The Role And Mechanism Of CircRTN4 In Glomerular Mesangial Cell Proliferation And Extracellular Matrix Deposition In Lupus Nephritis

Excessive proliferation of mesangial cells is one of the major pathological features of lupus nephrits（LN）.Proliferating mesangial cells lead to the progression of glomerulosclerosis in patients by releasing inflammatory factors and secreting extracellular matrix,seriously affecting prognosis.Therefore,it is urgent to explore the specific mechanism of glomerulosclerosis in lupus nephritis.Circular RNAs（circRNAs）,a novel type of noncoding RNA（nc RNA）,are covalently linked to make up a circular configuration via a connection with the 5′and 3′ends.Growing evidence showed that circRNA play important role in the multilevel regulation of gene expression.Emerging studies have suggested that deregulated circRNAs are involved in the development of lupus nephritis by regulating various biological processes.However,the potential mechanisms of circRNA in lupus nephritis,especially in the progression of mesangial cell dysfunction,have not been elucidated.This study was based on the pervious results of RNA microarray,LN patients,two types of lupus model mice,human renal mesangial cell lines（HRMCs）and THP1 cell lines were selected as subjects to explore the role and possible mechanism of circRTN4 in mesangial cell proliferation and ECM deposition in lupus nephritis,which will provide experimental basis for further elucidating the pathogenesis and seeking possible therapeutic targets of lupus nephritis.The experiment is divided into the following four parts:Part One CircRTN4 regulated mesangial cell proliferation and FN expression in lupus nephritisObjective:In this part,we verified the deregulated circRNAs in mesangial cells of lupus nephritis and explored the role of circRTN4 in mesangial cell proliferation and ECM deposition and its exact mechanism of regulating fibronectin（FN）expression.Methods:1.HRMCs treated with 5%LN plasma or control plasma were collected for circRNA microarray,and the expression and localization of circRTN4 in mesangial cells were detected by real-time PCR and RNA fluorescence in situ hybridization（FISH）.2.The renal biopsy of LN patients and the control renal tissues were used as study objects,RNA FISH was used to detect the expression of circRTN4;immunohistochemistry（IHC）was used to detect the expression of Cyclin D1and FN.3.HRMCs were used to detect the effects of circRTN4 on the proliferation level and ECM deposition of glomerular mesangial cells in lupus nephritis:（1）According to the circRTN4 sequence information,three si RNA were designed and transfected into mesangial cells,and the knockdown efficiency was detected by real-time PCR.（2）To observe the effect of circRTN4 on the proliferation and ECM deposition of glomerular mesangial cells:HRMCs were randomly divided into four groups:control plasma,LN plasma,LN plasma+sicircRTN4,LN plasma+si NC,and the proliferation level of mesangial cells was detected by flow cytometry（FCM）and Brd U assay,and the expression of FN m RNA and protein were detected by real-time PCR and western blot.（3）Circ Base and Target Scan were used to predict the target mi RNA of circRTN4 and FN.Three mi RNAs with higher reliability（mi R-607,mi R-647,mi R-513a-5p）were selected and infected with mesangial cells.The expression of FN m RNA and protein were detected by real-time PCR and western blot.4.293T cells were used as research objects to verify the binding site of circRTN4,mi R-513a-5p and FN by the dual-luciferase reporter gene assay.Results:1.Circ RTN4 was upregulated in mesangial cells of lupus nephritis:（1）RNA microarray results showed that there were 804 upregulated and512 downregulated circRNAs in LN plasma group（|Fold Change|≥2 and P<0.05）.（2）Real-time PCR confirmed that hsa＿circ＿0054595（circRTN4）was upregulated in the HRMCs of LN.（3）The circular form of circRTN4 was confirmed by DNA gel electrophoresis.（4）RNA FISH confirmed that circRTN4 was mainly located in the cytoplasm.2.Circ RTN4 was increased in the glomerulus of LN patients:RNA FISH showed that circRTN4 expression was remarkably increased in the glomerulus of patients with LN,and was localized to the cytoplasm of glomerular cells.3.Circ RTN4 regulated mesangial cell proliferation and ECM deposition:（1）IHC confirmed that the expression of Cyclin D1 and FN protein in the glomerulus of LN patients was significantly upregulated.（2）FCM and Brd U assay showed that the cell proliferation level was significantly increased in LN plasma group,while it was decreased after the knockdown of circRTN4.