The Effect And Mechanism Of Long Non-coding RNA PWARSN-Induced Pyroptosis Of Proximal Tubule Epithelial Cell By Regulating TXNIP In Diabetic Kidney Disease

Diabetic kidney disease（DKD）,one of severe microvascular complications of diabetes mellitus（DM）,has already been the primary cause of end stage renal diseases（ESRDs）around the world.Researchers used to focus on glomerular injury during the development of DKD,while accumulating evidences recently show that tubule lesion may play a more pivotal role in this process.Changed renal hemodynamics,lipid metabolism,persistent oxidative stress,as well as chronic inflammation all contribute to damages in morphology,structure and function of proximal tubule epithelial cells（PTECs）during the development of DKD.Irreversible death of PTECs could be the long-term result under hyperglycemic condition,along with tubulointerstitial fibrosis and clinical proteinuria.However,the pathophysiological mechanisms responsible for initiation and development of PTECs injury remain poorly understood.Hence,further studies are warranted to elucidate the detailed mechanisms in order to explore new therapy targets for DKD.Pyroptosis is a new-found form of programmed cell death initiated by aseptic inflammation,which mediated by Gasdermin family.Different from apoptosis and necrosis,pyroptosis is characterized by pores formation in membrane,which lead to subsequent plasma membrane rupture.During this process,NOD-like receptor thermal protein domain associated protein 3（NLRP3）will recruit the adapter protein which called apoptosis-associated speck-like protein containing a CARD（ASC）,and then activate Caspase-1.Activeted Caspase-1 is required for the processing of Gasdermin D production,which contributes to formation of pores subsequently.As a result,a great deal of soluble pro-inflammatory cytosolic contents,such as IL-1β and IL-18 will be released from pyroptosic cells,followed by activating local and systemic inflammation response.It has been identified that pyroptosis is involved in diverse chronic inflammatory diseases,while its role in PTECs injury during the development of DKD remains unclear.Thioredoxin-interacting protein（TXNIP）is a critical determinant in response to oxidative stress,which promotes the accumulation of reactive oxygen species（ROS）and finally induces inflammation processes.Notably,TXNIP has already been found that it involves in pyroptosis of kidney parenchymal cells in DKD in a NLRP3-dependent manner.Previously,we have found the elevated level of TXNIP in PTECs cultured under hyperglycemic condition.However,whether TXNIP can mediate PTECs pyroptosis and the exact upstream signaling pathway of regulating TXNIP expression in DKD needs to be further studied.In recent years,long non-coding RNA（lncRNA）has attracted great attention,which plays complicated roles in epigenetic control,regulation of transcription and translation through interaction with miRNA,mRNA and RNA binding protein.Studies concerning microvascular complications of DM have shown that lncRNAs are related to diabetic retinopathy and DKD,while the role of lncRNAs in pyroptosis of PTECs remains unclear and need further investigation.In present study,we firstly explored whether TXNIP induces the activation of NLRP3 inflammasome and PTECs pyroptosis in patients with DKD and diabetic mice.Through transcriptomic analysis,we identified a novel lncRNA,Prader Willi/Angelman region RNA,SNRPN neighbor（PWARSN）,which is a regulator of TXNIP in PTECs.Moreover,we demonstrated the role of PWARSN in PTECs pyroptosis by taking advantage of PWARSN-overexpressing models induced by renal cortex targeted injection in vivo.At last,bioinformatics and RNA binding protein immunoprecipitation（RIP）were used to elucidate the molecular mechanisms of PTECs pyroptosis under hyperglycemic condition.Part Ⅰ TXNIP induces the NLRP3 inflammasome activation and proximal tubule epithelial cells pyroptosis