The Long Noncoding RNA ALKBH3-AS1 Alleviates The Progression Of Lupus Nephritis By Inhibiting Th17 Differentiation

Background:Lupus nephritis is one of the most prevalent and dangerous complications of Systemic Lupus Erythematosus(SLE),an autoimmune disease affecting several organs.Particularly in lupus nephritis,T helper cell 17(Th17)is crucial to the pathophysiology of systemic inflammatory disease(SLE).Research has demonstrated that long noncoding RNA(lnc RNA)is connected to the onset and progression of SLE and has a role in Th17 cell differentiation.It is currently unknown,nevertheless,whether lnc RNA affects Th17 cell differentiation to affect the development and course of lupus nephritis.Objective:This paper employs human and mouse Th17 cells as the research target to examine the effects of the MDSC downstream molecule human ALKBH3-AS1(mouse Alkbh3os1)on Th17 differentiation,studying how ALKBH3-AS1 influences Th17differentiation and the molecular mechanism by which it contributes to the onset of illness progression using samples from SLE patients,lupus nephritis mice models,and in vitro investigations.Methods:1.Examining lnc RNA(ALKBH3-AS1)molecules that affect MDSC to promote the development of Th17 cells:human naive CD4~+T cells were separated into three groups to differentiate into Th17 cells,one for independent culture,one for co-cultivation with MDSC,and one for co-cultivation with MDSC and nor-NOHA(Arg-1inhibitor)group.Next,we examine differential genes using the Human Lnc RNA One Array Plus expression profile gene chip.2.Correlation between ALKBH3-AS1 expression in CD4~+T cells of SLE patients,Th17 cells,and disease activity:Peripheral blood PBMCs of SLE patients and normal control patients were isolated.Flow cytometer(FCM)was used to detect the proportion of Th17 cells and real-time fluorescence quantitative PCR(q PCR)was applied to detect the relative expression levels of ALKBH3-AS1 and IL17A.3.Whether and how ALKBH3-AS1(Alkbh3os1)regulated Th17 differentiation:a)The impact of ALKBH3-AS1(Alkbh3os1)on Th17 differentiation and TGF-β/SMAD signaling pathway:We created an ALKBH3-AS1(Alkbh3os1)overexpression plasmid and a short interfering RNA(si RNA)sequence;In vitro Th17 polarization experiment,FCM,q PCR and enzyme-linked immunosorbent assay(ELISA)were applied to investigate the effects of ALKBH3-AS1(Alkbh3os1)overexpression(OE)or knockdown on Th17 differentiation and TGF-β/SMAD signaling pathway.b)Whether ALKBH3-AS1 affected Th17 differentiation through regulating SMAD3 stability:Fluorescence in situ hybridization(FISH)experiment verified the cellular localization of ALKBH3-AS1 in human naive CD4~+T cells;RNA stability assay detected the impact of ALKBH3-AS1 on SMAD3 half-life;FCM and q PCR methods revealed the regulation of ALKBH3-AS1 on SMAD3 levels.The rescue experiment confirmed whether SMAD3 was necessary for ALKBH3-AS1 to regulate Th17differentiation and the TGF-β/SMAD signaling pathway.c)How ALKBH3-AS1 regulated SMAD3 expression:Dual-luciferase reporter gene assay was used to verify whether ALKBH3-AS1 directly bound to SMAD3;RNA pulldown experiment combined with mass spectrometry analysis were applied to determine ALKBH3-AS1 binding protein;To ascertain if the YTHDF2 protein and ALKBH3-AS1 bind to one another,Western Blot and RNA immunoprecipitation analyses were performed;The impact of ALKBH3-AS1 on the interaction between SMAD3 and YTHDF2 protein was confirmed by RIP tests.To confirm if YTHDF2 was necessary for the regulatory effects of ALKBH3-AS1 on SMAD3 half-life,Th17differentiation,and