| Objective: Systemic lupus erythematosus(SLE)is a chronic autoimmune disease that can affect multiple organs and tissues.Lupus nephritis(LN)is one of the most common complications of SLE.Despite the clinical application of new immunosuppressive agents and monoclonal antibodies in recent decades,the 10-year survival rate of SLE has exceeded 90%,but the incidence of LN remains high and at 31-82%.Moreover,10-30%of patients with LN(all types)and 44% of patients with type IV LN will develop into end stage renal disease(ESRD)within 15 years and require renal replacement therapy,which will bring huge economic burden to society.Therefore,elucidating the mechanisms of LN occurrence and development,and exploring precise target therapy may reduce the incidence of LN and slow or even prevent LN from progressing to ESRD.Circular RNAs(circRNAs)are newly discovered endogenous closed-loop single-stranded non-coding RNAs formed by backsplicing of mRNA precursors through covalent bond.circRNAs,which have a special structure and abundant expression model,have been proven to be involved in the development of many diseases.Current research focuses on tumors and cardiovascular diseases.circRNAs are rarely reported in kidney disease.This subject aims to 1)explore the expression profile of circRNAs in kidney tissue of LN patients and analyze the correlation between differentially expressed circRNAs and LN activity indicators;2)study the practical clinical application value of differentially expressed circRNAs;3)analyse whether differentially expressed circRNAs can be used as new targets for prevention and treatment of LN in animal models of LN.Methods: 1.Study on circRNAs expression profile in renal tissues of LN patients: A case-control study was used in this study.The renal tissues of 7 patients(6 females)with type IV LN diagnosed by renal biopsy were included as disease research groups,and 6(5females)renal carcinoma patients with renal tissue at least 5 cm from the cancer tissue served as a normal control(NC).The renal tissue RNA was extracted and circRNAs were enriched.The differentially expressed circRNAs in the two groups’ renal tissue were studied by high-throughput RNA sequencing(RNA-seq),and the differentially expressed circRNAs were verified using RT-qPCR.The bioinformatics database,TargetScan and miRanda were used to predict microRNAs(mi RNAs)regulated by differentially expressed circRNAs,and KEGG pathway and GO analysis were used to predict the biological functions and signaling pathways involved in differentially expressed circRNAs.We further analyzed the correlations between circHLA-C expression level,which had the greatest foldchange in LN,and clinical and pathological activity indicators in LN patients,and explored the expression change of miR-150 which has perfect binding sites to circHLA-C.2.The clinical significance of circHLA-C in peripheral blood mononuclear cells(PBMCs)from LN patients: 25 patients with LN confirmed by renal biopsy and 12 normal control were recruited.RNA in PBMCs and plasma were extracted,and the relative expression levels of circHLA-C were detected.The correlations between circHLA-C expression levels in PBMCs from LN patients and clinical indicators were analyzed.Receiver operating characteristic curve(ROC curve)analysis was used to evaluate the efficacy of circHLA-C as a diagnostic marker for LN in PBMCs.3.Using Fcgr2b-knockout(Fcgr2b-KO)spontaneous LN model mice,to explore the expression changes of four differentially expressed circRNAs with human-mouse homology obtained from RNA-seq and miR-150 and their potential basic application value: 40-week-old female Fcgr2b-KO mice and Fcgr2b-WT mice at the same age were used.Serum and kidneys were collected.Serum anti-nuclear antibody(ANA)and anti-dsDNA antibody,specific indicators for SLE were detected by enzyme-linked immunosorbent assay(ELISA).Urinary protein / creatinine ratio,which means SLE progress into LN were measured.Paraffin sections of kidney tissues were used to observe the morphological changes of kidney using PAS staining.RT-qPCR was used to detect the relative expression levels of inflammatory factors(Tnfα,Il-6,Il-1b and Il-10),fibrotic factors(Tgfβ,α-Sma,Fn and Col4),and 4 circRNAs(circZNF609,circFAM188 A,circUBR5 and circPDE4B)that have human-mouse homology and mi R-150.And the correlations between miR-150 and inflammatory and fibrotic factors were analyzed.Results: 1.Because LN is more common in women,the results in this section were compared between all women subjects.Compared with NC,there were 171 differentially expressed circRNAs in LN kidney