in diabetic kidney diseaseObjectiveTo observe the activation of TXNIP/NLRP3 inflammasome and proximal tubule epithelial cells pyroptosis in the kidneys of patients with DKD,diabetic mice,and human proximal tubule epithelial cells under high glucose condition.To explore the relationship between TXNIP and pyroptosis induced by the NLRP3 inflammasome preliminarily.Methods1.Human kidney specimens from patients with DKD（DKD group）and diabetes mellitus（Diabetes mellitus,DM group）were collected.The kidney specimens from unaffected portions of the kidney tumor with normal glucose tolerance（NGT）were served as normal controls（NC group）.Type 1 diabetic mouse models were established（DM group）,and C57BL/6J mice injected with citric acid buffer were served as normal controls（Normal control,NC group）.After 16 weeks,all the mice were killed and the kidney tissues were collected.The histopathological changes of renal and ultrastructural changes of renal tubules were observed by histopathological staining and transmission electron microscopy（TEM）.2.Immunohistochemical staining（IHC）was used to observe the protein expression and distribution of TXNIP and the NLRP3 inflammasome（NLRP3,caspase 1,and ASC）.3.Human proximal tubule epithelial cells（HK-2 cells）were cultured with normal glucose（5.6 mmol/L,NG group）and high glucose（30 mmol/L,HG group）for 0h,48 h,and 72 h.The pyroptosis and ultrastructural changes of HK-2 cells were observed by Incucyte（?） Live-Cell Imaging,TEM and scanning electron microscope（SEM）.4.TXNIP overexpression plasmid,TXNIP siRNA and negative control（NC）plasmid were constructed and transfected the HK-2 cells,which were divided into several groups as follows:normal glucose group（NG group）,high glucose group（HG group）,normal glucose+TXNIP overexpression negative control group（NG+NC group）,normal glucose+TXNIP overexpression plasmid group（NG+TXNIP group）,high glucose+TXNIP siRNA negative control group（HG+siNC group）,high glucose+TXNIP siRNA group（HG+siTXNIP group）,and mannitol hypertonic group（Hyperosmosis,HM group）.The protein expression level of TXNIP,NLRP3,Cleaved caspase-1,ASC,GSDMD,and IL-1β were detected by Western blot（WB）.Results1.Human kidney specimens:Compared with NC group and DM group,the infiltration of inflammatory cells in renal proximal tubules,atrophy of renal tubule epithelial cells,formation of vacuoles,thickening of renal tubular basement membrane and formation of interstitial fibrosis were observed in DKD group.TEM showed that there was no obvious abnormality in the structure of proximal tubular epithelial cells in NC group and DM group.However,in DKD group,the microvilli were disordered.Some mitochondrial structures disappeared with swelling as vacuolar.And autophagy also increased.2.Human kidney specimens:IHC showed that there was no significance in the protein expression of TXNIP,NLRP3,ASC and Caspase-1 in DM group compared with NC group（P>0.05）.Compared with NC group and DM group,the protein levels of TXNIP,NLRP3,ASC and Caspase-1 increased significantly in DKD group（P<0.05）.3.Kidney specimens of diabetic mice:Compared with NC group,the thickening of renal tubular basement membrane,infiltration of inflammatory cells,atrophy of epithelial cells,and formation of interstitial fibrosis were observed in DM group.TEM showed that in DM group,the microvilli of renal tubules were disordered.Some mitochondrial structures of tubular epithelial cells disappeared with swelling and bubbling.The broken ridges of mitochondrial outer membrane were disappeared.4.Kidney specimens of diabetic mice:IHC showed that the protein levels of TXNIP,NLRP3,Caspase-1 and ASC increased in renal tissues of DM group compared with NC group（P<0.05）.5.The results of Incucyte（?） Live-Cell Imaging,TEM and scanning electron microscope（SEM）showed that cytoplasmic translocated and accumulated on the membrane of HK-2 cells in the HG group,subsequently leading to cell characteristic swelling