TGF-β/SMAD signaling pathway,the rescue experiment employed the RNA stability experiment and the FCM method.4.The potential benefit of specifically targeting Alkbh3os1 in a pristane-induced lupus nephritis model:The overexpression plasmid Alkbh3os1 was packaged by the Adeno-associated virus(AAV);After intraperitoneal injection of Alkbh3os1overexpressed adeno-associated viral vector in SLE mice,Hematoxylin and eosin(HE),ELISA,FCM,and q-PCR methods were used to determine the regulatory effect of Alkbh3os1 on TGF-β/SMAD signaling pathway and the Th17 differentiation,as well as the therapeutic value of Alkbh3os1 on SLE.Results:1.According to RNA-seq data,when Th17 cell differentiation was stimulated by MDSC,ALKBH3-AS1 expression was downregulated;on the other hand,when nor-NOHA was given to prevent Th17 cell differentiation,the expression was elevated.2.The expression of ALKBH3-AS1 was reduced in CD4~+T cells of SLE patients,which was adversely linked with both the percentage of Th17 cells and SLEDAI.3.The molecular mechanism through which ALKBH3-AS1 regulated Th17differentiation:a)Overexpression of ALKBH3-AS1(Alkbh3os1)inhibited Th17 differentiation,whereas ALKBH3-AS1(Alkbh3os1)knockdown promoted Th17 differentiation.While ALKBH3-AS1(Alkbh3os1)knockdown activated the TGF-β/SMAD signaling pathway,overexpression of ALKBH3-AS1(Alkbh3os1)blocked the signaling pathway.b)ALKBH3-AS1 promoted the degradation of SMAD3,thereby suppressing the TGF-β/SMAD signaling pathway and Th17 cell differentiation.ALKBH3-AS1 mainly located in the cytoplasm of human naive CD4~+T cells;overexpression of ALKBH3-AS1 promoted SMAD3 degradation and decreased the expression of SMAD3;knockdown of ALKBH3-AS1 prolonged the half-life of SMAD3and increased the expression of SMAD3;The inhibitory effect of TGF-β/SMAD signaling pathway and Th17 cell differentiation induced by overexpression of ALKBH3-AS1 could be partially restored by overexpression of ALKBH3-AS1 and SMAD3.c)ALKBH3-AS1 acted as a scaffold molecule to promote the interaction between YTHDF2 protein and SMAD3,which in turn promoted the degradation of SMAD3,and eventually inhibited Th17 differentiation.ALKBH3-AS1 directly bound to SMAD3 and YTHDF2 protein;ALKBH3-AS1overexpression led to the inhibition of the TGF-β/SMAD signaling pathway and Th17differentiation,which could be restored by overexpressing ALKBH3-AS1 and knocking down YTHDF2.Overexpression of ALKBH3-AS1 promoted the interaction between YTHDF2 protein and SMAD3.4.Overexpression of Alkbh3os1 inhibited Th17 cell differentiation and alleviated disease progression in lupus nephritis miceIn PBMC and the spleen of lupus nephritis model,overexpression of Alkbh3os1inhibited the TGF-β/SMAD signaling pathway and Th17 differentiation.It also downregulated the expression of Il17a,Rorc,and Smad3 in the kidney,thereby mitigating renal pathological changes and nephritis symptoms in SLE mice.Conclusion:1.Th17 cell frequency and the severity of the disease are adversely connected with the downregulated expression of ALKBH3-AS1 in CD4~+T cells of SLE patients.2.ALKBH3-AS1(Alkbhos1)inhibits Th17 differentiation through TGF-β/SMAD signaling pathway;ALKBH3-AS1 serves as a scaffold to recruit SMAD3 and YTHDF2 protein,promoting the degradation of SMAD3 by YTHDF2 protein,eventually blocking the TGF-β/SMAD pathway and inhibiting Th17 differentiation.3.In lupus nephritis mice,Alkbh3os1 overexpression alleviates the pathological changes in kidney and clinical symptoms by inhibiting TGF-β/SMAD signaling pathway and Th17 differentiation.