tissue,with 142 up-regulated and 29 down-regulated.Ten differentially expressed circRNAs were selected and verified by RT-qPCR.Seven ofwhich were consistent with the results of RNA-seq.Bioinformatics website analysis showed that there were at least 5 different miRNAs binding sites in the top 20 circRNAs that were up-regulated and down-regulated,respectively.KEGG pathway and GO analysis showed that differentially expressed circRNAs may participate in the occurrence and development of LN through hypoxia inducible factor-1(HIF-1)and Neurotrophin signaling pathway.Compared with NC,the relative expression of circHLA-C in LN renal tissue was the highest of all differentially expressed circRNAs.And renal circHLA-C levels were positively correlated with the 24-hr urinary total protein excretion(r = 0.920,p = 0.009),serum creatinine(r = 0.756,p = 0.082),crescent glomeruli ratio(r = 0.929,p= 0.007)and renal activity index score(r = 0.884,p = 0.020).Compared with NC,miR-150 expression,downstream of circHLA-C in LN kidney tissues was down-regulated,and there was a negative trendency between miR-150 and circHLA-C(r= – 0.409,p = 0.211).2.Compared with NC,the expression of circHLA-C in PBMCs of LN patients was increased(1.91 ± 1.23 vs 1.00 ± 0.89,p <0.05),and there was no statistical difference in the expression of circHLA-C in plasma.The ROC curve was used to analyze the efficacy of circHLA-C in PBMCs in diagnosing LN.The area under the curve was 0.733(95% CI:0.563-0.903,p < 0.05).CircHLA-C in PBMCs of patients with LN was positively correlated with serum anti-dsDNA(r = 0.734,p < 0.0001),SLE disease activity index(SLEDAI)score(r = 0.742,p < 0.0001),24-hr urinary total protein excretion(r = 0.628,p < 0.001)and serum creatinine(r = 0.532,p < 0.01),and negatively correlated with estimated glomerular filtration rate(r = – 0.492,p < 0.05),complement C3(r = – 0.420,p < 0.05)and C4(r = – 0.4643,p <0.05).3.Female Fcgr2b-KO mice at 40 weeks of age showed spontaneous LN.Compared with Fcgr2b-WT mice of the same age,serum ANA and dsDNA antibodies were significantly increased(all p < 0.0001)and urinary protein / creatinine ratio was increased(p =0.0005),and typical LN-like changes were observed in renal pathology included diffuse glomerular proliferation and lobular changes.RT-qPCR results showed that compared with Fcgr2b-WT mice,Fcgr2b-KO mice renal inflammatory indicators(Tnfa,Il-6,Il-1b,and Il-10)and fibrotic indicators(Tgfb,a-Sma,Fn,and Col4)were significantly increased.Compared with Fcgr2b-WT mice,the 4 circRNAs(circ ZNF609,circFAM188 A,circUBR5,and circPDE4B)with human-mouse homology we detected in Fcgr2b-KO mice did not show differential expression.However,the expression of miR-150 in kidney tissue of Fcgr2b-KO mice was increased(1.53 ± 0.64 vs 1.00 ± 0.36,p <0.05),and the expression of miR-150 in kidney tissue were positively correlated with biochemical index dsDNA(r = 0.568,p = 0.009),ANA(r = 0.452,p = 0.045);and the inflammatory indicators Tnfa(r = 0.607,p = 0.005),Il6(r = 0.491,p = 0.028),Il1b(r =0.534,p = 0.015)and Il10(r = 0.530,p = 0.016);and the fibrotic indicators Tgfb(r =0.472,p = 0.036),α-Sma(r = 0.489,p = 0.029),Fn(r = 0.447,p = 0.048).Conclusions: 1.Our data are published,for the first time and provided analysis of the expression profiling of circRNAs in the kidneys of LN patients,as well as the interaction network between circRNAs and miRNAs in LN.There were significantly positive correlations between circHLA-C with the largest foldchange and LN disease activity indicators,indicating that circHLA-C plays an important role in the pathogenesis of LN.It is suggested that renal circHLA-C may be involved in the development of LN.2.The expression of circHLA-C in PBMCs of LN patients was increased,and there were correlations with multiple clinical LN activity indicators,suggesting that circHLA-C in PBMCs may be a new biomarker for the diagnosis of LN.3.Female Fcgr2b-KO mice at 40 weeks of age showed typical LN-like changes,while renal inflammation and fibrosis coexisted.However,the four circRNAs that were differentially expressed in the LN patients we tested did not show differential expression in the LN mouse model,suggesting the species specificity of circRNAs.4.The expression of miR-150 in Fcgr2b-KO mice was up-regulated,and the expression of miR-150 in kidney tissues was correlated with LN activity indicators,inflammatory indicators and fibrosis indicators,which supplements and confirms our we previous research in LN patients’ kidney tissue that miR-150 participates in the development of LN. |
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