（3）Real-time PCR and western blot results showed that compared with control plasma group,FN m RNA and protein levels were significantly increased in LN plasma group,while the expression of FN was decreased after the knockdown of circRTN4.4.Circ RTN4 regulated the expression of FN by binding mi R-513a-5p:（1）Bioinformatics analysis showed that mi R-607,mi R-647,mi R-513a-5p were target micro RNAs of circRTN4 and FN.Real-time PCR and western blot results showed that overexpression of mi R-513a-5p could reduce the m RNA and protein levels of FN.（2）Western blot results showed that mi R-513a-5p mimics reduced the protein expression level of FN,while overexpression of circRTN4 reversed this effect.（3）The dual-luciferase reporter gene experiment showed that mi R-513a-5p mimics could reduce the luciferase activity of plasmids containing the FN3’UTR sequence.After the mutation of this sequence,mi R-513a-5p mimics could not affect the luciferase activity.Moreover,mi R-513a-5p mimics significantly attenuated luciferase activity of plasmids containing circRTN4predictive binding site.After the mutation of this sequence,mi R-513a-5p mimics could not affect luciferase activity.Conclusion:Circ RTN4 was upregulated in the glomerular mesangial cells of LN,which promoted cell proliferation and regulated the expression of FN protein by binding mi R-513a-5p.Part Two Knockdown of circRTN4 in the kidney could improve the glomerular extracellular matrix deposition in LN miceObjective:In this study,local infection technology was used to knockdown the expression of circRTN4 in the kidney of BALB/c+P and MRL/lpr mice,and to verify the role and possible mechanism of circRTN4 in FN expression and renal injury of LN.Methods:（1）Female BALB/c mice aged 8 weeks were intraperitoneally injected with 0.5 m L pristane.After 24 weeks,serum anti-ds-DNA level and urinary protein level of the mice was upregulated,lymphoedema and joint lesions were observed,and the model was established.BALB/c+P mice were randomly divided into LN group（BALB/c+P group）,circRTN4 adeno-associated virus renal orthotopic injection group（BALB/c+P+circRTN4-R group）,adeno-associated virus control group（BALB/c+P+circ NC-R group）,each group with 6 mice.Female BALB/c mice of the same age were set as control group.（2）Female MRL/lpr mice aged 24 weeks were randomly divided into LN group（MRL/lpr group）,circRTN4 adeno-associated virus renal orthotopic injection group（MRL/lpr+circRTN4-R group）,adeno-associated virus control group（MRL/lpr+circRTN4-R group）,each group with 3 mice.Female MRL/MPJ mice of the same age were set as the control group.CircRTN4 adeno-associated virus and control adeno-associated virus were renally injected in both kidneys of the mice.24 h urine and blood samples were collected after 6 weeks and the mice were sacrificed to collect bilateral kidney tissue.Real-time PCR was used to observe the knockdown efficiency of AAV.The levels of urine protein,serum BUN and Scr were detected by biochemical kits.HE,PAS and Sirius red staining were used to observe the pathological morphology.Real-time PCR,western blot and immunofluorescence（IF）were used to detect the m RNA and protein level of FN in renal cortex of mice.Results:1.Adeno-associated virus infection effectively downregulated circRTN4expression in renal cortex of lupus mice:Real-time PCR showed that the expression of circRTN4 was reduced in the renal cortex of BALB/c+P+circRTN4-R and MRL/lpr+circRTN4-R groups.2.Knockdown of circRTN4 in kidney improved renal injury in LN mice:（1）Biochemical indicators tests showed that the levels of 24-h proteinuria,Scr and BUN in BALB/c+P group were significantly increased;24-h proteinuria and BUN levels in BALB/c+P+circRTN4-R group were decreased,whereas Scr level showed no significant difference.（2）Biochemical indicators tests showed that the levels of 24-h proteinuria,Scr and BUN in MRL/lpr group were increased;the level of 24-h proteinuria was decreased in MRL/lpr+circRTN4-R group.However,there was little difference in Scr and BUN between MRL/lpr+circRTN4-R group and MRL/lpr+circ NC-R group.3.Knockdown of circRTN4 expression in kidney alleviated glomerular cell proliferation and ECM deposition in LN mice:HE staining showed that compared with BALB/c mice,the glomerular volume and cell number was increased in BALB/c+P mice,which was decreased in BALB/c+P+circRTN4-R group.PAS and Sirius red staining showed remarkably mesangial expansion in BALB/c+P mice.However,the level of mesangial expansion and collagen composition was decreased in BALB/c+P+circRTN4-R mice.In addition,similar results were observed in MRL/lpr mice.4.Knockdown of circRTN4 expression in the kidney reduced the expression of FN m RNA and protein in glomerular of LN mice:Real-time PCR,western blot and IF showed that FN m RNA and protein levels in the renal cortex of BALB/c+P and MRL/lpr groups were significantly increased,which were