Mechanism Of LncRNA SOX2OT Inhibiting Mesangial Matrix Secretion In Diabetic Nephropathy

BackgroundDiabetic nephropathy(DN),the major microvascular complication of diabetes,has become the main cause of renal decompensation and renal failure worldwide.With the continuous increase of type 2 diabetes,the incidence of kidney diseases also increases.Due to the global prevalence,type 2 diabetes has led to more and more patients developing DN,and eventually end stage renal disease(ESRD).During the process of DN,many physiological and molecular changes have taken place in the kidney micro-environment,including oxidative stress,activation of certain signaling pathways,inflammation and increased accumulation of cellular matrix.Pathologically,DN mainly manifests as thickening of glomerular and tubular basement membrane,mesangial cell(MC)proliferation and extracellular matrix deposition,mesangial area enlargement,and progressive fibrosis of glomeruli and renal interstitium leading to sclerosis.In DN,the mesangial area is filled with a large amount of matrix components formed by matrix proteins,like Collagen(mainly types Ⅰ,Ⅳ,Ⅴ,Ⅵ),Laminin,and Fibronectin,which mainly derive from glomerular mesangial cells.Excessive deposition of matrix components in DN are caused by mesangial cell dysfunction,the unbalance production and degradation of extracellular matrix,which is a critical pathogenetic underpinnings for glomerular sclerosis.Although various molecules,signaling pathways and intrinsic kidney cells have been found to play a certain role in the pathogenesis of DN,the central link and key molecules affecting the excessive secretion of mesangial cell matrix have not yet been clarified.Long non-coding RNA(LncRNA)are non-protein coding transcripts with a length of more than 200 nucleotides,which play an important regulatory role in a variety of cell responses,development and disease processes.Studies have reported that lncRNA is an important participant in DN.We used db/db mice to construct a DN model(db/m mice as a control)in the early stage.Gene chip screened the differentially expressed lncRNA in the renal cortex and found that in addition to MALAT1,PVT1 and other lncRNA that have been reported in DN,The expression of lncRNA SOX2OT(Sox2 overlapping transcript)in DN is significantly reduced,but its relationship with kidney disease has not yet been reported.ObjectiveIn this study,we cultured human mesangial cell(HMC)in vitro:1.To detect the effects of high glucose-induced oxidative stress on the expression of lncRNA SOX2OT and related matrix proteins Fibronectin,Laminin and CollagenⅣ in HMC.2.To clarify whether oxidative stress regulates the expression of lncRNA SOX2OT through the transcription factor FoxO3a.3.To explore whether lncRNA SOX2OT regulates mesangial cell matrix secretion through Wnt/β-catenin pathway.MethodsHuman glomerular mesangial cell(HMC)were studied1.The cells were divided into four experimental groups:normal control group(glucose concentration:5.6mmol/L),osmotic pressure control group(5.6mmol/L glucose+34.4mmoI/L mannitol),20mmol/L high glucose group and 40mmol/L high glucose group,those were tested after 24h and 48h with different media.The total mRNA was extracted and the mRNA expression of lncRNA SOX2OT was detected by real-time fluorescent quantitative PCR.These experiments screen out the optimal stimulation time and concentration.After selecting the optimal stimulation time and acting on the above group for 24 hours,extract the total cell protein,and clarify the expression changes of matrix proteins Fibronectin,Laminin and Collagen Ⅳ at the protein level by Western blot.The kit detects the changes in the concentration of hydrogen peroxide(H2O2)and superoxide anion(O2·-)in the cell culture medium.2.The cells were divided into four experimental groups:normal control group(glucose concentration:5.6mmol/L),100μmo/L hydrogen peroxide group,200μmo/L hydrogen peroxide group,400μmo/L hydrogen peroxide Group,use different culture media for 30 and 60 minutes to test.The expression changes of lncRNA SOX2OT were detected by qRT-PCR.The above experiment screened out the optimal stimulation time and concentration.After selecting the optimal stimulation time of 60min and grouping as described above,Western blot clarified the expression changes of the matrix proteins Fibronectin,Laminin and Collagen Ⅳ at the protein level.3.By constructing an over-expressed plasmid vector for lncRNA SOX2OT,transfecting the over-expressed plasmid and the empty vector into human glomerular mesangial cells respectively,giving 40mmol/L high glucose stimulation for 24 hours,divided into 4 experimental groups:normal control