes and inhibition of phosphorylation of p38 MAPK contributes to alleviating podocyte injury induced by high glucose conditions.2.There is a positive interaction between lncRNA MIAT and p38 MAPK.