Silencing Of LncRNA PVT1 Improves Podocytes Damages And Apoptosis Induced By High Glucose

Background:Diabetic nephropathy(DN)has become one of the most common and serious complications of Diabetic patients.Once continuous proteinuria occurs in diabetic patients and renal function is impaired,the disease will be irreversible and eventually progress to end-stage renal disease,bringing huge social and economic burden.Epidemiological surveys show that the number of DN patients continues to increase worldwide.In 2019,there were 463 million people worldwide living with diabetes,accounting for 9.3%of the global adult population.Therefore,the prevention and treatment of diabetic nephropathy has become a focus of global attention.In order to delay the occurrence and progress of diabetic nephropathy,there is an urgent need to make breakthroughs in the pathogenesis and treatment of diabetic nephropathy.In the past,people mainly focused on the prevention and treatment of traditional factors such as smoking,hyperlipidemia,hypertension and hemodynamic changes.With the development of society,the role of genetic factors in the occurrence and development of diabetic nephropathy has been paid more and more attention.Long non-coding RNA(LncRNA),longer than 200 nucleotides,is non-protein transcripts that have become important modulators of various cellular responses,development,and disease processes.Studies have shown that LncRNA is not only involved in the occurrence of renal inflammation and fibrosis,but also can regulate the renal response to hyperglycemia and the progression of diabetic nephropathy.Long non-coding RNA plasmacytoma variant translocation 1(lncRNA PVT1)is the first non-coding RNA(ncRNA)associated with kidney disease.Early evidence suggests that the LncRNA PVT 1 gene is associated with kidney disease associated with type 1 and type 2 diabetes.Due to the accumulation of extracellular matrix(ECM)protein in renal cells in DN,LncRNA PVT 1 is relatively highly expressed,and its increased expression leads to increased fibrosis.In addition,LncRNA PVT1 was overexpressed in human mesangial cells(MCS)under the stimulation of high glucose.These suggest that LncRNA PVT1 is potentially involved in diabetic microvascular complications.Podocytes are a key component of the renal filtration barrier,and their loss of function is an important early pathological marker of diabetic nephropathy.Even some researchers put forward,diabetic nephropathy may also be a "podocyte disease".In addition,more and more basic experiments and clinical observations have shown that lncRNA plays an important role in podocyte injury and diabetic nephropathy.LncRNA miR210HG and KIAA1737-2 are closely related to podocyte injury induced by hypoxia.LncRNA cyp4b1-ps1-001 is associated with cell proliferation and fibrosis in diabetic nephropathy.Studies have shown that overexpression of lncRNA PVT1 can increase cell proliferation and inhibit apoptosis,but the role of lncRNA PVT1 in podocyte injury remains unclear.Based on the above background,this study aimed to analyze the relationship between LncRNA PVT1 and podocyte injury and apoptosis induced by high glucose,in order to provide a new idea for the research and treatment of diabetic nephropathy.Method:1.Verify the differential expression of lncRNA PVT11.1 To verify the differential expression of lncRNA PVT1 in normal glucose and high glucose concentrationsMPC5 cells were cultured in normal glucose(NG group,5.6 mmol/L)and high glucose(HG group,25 mmol/L),and the expression difference of lncRNAPVT1 in the two groups was detected by RT-qPCR assay;1.2 LncRNA PVT1 was differentially expressed in primary podocytes of normal mice and DN model mice.DN model mice were established as described in Method 2.1.1.Mice conforming to the DN model standard were killed,and the primary podocytes of mice were obtained.The expression difference of lncRNA PVT1 in the two groups was detected by RTqPCR.2.To determine the subcellular localization of lncRNA PVT 1LncRNA PVT1 was predicted to be mainly located in the nucleus by the website lncATLAS.To further verify this result,MPC5 cells in the NG group and the HG group were subjected to RNA fluorescence in situ hybridization(FISH)experiment.The subcellular localization of lncRNA PVT1 was determined and the transfected plasmid was constructed.3.Explore the