group,high glucose group,high glucose+empty carrier group,high glucose+SOX2OT over-expression group,after culturing for a period of time,qRT-PCR detects the expression changes of lncRNA SOX2OT.The kit detects the changes in the concentration of hydrogen peroxide(H2O2)and superoxide anion(O2·-)in the cell culture medium.4.The grouping is the same as 1 and 2,after using different media for a period of time,Western blot and qRT-PCR are used to detect the expression changes of FoxO3a at the mRNA and protein levels.5.By constructing an over-expressed plasmid vector for FoxO3a,transfecting the over-expressed plasmid and empty vector into human glomerular mesangial cells respectively,giving 40mmol/L high glucose stimulation for 24 hours,divided into 4 experimental groups:normal control group,high glucose group,high glucose+empty vector group,high glucose+FoxO3a over-expression group.Western blot was used to detect the expression changes of FoxO3a protein level,and qRT-PCR was used to detect the expression changes of lncRNA SOX2OT.6.The grouping is the same as 1.After using different media for 24 hours,Western blot was used to detect the protein level expression changes of the key protein in the Wnt/β-catenin pathway.7.The grouping is the same as 3.After a period of cell culture,Western blot confirmed the expression changes of the key protein in the Wnt/β-catenin pathway and the matrix proteins Fibronectin,Laminin,Collagen Ⅳ at the protein level.Results1.The effects of high glucose-induced oxidative stress on the expression of lncRNA SOX2OT and matrix protein in human mesangial cell.It was shown by qRT-PCR that,compared with the normal control group,the expression of lnRNA SOX2OT in the high glucose/hydrogen peroxide stimulation group was significantly reduced and concentration-dependent(*P<0.05).Western blot showed that compared with the normal control group,the expressions of matrix proteins Fibronectin,Laminin and Collagen Ⅳ in the mesangial cells of the high glucose/hydrogen peroxide stimulation group increased significantly and were concentration-dependent(*P<0.05).The test results of the kit showed that,compared with the normal control group,the concentration of H2O2 and O2·-in the high glucose group increased significantly and was concentration-dependent(*P<0.05).2.Oxidative stress regulates the expression of lncRNA SOX2OT through the transcription factor FoxO3a.After over-expression of lncRNA SOX2OT,qRT-PCR showed that compared with the normal control group,the level of lncRNA SOX2OT in the high glucose group was significantly reduced,and the concentrations of H2O2 and O2·-were significantly increased(*P<0.05).Compared with the high glucose+empty vector group,the level of lncRNA SOX2OT in the high glucose+SOX2OT over-expression group was significantly increased(*P<0.05),and there was no significant change both in the concentration of H2O2 and O2·-($P>0.05).Western blot and qRT-PCR showed that,compared with the normal control group,the expression of FoxO3a in the high glucose/hydrogen peroxide stimulation group was significantly reduced and concentration-dependent(*P<0.05).After over-expression of FoxO3a,Western blot showed that,compared with the high glucose+empty vector group,the FoxO3a protein level in the high glucose+FoxO3a over-expression group was significantly increased(*P<0.05),qRT-PCR showed that compared with the high glucose+empty vector group,the level of lncRNA SOX2OT in the high glucose+FoxO3a over-expression group was significantly increased(*P<0.05).3.LncRNA SOX2OT regulates mesangial cell matrix secretion through Wnt/β-catenin pathway.Western blot showed that,compared with the normal control group,the expression level of the key protein in the Wnt/β-catenin pathway,was significantly higher in the high glucose group(*P<0.05).After over-expression of lncRNA SOX2OT,Western blot showed that compared with the high glucose+empty vector group,the expression level of the key protein and matrix proteins Fibronectin,Laminin and Collagen Ⅳ in the high glucose+SOX2OT over-expression group were significantly decreased(*P<0.05).Conclusion1.Diabetic nephropathy induces oxidative stress in mesangial cells,which causes the transcription factor FoxO3a activity to decrease,and the expression of lncRNA SOX2OT decreases,thereby releasing the inhibitory effect on the Wnt/β-catenin pathway and causing hypersecretion of the matrix.2.Intervening lncRNA SOX2OT can alleviate the excessive secretion of mesangial cell matrix and the progression of renal fibrosis caused by diabetic nephropathy,which is expected to provide new ideas and targets for the early diagnosis and treatment of diabetic nephropathy.