and bubbling,even membrane rupturing.6.The results of WB showed that the protein levels of TXNIP,NLRP3,Cleaved caspase-1,ASC,GSDMD,and IL-1β increased in HG group compared with NG group（P<0.05）.Compared with NG group and NG+NC group,the protein levels of TXNIP,NLRP3,Cleaved caspase-1,ASC,GSDMD,and IL-1β increased in TXNIP overexpression group（P<0.05）.Compared with HG group and HG+siNC group,the levels of these proteins in TXN1P siRNA group were decreased（P<0.05）.ConclusionsTXNIP induces the activation of NLRP3 inflammasome and pyroptosis of PTECs in diabetic kidney disease.Part Ⅱ The role of lncRNA PWARSN in the pyroptosis of proximal tubule epithelial cells in diabetic kidney diseaseObjectives1.The transcriptome RNA microarray was performed to construct the differential expression profiles of lncRNA and mRNA in human proximal tubular epithelial cells and to screen the candidate lncRNAs,which were related to TXNIP/NLRP3 signaling pathway.To investigate preliminarily the expression and clinical significance of lncRNA PWARSN in diabetic kidney disease.2.To explore the role of PWARSN in pyroptosis of proximal tubular epithelial cells in vitro and in vivo.Methods1.LncRNA PWARSN promotes pyroptosis of human proximal tubular epithelial cells under high glucose condition through TXNIP/NLRP3 signaling pathway1.1 The lncRNA/mRNA differential expression profiles and lncRNA-mRNA network in human proximal tubular epithelial cells（HK-2 cells）cultured with normal glucose（NG）and high glucose（HG）were constructed.The TXNIP-related lncRNAs were screened by quantitative real-time PCR（RT-PCR）.1.2 Kidney specimens were collected from patients with DKD（DKD group,n=15）,patients with DM（DM group,n=22）and patients with normal glucose tolerance（NC group,n=32）.The expression and distribution of PWARSN in kidneys were detected by fluorescence in situ hybridization（FISH）;The expression levels of PWARSN,TXNIP and NLRP3 in renal tubular tissues were detected by RT-PCR,and the correlation between PWARSN,TXNIP and NLRP3 were analyzed.Receiver operating characteristic（ROC）was used to examined the diagnostic efficiency of PWARSN to distinguish DKD patients from DM patients.1.3 FISH and cytoplasmic and nuclear RNA isolation assay were used to detect the subcellular localization of PWARSN.1.4 PWARSN was knocked out（KO_PWARSN）in HK-2 cells by CRISPR/cas9 system.PWARSN was upregulated（OE_PWARSN）in HK-2 cells by lentivirus system.The cells were divided into several groups as follows:normal glucose group（NG group）,high glucose group（HG group）,normal glucose+PWARSN overexpression control group（NG+NC group）,normal glucose+PWARSN overexpression group（NG+OE_PWARSN group）,high glucose+PWARSN silencing control group（HG+siNC group）,high glucose+PWARSN knockout group（HG+KO_PWARSN group）.The expression level of PWARSN was detected by RT-PCR.1.5 The production of reactive oxygen species（ROS）in HK-2 cells was observed by fluorescence microscope;The ultrastructural changes of HK-2 cells were observed by TEM and SEM;The pyroptosis of HK-2 cells were observed by Incucyte（?） Live-Cell Imaging and propidium iodide（PI）staining.1.6 Cell pyroptosis and the protein levels of TXNIP,NLRP3,ASC,Cleaved caspase-1,GSDMD,IL-1β and FN in each group were detected by ROS,PI staining,and WB.Results1.LncRNA PWARSN promotes pyroptosis of human proximal tubular epithelial cells under high glucose condition through TXNIP/NLRP3 signaling pathway1.1 The differential expression profiles of lncRNA and mRNA in HK-2 cells under NG and HG condition were successfully established.The results of RT-PCR showed that the expression level of TXNIP mRNA in HG group was upregulated significantly compared with NG group（P<0.05）,which was consistent with the mRNA microarray results.1.2 LncRNA-TXNIP interaction network was successfully constructed.The results of RT-PCR showed that the expression of PWARSN（NR₀22011）in HG group was upregulated compared with NG