decreased after circRTN4 knockdown.Conclusion:Knockdown of circRTN4 expression in the kidney of BALB/c+P and MRL/lpr could suppress FN expression,alleviate glomerular cell ECM deposition,and improve renal function.Part Three Monocyte-derived exosomal circRTN4 promoted mesangial cell proliferation and FN expressionObjective:In this part,we analyzed the expression of circRTN4 in monocyte of LN and explored the role and mechanism of monocyte-derived exosomes in mesangial cell proliferation and FN expression.Methods:1.The renal biopsy of LN patients and the control renal tissues were obtained as study objects,and the infiltration of monocytes in kidney tissue was detected by IHC.2.To detect the effects of monocyte exosomes on mesangial cell proliferation and FN protein expression in LN:（1）Exosome identification:exosome morphology was observed by TEM.Western blot was used to detect the expression of exosome marker proteins CD9 and CD63.（2）PKH67 labeled THP1 exosomes were isolated and co-cultured with mesangial cells,and the uptake of exosomes by HRMCs was detected by IF.（3）To detect the effects of inhibiting monocyte-derived exosomes on mesangial cell proliferation and FN expression:THP1 cells were pretreated with GW4869 for 2 h and then stimulated by 5%control plasma or LN plasma for 8 h.The culture supernatant of THP1 was collected and made into LN plasma-treated conditioned medium（LM）to incubate HRMCs.HRMCs were randomly divided into control group,LM group,LM^GW4869group and LM^DMSOgroup.Brd U assay was used to detect the proliferation level.Real-time PCR was used to detect the expression of circRTN4 and FN.The level of FN in HRMCs culture supernatant was determined by ELISA.3.To detect the effects of monocyte-derived exosomal circRTN4 on mesangial cell circRTN4 expression:（1）The expression of circRTN4 in THP1,THP1 exosomes and peripheral blood monocytes of LN patients was detected by real-time PCR.The expression and localization of circRTN4 were detected by RNA FISH.（2）THP1 cells were randomly divided into control plasma group and LN plasma group.After 5%control plasma or LN plasma stimulation for 8 h,exosomes in the cultured supernatants of the two groups were extracted by centrifugation and named as THP1exo ^Conand THP1exo ^LN.HRMCs were randomly divided into control plasma group,LN plasma group,LN plasma+THP1exo ^LNgroup and LN plasma+THP1exo ^Congroup.After 5%control plasma or LN plasma stimulated for 8 h,the expression of circRTN4 in HRMCs was detected by real-time PCR.（3）THP1 cells were randomly divided into four groups:control plasma group,LN plasma group,LN plasma+sicircRTN4 group and LN plasma+si NC group.After 5%control plasma or LN plasma stimulation for 8 h,exosomes in the supernatant were extracted by centrifugation,and these exosomes were co-cultured with HRMCs for 8 h,the expression of circRTN4 in HRMCs was detected by real-time PCR.Results:1.Monocyte infiltration was observed in glomeruli of LN patients:IF showed that CD68 was significantly increased in the renal tissues of LN patients.2.Monocyte-derived exosomes promoted glomerular mesangial cell proliferation and FN expression of LN:（1）Identification of exosomes:The exosomes diameter was less than 150 nm observed by TEM.Exosome marker proteins CD9 and CD63 were identified by western blot.（2）The uptake of THP1 exosomes by HRMCs was increased in LN:IF showed that monocyte-derived exosomes were increased in HRMCs treated with LN plasma.（3）Inhibition of THP1 exosome reduced LM-induced mesangial cell proliferation:Brd U assay showed that HRMCs proliferation level was increased in the LM group,which was reduced by the exosome synthesis and release inhibitor（GW4869）.（4）Inhibition of THP1 exosome reduced the expression of FN in LM cultured HRMCs:Real-time PCR and ELISA showed that FN expression in LM group was significantly increased compared with control group.GW4869 inhibited LM-induced increase of FN synthesis and secretion in HRMCs.（5）Inhibition of THP1 exosome downregulated LM-induced circRTN4expression in HRMCs:Real-time PCR results showed that circRTN4 expression was significantly increased in LM group.However,it was decreased in LM^GW4869group.3.THP1 derived exosomal circRTN4 could regulate the expression of circRTN4 in HRMCs:（1）Real-time PCR results confirmed that circRTN4 expression was upregulated in THP1,THP1 exosomes and PBMCs of LN patients;RNA FISH results showed that circRTN4 was mainly located in the cytoplasm（2）Real-time PCR results showed that compared with control plasma group,the expression of circRTN4 in HRMCs was increased induced by LN plasma,which was much higher in LN plasma+THP1exo ^LNgroup.