upregulation of lncRNA PVT1 to cause podocyte injuryMPC5 podocytes were transfected with OE-NC and OE-PVT1 plasmids and cultured in NG.The expressions of podocyte marker proteins such as synaptopodin and podocin in the two groups were observed by immunofluorescence staining.The primary podocytes of mice were obtained by the method described in Method 2.3.2.OE-NC and OE-PVT1 plasmids were transfected into primary podocytes of mice and cultured in NG.The expressions of synaptopodin and podocin in the two groups were observed by immunofluorescence staining.4.To explore the improvement of podocyte injury induced by HG stimulation by downregulating lncRNA PVT1MPC5 podocytes were transfected with sh-NC and sh-PVT1 plasmids and cultured in HG respectively.The expressions of synaptopodin and podocin in the two groups were observed by immunofluorescence staining.The primary podocytes of mice were obtained by the method described in Method2.3.2.And sh-NC and sh-PVT1 plasmids were transfected into primary podocytes of mice and cultured in HG respectively.The expressions of synaptopodin and podocin in the two groups were observed by immunofluorescence staining.5.Explore the up-regulation of lncRNA PVT1 to induce podocyte apoptosisMPC5 podocytes and mouse primary podocytes were transfected with OE-NC and OE-PVT1 plasmids(as per Method 2.3.2)respectively,and cultured in NG.The apoptosis and apoptosis rate of MPC5 podocytes and primary mouse podocytes were observed by flow cytometry after upregulation of lncRNA PVT1;Western blot analysis was performed to observe the expression levels of antiapoptotic related protein Bcl-2 in MPC5 podocytes and primary mouse podocytes after upregulation of lncRNA PVT1,as well as the expression levels of apoptotic related protein Bax and cleaved caspase-3.6.To explore the effect of downregulating lncRNA PVT1 on podocyte apoptosis induced by HGMPC5 podocytes and primary mouse podocytes were transfected with sh-NC and sh-PVT1 plasmids(as per Method 2.3.2),respectively,and cultured in HG.By flow cytometry,apoptosis and apoptosis rate of MPC5 podocytes and primary mouse podocytes after down-regulation of lncRNA PVT1 were observed;Western blot analysis was performed to observe the expression levels of antiapoptotic related protein Bcl-2 in MPC5 podocytes and primary mouse podocytes after down-regulation of lncRNA PVT1,as well as the expression levels of apoptotic related protein Bax and cleaved caspase-3.Result:1.LncRNA PVT1 was highly expressed in MPC5 podocytes stimulated by high glucose.2.LncRNA PVT1 was highly expressed in podocytes of diabetic nephropathy mice.3.LncRNA PVT1 was mainly localized in the nucleus.4.The up-regulation of LncRNA PVT1 resulted in a significant decrease in the expression levels of synaptopodin and podocin in MPC5 podocytes and mouse primary podocytes.The fibrous structure of synaptopodin protein was destroyed,and podocin protein appeared wrinkle and perinuclear expression.5.The down-regulation of LncRNA PVT1 led to the up-regulation of synaptopodin and podocin expression in MPC5 podocytes and mouse primary podocyte marker proteins under the stimulation of high glucose,and the fibrous structure of synaptopodin protein was restored,and podocin protein was evenly distributed in the cytoplasm.6.Upregulation of LncRNA PVT1 resulted in increased apoptosis of MPC5 podocytes and primary mouse podocytes,and the apoptosis rate was significantly increased.7.The down-regulation of LncRNA PVT1 improved the apoptosis of MPC5 podocytes and primary mouse podocytes induced by high glucose(HG),and the apoptosis rate was significantly reduced.8.Upregulation of LncRNA PVT1 resulted in decreased expression of antiapoptosis-related protein Bcl-2 in MPC5 podocytes and mouse primary podocytes,and significantly increased expression of apoptosis-related protein Bax and cleaved caspase-3.9.Down-regulation of LncRNA PVT1 led to increased expression of antiapoptosis-related protein Bcl-2 in MPC5 podocytes and mouse primary podocytes under high glucose stimulation,and significantly decreased expression of apoptosis-related protein Bax and cleaved caspase-3.Conclusion:LncRNA PVT1 was highly expressed in DN,and the up-regulation of LncRNA PVT1 led to podocyte injury and apoptosis,while the down-regulation of LncRNA PVT1 improved the injury and apoptosis of podocytes stimulated by HG.