The Expression And Clinical Significance Of Hsa_circ₀007379/miR-7977/NR4A1 In Lupus Nephritis

BackgroundSystemic lupus erythematosus?SLE?is a multi-systemic autoimmune disease characterized by loss of tolerance to autoantigens,production of autoantibodies,and deposition of immune complexes,leading to damage of multiple organs including the kidneys.Lupus nephritis?LN?is one of the most common and serious complications,which seriously affects the prognosis and quality of life of patients.Although the etiology of LN is unclear,the present studies have shown that the pathogenesis of LN is associated with genetic factors.With the development of high-throughput sequencing and microarray technology,non-coding RNA represented by microRNA?miRNA?,long-chain non-coding RNA and circular RNA?circRNA?is a hot research topic.MiRNA is a 18 to 22 nucleotide long single-stranded RNA molecule that regulates gene expression post-transcriptionally by inducing degradation of mRNA or inhibiting translation of mRNA.CircRNA is a type of endogenous ncRNAs characterized by a covalent closed loop without 5'caps and 3'tails,and is mainly located in the cytoplasm.Unlike linear non-coding RNAs,circular RNAs are highly conserved and tissue-specific,with greater stability and higher expression abundance in some organs of eukaryotic animals.In recent years,the researches of circRNA have been progressing rapidly.As for the physiological function of circRNAs,they are involved in acting as miRNA molecular sponges,regulating transcription,splicing and expressing the parental gene,forming RNA-protein complexes,and serving as competing endogenous RNAs?ceRNAs?regulating gene expression past-transcriptionally.Some circRNAs and miRNAs were identified to be differentially expressed in patients with autoimmune diseases compared with healthy controls.We think circRNAs and miRNAs might be differentially expressed in LN,and the present studies on their expression and function were still limited.In this study,we investigate the the role of circRNAs as ceRNAs in the progression of LN,and provides new targets and strategies for the diagnosis and treatment of LN.ObjectiveTo identify differentially expressed circRNA,miRNA and mRNA by high-throughput sequencing technology,constructing a circRNA-miRNA-mRNA molecular regulatory network,verifying the expression of candidate circRNA,miRNA and mRNA.To analyze the correlation between the expression levels and LN clinical characteristics.To explore its role in the progression of LN,and provide a new idea for the diagnosis and treatment of LN.MethodsIn this study,three renal tissues of active LN patients were collected as case group and three normal renal tissues of patients with radical nephrectomy were collected as control group.We measured the circRNAs,miRNAs and mRNAs expression profiles in kidney tissues of LN patients with RNA sequencing?RNA-Seq?.Then we performed bioinformatics analysis to explore the potential ceRNA for differentially expressed circRNAs,miRNAs and mRNAs.Another ten renal tissues with LN and five normal renal tissues,ten peripheral blood samples with LN and ten healthy peripheral blood samples from healthy volunteers were collected.We performed quantitative real-time polymerase chain reaction?qRT-PCR?to verified candidate circRNA,miRNA and mRNA in kidney tissues and peripheral blood.And we assessed the correlation between RNAs and clinical variables.Results1.A total of 159 circRNAs?73 circRNAs were upregulated,and 86 circRNAs were downregulated?,159 miRNAs?84 miRNAs were upregulated,and 75 miRNAs were downregulated?,and 1089 mRNAs?565 mRNAs were upregulated,and 524 mRNAs were downregulated?were differentially expressed between LN patients and normal controls with RNA-Seq.Among them,hsa_circ₀007379 and NR4A1 mRNA were downregulated,while miR-7977 was upregulated.2.The circRNA-miRNA-mRNA regulatory network were constructed on the basis of hsa_circ₀007379-miR-7977-NR4A1 by correlation analysis.The KEGG Pathway analysis revealed PI3K-Akt signaling pathway and MAPK signaling pathway related to the functions of NR4A1 in patients with LN.We predicted the low expression of hsa_circ₀007379 competed together with miR-7977 to influence the expression of NR4A1,and regulated PI3K-Akt signaling pathway and MAPK signaling pathway to affect the pathogenesis of LN.3.We selected hsa_circ₀007379,miR-7977 and NR4A1 for further validated in another group of kidney tissues and peripheral blood samples by qRT-PCR.Compared with control group,the expression of miR-7977 was increased and the expression of NR4A1 was decreased both in kidney tissues and peripheral blood samples in LN patients,but hsa_circ₀007379 only revealed same changes in kidney tissues.4.The expression levels of hsa_circ₀007379,miR-7977 and NR4A1 mRNA were not correlated with urinary total proteinuria per day,serum creatinine,estimated glomerular filtration rate,activity index,chronicity index,SLEDAI score and erythrocyte sedimentation rate.The expression level of NR4 A mRNA was positively correlated with white blood cells and complement 3.Conclusion1.The hsa_circ₀007379-miR-7977-NR4A1 ceRNA network was a potentially regulatory mechanism involved in the pathogenesis of LN.2.miR-7977 and NR4A1 can serve as biomarkers to provide early warning for clinical diagnosis and treatment in LN.