（3）Real-time PCR results showed that circRTN4 expression in HRMCs was significantly increased in THP1exo ^LNgroup,whereas knockdown of circRTN4 in monocytes weakened this effect.Conclusion:Upregulated circRTN4 in monocyte-derived exosomes could be internalized by mesangial cells,thus leading to upregulation of circRTN4 in mesangial cells,accompanied by increasing cell proliferation and excessive synthesis and secretion of FN.Part Four Knockdown of monocyte circRTN4 expression improved glomerular cell proliferation and extracellular matrix deposition in LN miceObjective:In this study,tail vein injection technology was used to knockdown the expression of circRTN4 in peripheral blood monocytes of BALB/c+P and MRL/lpr mice,and to verify the effect of monocyte-derived circRTN4 on mesangial cells dysfunction and renal injury in LN mice.Methods:（1）Female BALB/c mice aged 8 weeks were intraperitoneally injected with 0.5 m L pristane.After 24 weeks,the identification of model was the same as that in part II.BALB/c+P mice were randomly divided into LN group（BALB/c+P group）,circRTN4 adeno-associated virus tail vein injection group（BALB/c+P+circRTN4-V group）,control adeno-associated virus tail vein injection group（BALB/c+P+circ NC-V group）,each group with 6 mice.Female BALB/c mice of the same age were set as control group.（2）Female MRL/lpr mice aged 24 weeks were randomly divided into LN group（MRL/lpr group）,circRTN4 adeno-associated virus tail vein injection group（MRL/lpr+circRTN4-V group）,control adeno-associated virus tail vein injection group（MRL/lpr+circ NC-V group）,each group with 3 mice.Female MRL/MPJ mice of the same age were set as the control group.CircRTN4 adeno-associated virus and control adeno-associated virus were injected through the tail vein of mice.24 h urine and blood samples were collected after 6 weeks and the mice were sacrificed to collect bilateral kidney tissue.The knockdown efficiency of circRTN4 in peripheral blood monocytes and serum exosomes was detected by RNA FISH and real-time PCR.The levels of urine protein,BUN and Scr were detected by biochemical kits.The histopathological observation was carried out by HE,PAS and Sirius red staining.Real-time PCR,western blot,IF or IHC were used to detect the m RNA and protein expression levels of FN and Cyclin D1 in the renal cortex of mice.Results:1.The expression of circRTN4 in PBMCs and serum exosomes of mice was downregulated by circRTN4 r AAV:（1）RNA FISH results showed that the circRTN4 was mainly located in the cytoplasm.And the expression of circRTN4 in monocytes was significantly decreased in BALB/c+P+circRTN4-V and MRL/lpr+circRTN4-V groups.（2）Real-time PCR results showed that compared with BALB/c and MRL/MPJ groups,circRTN4 expression in serum exosomes was significantly increased of BALB/c+P and MRL/lpr groups,whereas it was decreased in BALB/c+P+circRTN4-V and MRL/lpr+circRTN4-V groups.2.Knockdown of circRTN4 in monocyte improved renal injury in LN mice:（1）Biochemical indicators tests showed that the levels of 24-h proteinuria,Scr and BUN in BALB/c+P group were significantly increased.24-h proteinuria and BUN levels in BALB/c+P+circRTN4-V group were decreased,whereas the level of Scr showed no significant difference.（2）Biochemical indicators tests showed that the levels of 24-h proteinuria,Scr and BUN in MRL/lpr group were increased;the level of 24-h proteinuria was decreased in MRL/lpr+circRTN4-V group.However,there was little difference in Scr and BUN between MRL/lpr+circRTN4-V group and MRL/lpr+circ NC-V group.3.Knockdown of monocyte circRTN4 expression alleviated renal pathological changes in LN mice:HE staining showed that compared with BALB/c mice,the glomerular volume and cell number was increased in BALB/c+P mice,which was decreased in BALB/c+P+circRTN4-V group.PAS and Sirius red staining showed remarkably mesangial expansion in BALB/c+P mice.However,the level of mesangial expansion and collagen composition was decreased in BALB/c+P+circRTN4-V mice.In addition,similar results were observed in MRL/lpr mice.4.Knockdown of monocyte circRTN4 expression improved glomerular cell proliferation in LN mice:Western blot and IHC results showed that the expression of Cyclin D1protein was significantly increased in renal cortex BALB/c+P and MRL/lpr mice,which was downregulated in BALB/c+P+circRTN4-V and MRL/lpr+circRTN4-V mice.5.Knockdown of monocyte circRTN4 expression improved glomerular ECM deposition in LN mice:Real-time PCR and IHC showed that FN m RNA and protein levels in the renal cortex of BALB/c+P and MRL/lpr groups were significantly increased,which were decreased after knockdown of circRTN4 expression in monocyte.Conclusion:Knockdown of monocyte circRTN4 in BALB/c+P and MRL/lpr mice could improve the glomerular cell proliferation and ECM deposition and alleviated renal pathological changes.