A Study Of Circ-FAM169A Regulating Lumbar Intervertebral Disc Degeneration Via Sponging MiR-583

【Objective】Low back pain(LBP)is one of the most not uncommon reasons for chief complaint in orthopedic clinic.According to a report,70%-85% of worldwide people experience LBP at least once in their lifetime,and 5% of them develop into chronic LBP ultimately,which has seriously affected the living quality of patients and increased the economic burden of patients.Increasingly researches have indicated that symptomatic intervertebral disc degeneration(IDD)is mostly related to LBP,accounting for about 40% of all LBP-causing factors.Increasingly researches have reported that non-coding RNAs(ncRNAs)including miRNAs,and emerging circRNAs play an significant role in the occurrence and progression of IDD.The most explored function of circRNAs is as master regulators of gene expression to sponge or sequester microRNA(miRNA)in the cytoplasm.It had been reported that circRNAs mediate nucleus pulposus cells(NPCs)apoptosis as well as regulate the expression of inflammatory cytokines、matrix metalloproteinase(MMP)、a disintegrin and metalloproteinase with thrombospondin motifs(ADAMTS)、various apoptosis proteins and extracellular matrix(ECM)to participate in the modulation of the pathological process of IDD.However,the specific mechanism for circRNAs regulating IDD remain unclear.This investigation aimed to find novel pivotal circRNA and miRNA and to explore their regulatory mechanisms,which can provide new strategy for the treatment of IDD.【Method】1.Based on our team previous bioinformatics analysis,identify pivotal circRNA and corresponding pivotal miRNA as subjects.2.In NPCs,the fluorescence in situ hybridization(FISH)assay was used to detect whether the pivotal circRNA and miRNA are located in the nucleus or cytoplasm of NPCs.3.Luciferase reporter assay was used to validate the effectiveness of binding sites of circRNA and miRNA in NPCs.4.The level of pivotal circRNA were overexpressed in NPCs.【Result】1.According to our team’s previous bioinformatics analysis,the molecular of circ-FAM169 A and miR-583 were screened for further research.2.The molecular of circ-FAM169 A and miR-583 coexist in the cytoplasm of NPCs was confirmed by FISH.3.The luciferase reporter assay reveled that when circ-FAM169 A and miR-583 were co-transfected in NPCs,the miR-583 mimic and miR-583 inhibitor groups were significantly different from the negative control group(P< 0.01).When transfected with circ-FAM169A-mut1,there was no difference in statistical analysis;while transfected with circ-FAM169A-mut2,the statistical analysis of the results was still significantly different.It is speculated that miR-583 interacts with circ-FAM169 A at the first position.4.In this study,the overexpression(OE)vector of circ-FAM169 A was successfully constructed,the expression of circ-FAM169 A in NPCs was up-regulated by 4712.81 times compared with the control group(P<0.01).This results indicate that circ-FAM169 A OE can be used in the next functional verification experiments 【Conclusion】1.This study identified circ-FAM169 A and miR-583 as pivotal circRNA and pivotal miRNA that regulat the process of IDD,respectively.2.The study implied that the circ-FAM169A-miR-583 axis may play an crucial role in modulating the process of IDD.3.This result provides a theoretical basis for further research on the pathogenesis and clinical intervention of IDD.