LncRNA GAS5 Exacerbate Renal Tubulointerstitial Fibrosis By Sponged MiR-96-5p In Diabetic Kidney Disease

Background:Diabetic kidney disease(DKD)is not only a major complication of diabetes,but also the leading cause of chronic kidney disease(CKD)especially end-stage renal disease(ESRD)worldwide.Renal fibrosis is the final pathological mechanism of chronic kidney disease,including DKD,shown as:glomerular sclerosis,renal tubular atrophy and renal interstitial fibrosis.Although glomerular lesions are unarguably of great importance and significance in DKD,diabetic tubular injury is also an essential component of DKD pathology and actually the major determinant of renal prognosis in diabetes.Targeted attempts to identify and treat early tubular injury may prevent or delay diabetic nephropathy.Mammalian genome and transcriptome research data show that protein-coding genes account for only a very small proportion(1%-2%),and a large proportion of the genome generates message which is not coded into protein,This is called non-coding RNAs(IncRNAs),they are a kind of long chain noncoding RNA transcriptions longer than 200 nucleotides,but in the form of RNA to participate in a variety of important biological processes.The growth arrest-specific 5(GAS5),plays important roles in several biological processes,including cell growth arrest,cell proliferation,apoptosis and autophagy.As GAS5 level was differential expressed in plasm of db/db mice and diabetes patients.GAS5 was found to play a role in TGF?-SMAD signaling.TGF-?1 signaling is important in the development and progression of DKD.GAS5 plays a suppressive role in cardiac fibrosis and liver fibrosis.The role of GAS5 on renal fibrosis and DKD still need to be elucidated.In this study,we aim to find the influence of renal fibrosis on GAS5 expression,the role of GAS 5 in TGF?1 treated HK-2 cells,and provide mechanistic insights into the epigenetic regulation of expression.We hope to find a new method for treatment of diabetic nephropathy through adjusting GAS5 expressionMethods:1.HDF/STZ induced C57BL/J mice was used as DKD animal model,homologous normal mice were defined as control group.HE and Masson's trichrome stainning was used to observe morphological changes of Kidney.Immunohistochemistry was used to observe expression of TGF?1,COL1,FN1,CTGF protein in renal tissues.RT-qPCR and in situs hybridization(ISH)were used to test the mRNA expression of Gas5.2.To study the renal fibrogenic effect in tubular epithelial,human renal proximal tubular(HK-2)cells were treated by TGF-?1(1 ng/mL)in vitro for 48 hours.RT-qPCR and western Blot analysis were used to test the mRNA and protein expression of COL 1,FN1 and CTGF.3.To examine the role of GAS5 in renal fibrosis,cells were transfected with siRNA against human GAS5 or their negative controls.The cells were then stimulated with TGF-?1(1 ng/mL)for 48 hours.RT-qPCR and Western Blot analysis were used to test the mRNA and protein expression of COL1,FN1,CTGF.4.The localization of GAS5 and miR-96-5p in the HK-2 cells was detected by cytoplasm/nucleus fraction isolation.Bioinformatic analysis was used to select the candidate gene for potential target regulation of GAS5.miR-96-5p was determined through RT-qPCR analysis of HK-2 cells treated with siRNA against human GAS5.RIP and Dual luciferase reporter assays were used to confirm the interaction of GASS with miR-96.The GAS5 expression was analyzed after transfected with miR-96-5p mimic/inhibitor or their negative controls5.To examine the role of miR-96-5p in renal fibrosis,cells were transfected with miR-96-5p mimic/inhibitor or their negative controls.RT-qPCR and Western Blot analysis were used to test the mRNA and protein expression of COL1,FN1,CTGF.Bioinformatic analysis was used to identify miR-96-5P could target FN13'UTR.Dual luciferase reporter assays were used to confirm the interaction of them.Results:1.HE staining and Masson staining demonstrated that the kidney of 12-weeks-age HDF/STZ induced C57BL/J mice revealed the pathological feature of DKD.Immunohistochemistry analysis of renal tissues revealed that expression of TGF-?1,COL1,FN1 and CTGF was significantly elevated in the DM group.GAS5 levels was dramatically increased too by RT-qPCR and ISH.2.It was shown that HK-2cells induced by TGF-?1 significantly increased levels of COL1,FN1,CTGF by RT-qPCR and western blot analysis in HK-2 cells.GAS5 mRNA levels was dramatically increased too after induced by TGF-?1.3.Compared with their negative controls,cells transfected with siRNA against human GAS5 significantly decreased levels of COL1,FN1,CTGF by RT-qPCR and western blot analysis in HK-2 cells induced by TGF-?1.4.By cell cytoplasm/nucleus fraction isolation analysis,we found that both lncRNA GAS5 and miR-96-5p were localized mainly in the cytoplasm of HK-2 cells.the results revealed five miRNAs with the highest scores for binding to IncRNA GAS5 in kidney tissues.We designed qPCR primers for them to test whether they were regulated by GAS5.The expression of miR-96-5P were significantly increased in the GAS5 knockdown group.The luciferase reporter gene assay revealed that miR-96-5p mimics effectively decreased the luciferase activity in wild type GAS5.These results suggest a direct binding and interaction between IncRNA GAS 5 and miR-96-5p.In addition,endogenous AGO2 could bind directly with GAS 5 and miR-96-5P.We found that miR-96-5p mimic downregulated GAS5 expression,while the miR-96-5p inhibitor increased GAS5 expression in vitro.5.Overexpression of miR-96-5p alleviated the increase in FN1,COL1,and CTGF induced by TGF-?1,while miR-96-5p inhibitor increased expression of FN1,COL1,and CTGF in mRNA and protein levels.Bioinformatics approach suggested that there was a direct binding site between miR-96-5p and the target FN1.The luciferase reporter gene assay revealed that miR-96-5p mimic effectively decreased the luciferase activity in wild type FN1 but had no effect on mutant FN1.Luciferase activity assay suggest a direct binding and interaction between FN1 and miR-96-5p.Conclusion:1.LncRNA GAS5 in renal tissue is increased under the condition of diabetes,which may promote tubulointerstitial fibrosis.2.LncRNA GAS5 sponges miR-96-5P in human renal tubular epithelial cells,reduces the inhibitory effect of miR-96-5P on target gene FN1,and plays a role in promoting tubulointerstitial fibrosis.