MicroRNA-96 Regulates Nucleus Pulpocyte Proliferation By Targeting The ARID2/AKT Pathway

Objective: Low back pain(LBP)is one the most common musculoskeletal disorder,which inflicts a huge burden on economies and health care system.The causes of low back pain is complex,and intervertebral disc degeneration is considered to be a significant contributor to the development of this pain.The underlying cellular and mechanisms of IDD remain largely unknown,a various of etiological factors has been suggested to influence its etiology,including aging,genetic predisposition and environmental factors,and recently several studies have shown that the formation of nucleus pulposus(NP)cell clusters and the proliferation of fibrocartilaginous tissue play a important role in IDD.Thus far,the precise cause of increased NP cell proliferation in IDD is still unclear.Methods: The current study was conducted a total of human lumbar NP specimens were obtained from patients with scoliosis undergoing discectomy [n =17,with an average age of 27.9 years(24-36)] of the intervertebral disc nucleus pulposus tissue.And intervertebral disc degeneration patients(n=30,the average age of 39.4 years(37-56)] of the intervertebral disc nucleus pulposus tissue.We first isolated from fresh human nucleus pulposus cells of extracting total RNA was isolated,in after the miRNA RNA markers and hybridization,analysis on the chip.Relative expression of microRNAs in real time quantitative PCR verification.MiR-96 control target protein to miRNAs online databases and computer analysis to predict.MiR-96 and potential target protein 3 'UTRs role by dual luciferase report system is analyzed.We on in vitro cultured human nucleus pulposus cells transfection expression Has.MiR-96 mimic that miR-96,at the same time transfection anti-miR-96 implementation miR-96.By Western blot(Western B100 detection of nucleus pulposus cells pre-AKT,AKT,Cyclin D1 and ARID2 expression level,analysis of nucleus pulposus cells proliferation rate with CCK8 reagents and absorbance changes.We test control group by transmission electron microscopy and the proliferation of nucleus pulposus of intervertebral disc degeneration group,at the same time we through transfection ARID2 plasmid validation ARID2 can affect nucleus pulposus cells proliferation through AKT pathway.Results: Through this experiment,we found that intervertebral disc degeneration of nucleus pulposus cells expression of microRNAs-96.Real-time quantitative reverse transcription polymerase chain reaction PCR polymer further confirmed that the rise in miR-96.Biological information method to predict ARID2 for potential target protein of miR-96.MiR-96 can be by direct effects on the 3 'UTRs and inhibiting ARID2 expression,and its target protein in 3' UTR with miR-96 binding sites of mutation can stop the inhibition.ARID2/ AKT signaling pathways regulating cell growth,proliferation,migration and adhesion,etc.Human nucleus pulposus cells increase caused by miR-96 in the pre-AKT,and Cyclin D1 increases and ARID2 downgrade.Cut the expression of miR-96 and can lead to the pre-AKT,and Cyclin D1 cut and ARID2 raised.MiR-96 cut reduce ARID2/AKT mediated proliferation,while the rise in miR-96 increase ARID2/AKT mediated proliferation.ARID2/AKT pathway activation cell proliferation through inner and outer channels.Conclusion: This study for the first time on the molecular mechanisms underlying apoptosis increase in intervertebral disc degeneration and miRNAs expression spectrum study.At the same time,we identified ARID2 for miR-96 control new target protein.Our research shows that in human intervertebral disc degeneration,increase of miR-96 through to the target protein ARID2 and pre-AKT regulation to promote Cyclin D1 mediated proliferation,prompt miR-96 role in the etiology of the intervertebral disc degeneration.And,degeneration of intervertebral disc nucleus pulposus cells mainly into living cells,we provide a direct basis in the body,to confirm human nucleus pulposus cells proliferation function,help to understand the degeneration of nucleus pulposus cells cluster formation mechanism;Therefore,in order to we can be treated by slow virus mediated miR-96 keyboard degeneration provides new train of thought and ideas.