group（P<0.05）.1.3 Inhibition of PWARSN in HG-induced HK-2 cells reduced the expression of TXNIP mRNA and protein（P<0.05）.1.4 PWARSN was mainly distributed in renal tubules of patients with DKD.RT-PCR showed that compared with NC group,there was no significant difference in the expression levels of PWARSN,TXNIP and NLRP3 in DM group（P>0.05）,while the expression levels of PWARSN,TXNIP and NLRP3 in DKD group were significantly increased compared with DM group（P<0.05）.Spearman correlation analysis showed that the expression of PWARSN was positively correlated with the expression of TXNIP（P<0.05）,as well as the expression of NLRP3（P<0.05）.ROC analysis showed when the cutoff is 3.096,the specificity was 0.68 and the sensitivity was 0.93,the area under the ROC was 0.836 for PWARSN to distinguish DKD patients from DM patients.1.5 PWARSN was localized in the cytoplasm and nucleus in HK-2 cells（P<0.05）.1.6 The results of RT-PCR showed that the expression of PWARSN in HG group was increased compared with NG group（P<0.05）,and there were no significant changes in the expression of NG+NC group（P>0.05）.Compared with NG+NC group,the expression of PWARSN was increased significantly in NG+OE_PWARSN group（P<0.05）.Compared with HG group,there was no significant difference in the expression of PWARSN in Hg+siNC group（P>0.05）.However,PWARSN was successfully knocked out in KO_PWARSN group（P<0.05）.1.7 Compared with NG+NC group,overexpressing PWARSN promoted intracellular ROS accumulation;Compared with HG+ siNC group,knocking out PWARSN inhibited the production of ROS.1.8 TEM and SEM showed that cytoplasmic distribution was translocated to the plasma membrane of HK-2 cells in the HG+siNC group,subsequently leading to cell swelling and bubbling,even rupturing.The above phenomenon was significantly improved by knocking out PWARSN.1.9 Incucyte（?） Live-Cell Imaging and PI staining showed that compared with NG group,cell pyroptosis was increased in HG group.Compared with NG+NC group,cell pyroptosis was increased in NG+OE_PWARSN group.Compared with HG and HG+siNC groups,knocking out PWARSN reduced cell pyroptosis.1.10 Compared with NG group,the protein levels of TXNIP,NLRP3,ASC,Cleaved caspase-1,GSDMD,IL-1β and FN were increased in HG group（P<0.05）,but there were no significant changes in NG+NC group（P<0.05）.Compared with NG+ NC group,the levels of these proteins were increased in NG+OE_PWARSN group（P<0.05）.Compared with HG group and HG+siNC group,the levels of those proteins in KO_PWARSN group were decreased（P<0.05）.1.11 The results of ROS,PI staining and WB showed that TXNIP overexpression reversed the inhibitory effect of KO_PWARSN on cell pyroptosis.Methods2.LncRNA PWARSN promotes pyroptosis of mouse renal proximal tubule epithelial cells in vivo2.1 Mouse renal tubule epithelial cells（mRTECs）were cultured and transfected with PWARSN overexpression plasmid.The cells were divided into several groups as follows:NG group,HG group,normal glucose+PWARSN overexpression plasmid control group（NG+NC group）,normal glucose+PWARSN overexpression plasmid group（NG+OE_PWARSN group）.2.2 The expression levels of PWARSN and TXNIP were detected by RT-PCR;The co-localization of PWARSN and TXNIP were observed by fluorescence in situ hybridization-immunofluorescence double staining（FISH-IF）;The protein binding ability of TXNIP and NLRP3 was detected by Co-immunoprecipitation（Co-IP）.2.3 The pyroptosis of mRTECs was observed by Incucyte（?） Live-Cell Imaging and PI staining.2.4 The expression levels of TXNIP,NLRP3,ASC,Cleaved caspase-1,GSDMD and IL-1β in each group were detected by WB.2.5 The SPF grade healthy male C57BL/6J mice were fed for 2 weeks after the adaptive feeding,and STZ was injected intraperitoneally to construct type 1 diabetic mice（DM group）.Human PWARSN overexpressing adeno-associated virus was constructed and was injected into the renal cortex of wild type C57BL/6J mice（PWARSN group）.All the mice were