Long Noncoding RNA Lnc-TSI Inhibits Renal Fibrogenesis By Negatively Regulating The TGF-β/Smad3 Pathway

BackgroundChronic kidney disease(CKD)has become a "public health problem" all over the world.In 2010,2.6 million uremic patients worldwide were relied on dialysis or kidney transplantation for life support due to renal dysfunction and 2.3-7.1 million patients worldwide died prematurely due to unavailable dialysis or kidney transplantation treatment.Therefore,any strategy that can prevent or delay the progression of chronic kidney disease to uremic has extremely important social and economic significance.The discovery of new targets to intervene renal fibrosis is the foundation in the establishment of clinical prevent:ion strategies.Renal fibrosis is the common outcome of almost all progressive chronic kidney diseases.TGF-β has been recognized as one of the most vital mediator in the pathogenesis of renal fibrosis and plays an important role in promoting fibrosis in a variety of renal cells,including tubular epithelial cells(TECs).TGF-β1 initiates its cellular actions by binding with the TGF-β type Ⅱ receptor,which activates the TGF-β type Ⅰ receptor(TβRI),resulting in the phosphorylation of Smad2/3.The activated Smad complex then translocates into the nucleus and regulates the transcription of profibrotic genes.Smad3 phosphorylation induced by interacting with TβRI has been recognized as a crucial step in TGF-β1/Smads signaling.In the past,researches on renal fibrosis have mainly focused on the coding genes of key proteins,that is,the mutations of traditional key genes lead to the changes of the encoded proteins and cause the biological disfunction.However,with the development of molecular biological mechanisms,epigenetic regulation has been found to play an important role in the development of renal interstitial fibrosis.Epigenetic phenomena mainly include genomic imprinting,DNA methylation,chromatin remodeling and noncoding RNAs(ncRNAs)etc.Long noncoding RNAs(lncRNAs)are a heterogeneous class of long transcripts(>200 nucleotides[nt])without protein-coding potential.In recent years,with the popularization and application of second-generation sequencing technology,lncRNA,which was originally considered as the "noise" of transcriptome,has been proved to have important biological functions under physiological and disease conditions.It is evident that lncRNAs have a wide range of biological functions,and their aberrant expression has been associated with diverse pathological settings,including cancer,metabolic,and cardiovascular diseases,lncRNAs can regulate gene expression at multiple levels,thus affecting the pathophysiological process of diseases.Previous studies on lncRNA in renal diseases mainly focus on renal inflammation,diabetic nephropathy and renal fibrosis.However,most lncRNAs in these studies were found in mouse models,and only a few were found in human kidneys.Unlike other noncoding RNAs such as microRNAs and small-nucleolar RNAs,which exhibit high degrees of conservation across diverse species,lncRNAs often lack strong conservation.Therefore,the biological roles of lncRNAs in regulating human TIF remain elusive,and more detailed studies are needed to unravel the molecular functions of lncRNAs in this settingMethods1.TGF-β1 was applied to induce profibrotic changes in renal tubular epithelial cells,and lncRNA expression profile chips were used to screen differentialy expressed lncRNAs in TGF-β1 stimulated cells.2.The full length of the lnc-TSI transcript was determined and obtained through 5’RACE and 3’RACE assays,the conservation across diverse species and the coding capacity of lnc-TSI were analyzed through bioinformatics analysis.3.Bioinformatics analysis was applied to predict Smad3 that may bind to the promoter of Inc-TSI,and ChIP-qPCR was used to verify the interaction between Smad3 and the promoter of Inc-TSI.4.Specific mRNA or lncRNA were knocked down with siRNAs or lentivirus,and lnc-TSI was also knocked out with CRISPR/cas9 system to observe the corresponding biological effects.5.Lnc-TSI overexpression was accomplished by pcDNA3.1 vector in human and mouse renal tubular epithelial cells.6.Lnc-TSI interacting proteins were determined by RNA pull-down and mass spectrometry.7.The binding of lnc-TSI and its interacting proteins was verified by RNA pull-down and RNA immunoprecipitation.8.Protein-protein interactions were analyzed by co-immunoprecipitation.9.Lnc-TSI was specifically expressed in the kidneys of mice by injecting lnc-TSI plasmid via tail vein or lnc-TSI adeno-associated virus via kidney vein,the effect of lnc-TSI on the progression of renal interstitial fibrosis was investigated.10.To investigate the expression pattern of Inc-TSI in human tubulointerstitial fibrosis and its relationship with the progression of tubulointerstitial fibrosis,lnc-TSI was detected in a clinical cohort of patients with chronic kidney disease.ResultsWe identified a poorly-conserved and kidney-enriched long noncoding RNA in TGF-β1-stimulated human tubular epithelial cells and fibrotic kidneys,which we termed TGF-β/Smad3 interacting long noncoding RNA(lnc-TSI).Lnc-TSI was transcriptionally regulated by Smad3 and specifically inhibited TGF-β-induced Smad3 phosphorylation and downstream profibrotic gene expression.Lnc-TSI worked by binding with the MH2 domain of Smad3,blocking the interaction of Smad3 with TGF-β receptor I independent of Smad7.Delivery of human lnc-TSI into unilateral ureteral obstruction(UUO)mice and ischemic reperfusion injury(IRI)mice,two well-established models of renal fibrosis,inhibited phosphorylation of Smad3 in the kidney and attenuated renal fibrosis.In a cohort of 58 patients with biopsy-confirmed IgA nephropathy(IgAN),lnc-TSI renal expression negatively correlated with the renal fibrosis index(r=-0.56,P<0.001)after adjusting for cofounders.In a longitudinal study,32 IgAN patients with low expression of renal Inc-TSI at initial biopsy had more pronounced increases in their renal fibrosis index and experienced stronger declines in renal function at repeat biopsy at a mean 48 months to follow-up.These data suggest that Inc-TSI reduced renal fibrogenesis through negative regulation of the TGF-β/Smad pathway.ConclusionIn conclusion,our study identified a kidney-enriched lncRNA that function as an endogenous inhibitor of TGF-β1/Smad3 pathway and regulated renal fibrogenesis.These findings provide novel information for understanding the mechanisms underlying TGF-β1-mediated tissue fibrosis.Our work hints at the possibility of a new therapeutic target to halt the most damaging process in kidney diseases,though this remains to be confirmed by future studies.