| BackgroundDiabetic nephropathy(DN)has now surpassed primary glomerular disease and has become the main cause of chronic kidney disease in Urban inpatients of China.The pathogenesis of DN is very complex and involves many factors.Once it progresses to the end stage,it is more difficult to treat than other kidney diseases.At present,DN is considered to be the disease related to podocyte injury.The development of DN is inseparable with the loss of podocytes and the damage of glomerular filtration barrier function.Recent studies have shown that long non-coding RNA(LncRNA)plays an important role in the development of DN,such as response to risk factors such as hyperglycemia,hypertension and hyperlipidemia.lncRNA is a non-protein-encoded transcript longer than 200 nucleotides,and its potency has been recognized in diseases such as glomerular disease,inflammation,fibrosis,renal ischemic injury,kidney transplantation and kidney cancer.The dysregulation of LncRNA involves the early and developmental stages of DN.MicroRNA(miRNA)is also a non-coding RNA,which is closely related to the development of DN.It directs the RNA-induced silencing complex(RISC)to the target gene,thereby inhibiting gene expression.One of the main mechanisms by which LncRNA works is the ceRNA mechanism,which binds to the target site of the miRNA effect complex,causing it to lose its regulatory function,thereby upregulating the expression of the miRNA target gene.The microarray screening showed that the expression of lncRNA DLX6-AS1 in DN patients was higher than that in the normal control group(highly homologous to mouse Dlx6-os1),and the difference was statistically significant.At the same time,miR-346 expression was down-regulated.Bioinformatics suggests that DLX6-AS1may contain a binding site for miR-346.The development of DN may be related to the lncRNA–miRNA competitive ceRNA mechanism.Some studies have shown that miR-346 regulates the expression of GSK-3?and activates the Wnt/?-catenin pathway that regulates the differentiation of human bone marrow mesenchymal stem cells.However,whether miR-346 targets GSK-3?in DN and the regulatory function is unclear.In this study,we will investigate the role of LncRNA Dlx6-os1 in the regulation of GSK-3?expression by binding to miR-346 in type 1 diabetic nephropathy mice,and to play a ceRNA mechanism and participate in podocyte injury.Objective1.To clarify the expression of LncRNA Dlx6-os1 in STZ model mice;2.To investigate the alleviation of podocyte injury by LncRNA Dlx6-os1 shRNA in STZ model mice;3.To investigate the damage of LncRNA Dlx6-os1 overexpressing lentivirus on podocytes in normal mice.Methods6-week-old SPF-class healthy male C57BL/6J mice were adaptively feed in the IVC system for 1 week,freely supplied with food and water,and kept at a constant temperature and humidity of 25?.Switching lights every 12 hours to simulate day and night environment.A low-dose multiple injection of streptozotocin(STZ)was used.The specific method was as follows:the day before the induction,fasting,but privode water,and after 12 hours,the mice were intraperitoneally injected with 55mg/kg STZ-citrate buffer.Once a day for 5 consecutive days.One week after the last STZ injection,mice with non-fasting blood glucose less than 15 mmol/L were eliminated.The normal control group was intraperitoneally injected with the corresponding volume of sodium citrate buffer.All mice were randomly divided into three parts according to the experimental content.The first part was the injury group,(1)CON 8w group:8 weeks after injection of sodium citrate buffer;(2)CON 16w:16weeks after injection of sodium citrate buffer;(3)STZ 8w group:8 weeks after STZ injection;(4)STZ 16w group:16 weeks after STZ injection.The second part is the Dlx6-os1 overexpression group,(1)CON+saline group:CON mice was injected with saline of Dlx6-os1 overexpressing lentivirus,at a dose of 100 ul;(2)CON+con-LV group:CON mice was injected with Dlx6-os1 control overexpressing lentivirus,at a dose of 100 ul,titer of 1×10~8TU/per;(3)CON+Dlx6-os1 4w group:4 weeks after injection of Dlx6-os1 overexpressing lentivirus on CON mice,at a dose of 100 ul,titer of 1×10~8TU/per;(4)CON+Dlx6-os1 12w group:12 weeks after injection of Dlx6-os1 overexpressing lentivirus