divided into the following groups:normal control group（NC group）,diabetes mellitus group（DM group）,saline injection group（saline group）,Vector injection group（Vector group）,and PWARSN adeno-associated virus injection group（PWARSN group）.2.6 The expression of PWARSN in mice in each group was detected by RT-PCR;The expression and distribution of PWARSN in renal tissues were observed by FISH-IF staining.2.7 The levels of blood glucose,body weight,kidney weight/body weight ratio,serum creatinine（Scr）,blood urea nitrogen（BUN）,24h urinary total protein（24h-UTP）and KIM-1 were detected.2.8 The histopathological changes of renal tubules were observed.2.9 The expression of TXNIP,NLRP3 and ASC in renal tubules of mice in each group were detected by RT-PCR and IHC staining.Results2.LncRNA PWARSN promotes pyroptosis of mouse renal proximal tubule epithelial cells in vivo2.1 RT-PCR showed that compared with NG group and NG+NC group,the expression of PWARSN and TXNIP mRNA were increased in mRTECs in NG+OE_PWARSN group（P<0.05）.The co-localization of PWARSN and TXNIP was increased in NG+OE_PWARSN group.PWARSN increased the binding ability of TXNIP and NLRP3.2.2 Incucyte（?） Live-Cell Imaging and PI staining showed that compared with NG+NC group,PWARSN promoted cell pyroptosis.2.3 Compared with NG and NG+NC group,PWARSN upregulated the expression levels of TXNIP and NLRP3 related proteins（P<0.05）.2.4 RT-PCR showed that compared with NC group,saline group and vector group,PWARSN was increased in renal tubules at 4 weeks,8 weeks,12 weeks and 16 weeks after PWARSN injection,and the expression of PWARSN was highest at 8 weeks（P<0.05）.FISH-IF staining showed that PWARSN was mainly distributed in renal tubules.2.5 Blood glucose,body weight and renal function in mice:compared with NC group,the levels of blood glucose,Scr,BUN,24 h-UTP and serum KIM-1 were increased in DM group.The body weight was decreased（P<0.05）.Compared with 8W-saline group and 8W-vector group,Scr,BUN,24 h-UTP and serum KIM-1 were increased in 8W-PWARSN group,but there were no significant changes in body weight and blood glucose（P>0.05）.2.6 Compared with NC group,the thickening basement membrane of proximal tubules,inflammatory cell infiltration,epithelial cell atrophy and tubulointerstitial fibrosis were observed in DM group,and the similar histopathological changes were observed in 8W-PWARSN group.TEM showed that compared with NC group,the microvilli of renal tubules in DM group were disordered.Some mitochondrial structures of tubular epithelial cells were disappeared and mitochondria swelled in vacuole shape.Compared with NC group,8W-saline group and 8W-vector group,the similar ultrastructural changes of renal tubular epithelial cells were observed in 8W-PWARSN group.2.7 RT-PCR and IHC staining showed that the expression levels of TXNIP,NLRP3 and ASC were increased in renal tubules in DM group compared with NC group（P<0.05）,and the above similar changes were observed in 8W-PWARSN group（P<0.05）.ConclusionsLncRNA PWARSN regulates proximal tubular epithelial cells pyroptosis through TXNIP/NLRP3 signaling pathway in diabetic kidney disease in vitro and in vivo.Part Ⅲ The mechanism of lncRNA PWARSN-mediated pyroptosis of proximal tubule epithelial cells in diabetic kidney diseaseChapter 1 LncRNA PWARSN sponges miR-372-3p in the cytoplasm to regulate TXNIPObjectivesTo explore the detailed molecular mechanism of cytoplasmic lncRNA PWARSN regulating TXNIP/NLRP3 signaling pathway in proximal tubule epithelial cells.Methods1.LncRNA regulates miR-372-3p1.1 PWARSN-miRNAs-TXNIP endogenous competitive RNA（ceRNA）network was established.And the expression levels of candidate miRNAs in HK-2 cells were detected by RT-PCR.1.2 The co-localization of PWARSN and miR-372-3p in HK-2 cells were detected by FISH assay.1.3 The endogenous binding between PWARSN and miR-372-3p was detected by RNA immunoprecipitation（RIP）-qPCR and dual luciferase reporter