Research On Mechanism Of Hsa_circ₀123190 Sponging Hsa-miR-483-3p To Regulate Renal Fibrosis In Lupus Nephritis

BackgroundLupus nephritis（LN）is one of the most common secondary glomerular diseases in China,which is also an important cause of renal failure and mortality of patients with systemic lupus erythematosus（SLE）.Although the treatment of glucocorticoids and immunosuppressive agents can improve the survival rate of LN patients,20%of them can develop to end-stage kidney disease（ESRD）in 10 years.At the meanwhile,the prognosis of each LN patient is significantly different.Therefore,for improving the survival of LN patients,it is essential to upgrade better clinical evaluation system and accurate treatment.Circular RNA（circRNA）is a new type of identified non-coding RNAs,which was found to be involved in the occurrence and development of several diseases.Due to the tissue specificity and expression stability,circRNAs can function as biomarkers for early diagnosis and diseases surveillance.In addition,circRNAs have a variety of biological functions,particularly acting as competitive endogenous RNA molecules in many diseases.In other words,due to the abundant miRNA binding sites,circRNAs can function as miRNA sponges to regulate expression of target genes.Recent studies demonstrated that circRNAs played an important role in various kidney diseases,as ceRNAs and potential biomarkers.However,the function and mechanism of circRNAs in LN are unclear.Objectives1.To identify the candidate circRNA which is differently expressed in kidney tissues of patients with LN by bioinformatic analysis of circRNA expression profile.2.To analyze the clinical significance of candidate circRNA and explore a potential biomarker for diagnosis and treatment in LN.3.To discover the molecular mechanism of candidate circRNA involved in the pathogenesis of LN and provide new ideas for the development of therapeutic drugs.Methods1.Samples collection:A total of 10 cases of kidney tissues from LN patients were collected.And 5 kidney tissues at least 5 cm from the edge of tumor in patients undergoing radical nephrectomy which concurrently confirmed by microscope were taken as normal controls.In addition,peripheral blood samples from 10 patients with LN and 10 healthy volunteers were obtained.2.The candidate circRNA selection:circRNA expression profile of kidney tissues from 3 patients with LN and 3 normal controls via high-throughput sequencing was analyzed.There were four differently expressed circRNA screened out and verified by real-time fluorescence quantitative PCR（qRT-PCR）in larger sample size.The circRNA with the most obviously different expression was selected as the next research object.3.The downstream target molecules of circRNA:The interaction between candidate circRNA and miRNA,or miRNA and mRNA,were determined by bioinformatic analysis and double luciferase reporting experiments.The expression of target miRNA and mRNA were verified by qRT-PCR,and the clinical significance was analyzed.4.Assessment of renal fibrosis in LN:The fibrosis degree of kidney tissues in LN was evaluated by Masson-trichome staining compared with normal controls.Furthermore,the fibrosis-related proteins transforming growth factor-β1（TGF-β1）andα-smooth muscle actin（α-SMA）were stained by immunohistochemistry and semi-quantitative analysis with H-Score.5.The expression of circRNA in peripheral blood and its clinical correlation:The expressions of the circRNA in peripheral blood of patients with LN and healthy controls were verified by qRT-PCR.The correlation of the circRNA with clinical characteristics was analyzed and the important utility of it was further explored by receiver operating characteristic（ROC）curve analysis.Results1.159 circRNAs were significantly dysregulated in LN patients compared with normal controls.The expression of hsa_circ₀123190 and hsa_circ₀000660 was significantly decreased in kidney tissues of LN patients by qRT-PCR（P<0.05）.2.There was a binding relationship between hsa_circ₀123190 and hsa-miR-483-3p.The expression of hsa-miR-483-3p was upregulated in kidney tissues of LN（P<0.05）.3.The level of APLNR mRNA was significantly downregulated in kidney tissues of LN.And hsa-miR-483-3p was interacted with APLNR mRNA（P<0.05）.4.The level of APLNR mRNA was positively associated with renal chronicity index and C-reactive protein（P<0.05）.5.APLNR can regulate the expression of TGF-β1.In this study,the results of semi-quantitative analysis and immunohistochemical staining showed the renal expression of TGF-β1 was increased in LN group compared with normal controls（P<0.05）.6.The expression of hsa_circ₀123190 in peripheral blood was also significantly downregulated in LN group and negatively correlated with serum creatinine level（P<0.05）.Conclusion1.Hsa_circ₀123190 could function as a ceRNA to regulate APLNR expression involved in renal fibrosis by sponging hsa-miR-483-3p in LN.2.Peripheral blood hsa_circ₀123190 acts as a potential and noninvasive biomarker for LN and provides application value for early diagnosis and disease surveillance.