on CON mice,at a dose of 100 ul,titer of1×10~8TU/per.The third part was Dlx6-os1 knockdown group,(1)STZ+saline group:STZ mice was injected with saline of Dlx6-os1 shRNA lentivirus,at a dose of 100 ul;(2)STZ+con-shRNA group:STZ mice was injected with Dlx6-os1 shRNA unrelated sequence lentivirus,at a dose of 100 ul,titer of 1×10~8TU/per;(3)STZ+Dlx6-os1shRNA 4w group:4 weeks after injection of Dlx6-os1 shRNA lentivirus on STZ mice,at a dose of 100 ul,titer of 1×10~8TU/per;(4)STZ+Dlx6-os1 shRNA 12w group:12weeks after injection of Dlx6-os1 shRNA lentivirus on STZ mice,at a dose of 100 ul,titer of 1×10~8TU/per.Results1.The blood glucose and urine protein in STZ mice increased with time,and were higher than those in CON mice at the same age(p<0.05).After injection of Dlx6-os1 overexpression lentivirus,the urinary protein was increased in normal mice(p<0.05),but there was no significant change in blood glucose(p>0.05).After injection of Dlx6-os1 shRNA lentivirus in STZ model mice,the level of urinary protein was lower than that of STZ mice at the same time(p<0.05),but there was no significant change in blood glucose level(p>0.05).2.FISH showed that with decreased expression of podocyte marker protein Podocin,the expression of Dlx6-os1 was increased in STZ model mice.After injection of Dlx6-os1 overexpression lentivirus,the expression of Podocin was decreased in CON mice,while the expression of Dlx6-os1 was increased.After injection of Dlx6-os1 shRNA lentivirus in STZ model mice,the expression of Podocin was up-regulated in STZ mice and the expression of Dlx6-os1 was down-regulated.3.PAS and electron microscopy showed that the STZ model mice gradually showed pathological changes such as mesangial proliferation,sclerosis,foot process fusion and basement membrane thickening compared with normal mice.Similar pathological changes occurred in Dlx6-os1 overexpressing lentivirus injected normal mice,but to a lesser extent than STZ model mice.Glomerular injury was alleviated after injection Dlx6-os1 shRNA lentivirus in STZ model mice.4.Western blot showed that podocyte injury and inflammation were significantly observed in STZ model mice compared with CON mice,which showed the expression of Podocin was decreased,while the expression of IL-17,Claudin-1,B7-1and CCL-2 was increased.Immunofluorescence also showed that the expression of Podocin and Synapotodin was decreased in STZ model mice,while the expressions of IL-17,Claudin-1 and B7-1 were increased.After injection of Dlx6-os1overexpression lentivirus,podocytes were damaged in CON mice.Western blot showed a decrease in Podocin expression,while IL-17,Claudin-1,B7-1 and CCL-2increased.Immunofluorescence also showed that the expression of Podocin and Synapotodin was decreased in CON mouse,and the expression of IL-17,claudin-1and B7-1 was increased.After injection of Dlx6-os1 shRNA lentivirus in STZ model mice,the podocyte injury was alleviated.Western blot showed the expression of Podocin was increased,while the expression of IL-17,Claudin-1,B7-1 and CCL-2was decreased.Immunofluorescence also showed the expression of Podocin and Synaptodin in STZ model mice was increased,and the expression of IL-17,claudin-1and B7-1 was decreased.5.Western blot showed that the expression of GSK-3?was increased and the expression of pSer9GSK-3?was decreased in STZ model mice compared with CON mice.After injection of Dlx6-os1 overexpression lentivirus,it also showed an increase in GSK-3?expression and a decrease in pSer9GSK-3?expression in CON mice.After injection of Dlx6-os1 shRNA lentivirus,GSK-3?expression was decreased and pSer9GSK-3?expression was increased in STZ model mice.Conclusion1.The expression of LncRNA Dlx6-os1 increased with the damage of glomerular podocytes in the kidney tissue of STZ model mice.Up-regulation of Dlx6-os1 in normal mice can cause glomerular podocyte injury and urine protein production;Down-regulation of Dlx6-os1 in STZ model mice can reduce glomerular podocyte injury and reduce urine proteinuria;Suggesting that LncRNA Dlx6-os1 may be a biomarker and potential therapeutic target for DN.2.LncRNA Dlx6-os1 may cause podocyte injury by regulating the expression of GSK-3?in STZ model mouse kidney tissue. |
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