assay.2.LncRNA PWARSN sponges miR-372-3p in the cytoplasm to regulate TXNIP2.1 The endogenous binding between TXNIP and miR-372-3p was detected by dual luciferase reporter assay.2.2.MiR-372-3p mimics,miR-372-3p inhibitor,NC mimics and NC inhibitor were constructed and were transfected into HK-2 cells.The cells were divided into the following groups:NG group,HG group,NG+NC inhibitor group,NG+miR-372-3p inhibitor group,HG+NC mimics group,and HG+miR-372-3p mimics group.The protein levels of TXNIP and NLRP3 were detected by WB.2.3 MiR-372-3p mimics or inhibitor were transfected into OE_PWARSN and KO_PWARSN HK-2 cells,respectively.The cells were divided into the following groups:NG group,HG group,NG+OE_PWARSN group,NG+OE_PWARSN+miR-372-3p mimics group,HG+KO_PWARSN group,HG+KO_PWARSN+miR-372-3p inhibitor group and NC group.The protein levels of TXNIP were detected by WB.Results1.LncRNA PWARSN regulates miR-372-3p1.1 PWARSN-miRNAs-TXNIP ceRNA network was successfully established.RT-PCR showed that compared with NG group,the expression of miR-17,miR-320d-3p,miR-372-3p,and miR-520c-3p were decreased,while miR-20a-5p and miR-373-3p were increased in HG group;Compared with NG+NC group,overexpressing PWARSN upregulated miR-372-3p;However,compared with HG+siNC group,knocking out PWARSN inhibited the expression of miR-372-3p（P<0.05）.1.2 FISH assay showed that PWARSN and miR-372-3p were co-located in the cytoplasm.1.3 RIP-qPCR and dual luciferase reporter assay verified the endogenous binding between PWARSN and miR-372-3p.2.LncRNA PWARSN sponges miR-372-3p in the cytoplasm to regulate TXNIP2.1 Dual luciferase reporter assay verified the endogenous binding between miR-372-3p and TXNIP.2.2.WB showed that compared with NG group,the protein levels of TXNIP and NLRP3 in HG group were increased（P<0.05）,and there was no significant change in the protein level of NG+NC inhibitor group（P>0.05）.Compared with NG group and NG+NC inhibitor group,the protein levels of TXNIP and NLRP3 were increased in NG+miR-372-3p inhibitor group（P<0.05）.Compared with HG group and HG+siNC group,the levels of these proteins in HG+miR-372-3p mimics group were decreased（P<0.05）.2.3 The results of WB showed that compared with HG+siNC group,the protein level of TXNIP was decreased in HG+KO_PWARSN group,while miR-372-3p inhibitor could reverse the level of TXNIP.Compared with NG group,the protein level of TXNIP was increased in NG+OE_PWARSN group,while miR-372-3p mimics could reverse the protein level of TXNIP.Chapter 2 Nuclear PWARSN promotes RBMX degradation via the ubiquitination-proteasome pathway to regulate TXNIPObjectivesTo explore the molecular mechanism of nuclear lncRNA PWARSN regulating TXNIP/NLRP3 signaling pathway in proximal tubule epithelial cells.Methods1.Nuclear PWARSN promotes RBMX degradation via the ubiquitinationproteasome pathway1.1 LncRNA PWARSN binding protein profile was constructed in HK-2 cells under high glucose condition.1.2 ChIRP-WB and RNA binding protein immunoprecipitation（RIP）were performed to examine the interaction between RBMX and PWARSN.1.3 FISH-IF staining was performed to observe the co-localization of PWARSN and RBMX;The expression of RBMX in HK-2 cells were detected by RT-PCR and WB.1.4 Protein synthesis inhibitor cycloheximide（CHX）and proteasome inhibitor MG-132 and dimethyl sulfoxide（DMSO）were added to HK-2 cells respectively,the protein level of RBMX in each group was detected by WB.1.5 Immunocoprecipitation（IP）-WB assay was performed to detect the effect of PWARSN on the ubiquitination level of RBMX protein in HK-2 cells.1.6 catRAPID software（http://service.tartaglialab.com/page/catrapid_group）was used to predict the binding region between PWARSN and RBMX.1.7 The expression plasmids of different regions of PWARSN were constructed,and RNA pull down truncation experiment was performed to determine the specific binding region of PWARSN and RBMX.PWARSN overexpression mutated plasmid at the binding site of PWARSN and RBMX was constructed and transfected into HK-2 cells.The protein levels of RBMX and TXNIP were detected by WB.2.Nuclear PWARSN regulates TXNIP through RBMX2.1 Luciferase promoter activity experiment was performed to detect the regulatory effect of RBMX on TXNIP promoter activity and to identified the specific region of RBMX regulating TXNIP promoter activity.2.2 Chromatin immunoprecipitation（ChIP）-qPCR assay was performed to explore the regulatory effect of RBMX on the enrichment of H3K9me3 in the TXNIP promoter region.2.3 The RBMX overexpression plasmid and siRNA were constructed and transfected with HK-2 cells.The protein level of TXNIP in HK-2 cells was detected by WB.2.4 The miRNAs production blocker Dicer siRNA and RBMX siRNA were transfected into KO_PWARSN HK-2 cells and the protein levels of RBMX and TXNIP were detected by WB.Results1.Nuclear PWARSN promotes RBMX degradation via the ubiquitinationproteasome pathway1.1 LncRNA PWARSN binding protein profile was successfully constructed in HK-2 cells under high glucose condition.Combined with relevant literatures,we chose RBMX（heterogenetic nuclear ribonucleoprotein,hnRNP G）for further study.1.2 ChIRP-WB and RIP-qPCR confirmed that PWARSN could bind to RBMX protein.1.3 PWARSN and RBMX were co-located in the nucleus.The results of RT-PCR and WB showed that compared with NG group,the expression levels of RBMX mRNA and protein in HG group were significantly increased（P<0.05）.Compared with NG+NC group,there was no significant change in the expression level of RBMX mRNA in NG+ OE_PWARSN group（P>0.05）,but the protein level was decreased.Compared with HG+siNC group,there was no significant change in the expression level of RBMX mRNA in HG+KO_PWARSN group（P>0.05）,but the protein level was increased.1.4 Compared with HG+DMSO group,the protein level of RBMX was decreased in HG+CHX group and HG+siNC+CHX group.Compared with HG+CHX group and HG+siNC+CHX group,the protein level of RBMX was increased in HG+KO_PWARSN+CHX group.1.5 Compared with NG+NC+DMSO group,the protein level of RBMX was decreased in NG+OE_PWARSN+DMSO group,whereas the protein level of RBMX was increased in NG+NNC+MG-132 group.Compared with NG+OE_PWARSN+DMSO group,the protein level of RBMX was increased in NG+OE_PWARSN+MG-132 group.1.6 Compared with NG+NC group,the ubiquitination level of RBMX protein was increased in NG+OE_PWARSN group.Compared with HG+siNC group,the ubiquitination level of RBMX protein was decreased in HG+KO_PWARSN group.1.7 catRAPID software showed that there was obvious enrichment of RBMX protein in the 1100 nt～1300 nt and 1450 nt～1700 nt of PWARSN.RNA pull down truncation experiment showed that RBMX protein was significantly enriched in the 1000 nt～1250 nt and 1450 nt～1797 nt of PWARSN.1.8 Overexpressing PWARSN downregulated the protein level of RBMX and upregulated the protein level of TXNIP,while P WARSN mutant plasmid had no effect on their protein levels.2.Nuclear PWARSN regulates TXNIP through RBMX2.1 Luciferase promoter activity experiment showed that overexpressing RBMX or knocking out PWARSN could inhibit TXNIP promoter activity（P<0.05）.The truncation of luciferase promoter activity assay showed that RBMX was mainly bound to-700～-300 bp in TXNIP promoter region（P<0.05）.2.2 ChIP-qPCR showed that overexpressing RBMX could increase the enrichment of H3K9me3 in TXNIP promoter（P<0.05）.2.3 WB showed that overexpressing RBMX reduced the protein level of TXNIP.Inhibiting RBMX increased the protein level of TXNIP.However,inhibiting RBMX in KO_PWARSN cells could reverse the inhibitory effect of knocking out PWARSN on TXNIP expression.Conclusionsl.LncRNA PWARSN sponges miR-372-3p to regulate TXNIP in the cytoplasm of proximal tubule epithelial cells.2.LncRNA PWARSN in the nuclear of proximal tubule epithelial cells promotes RBMX degradation via the ubiquitination-proteasome pathway to regulate TXNIP.