Study On The Effect And Mechanism Of MiR-640 In Degeneration Of The Intervertebral Disc

【Background】Intervertebral Disc Degeneration is the most common reason for lower lumbar pain,and an initiating factor in spinal degenerative diseases.There are 632 million people attacked by this disease and severe cases can be lead to the loss of labor ability which influences the health of human beings seriously.At present,the main treatments for IDD are conservative treatment and surgical treatment.But both of the methods are not able to interrupt or reverse the process of intervertebral disc degeneration.Intervertebral disc degeneration is a progressive disease,therefore,how to interrupt or even reverse the process of intervertebral disc degeneration effectively are the key elements to treat IDD.Deeply revealing the molecular mechanism of intervertebral disc and looking for the key molecules of postponing or even reversing the intervertebral disc degeneration have significant implications for the treatment of IDD.【Objectives】After reading literatures and comparative analysis of databases,we found that miR-640 is high expression in the degeneration of intervertebral disc tissues.Then,we designed experiments to verify the conclusion.RT-PCR and IHC were used to detect the expression quantity of miR–640 between obviously degenerated intervertebral disc specimens and relatively normal specimens.The results showed that the expression quantity of miR–640 in obviously degenerated intervertebral disc specimens is much higher than the relatively normal specimens.So we speculate that miR–640 has played an important role in the pathophysiological process of IDD.Therefore,the main purpose of this study is regarding the function and regulation mechanism of miR-640 as the breakthrough point,and utilize lentivirus infection,dual-luciferase reporter systerm,RNAi technology,et,al.to reveal the mechanism of miR-640 in IDD and nucleus pulposus cells in the process of senescence and apoptosis.Meanwhile,enrich the study of the theory of IDD and provide potential drug targets for clinical prevention and treatment of the IDD.【Methods】 1.The cultivation of the human nucleus pulposus cells.2.Extract the total RNA of human intervertebral disc tissue and NP cells,and detect the expression of miR – 640.3.Detect the expression of inflammatory factors in human intervertebral disc tissue and NP cells.Study the interaction between the miR – 640 and inflammatory factors.4.Study the interaction between the miR – 640 and NF-κB by dual-luciferase reporter system and immunocytochemistry.5.Predict the downstream target molecules of miR – 640 by bioinformatics methods,and research the interaction between the miR – 640 and the downstream target molecules.6.SA-β-gal was used to detect cell senescence of NP cells.7.Flow cytometry was used to analysis the cell cycle.CellTiter-Glo kits was used to detect the cell viability.【Results】1.The expression of miR-640 is higher in intervertebral disc degeneration tissues.2.Inflammatory factors can promote the expression of miR – 640 by NF-κB signal pathway.3.miR-640 can activate the NF-κB signal pathway by suppressing the LRP1.This may become a part of positive feedback loop.4.miR-640 can suppress the activation of WNT signal pathway by suppressing the β-catenin and EP300,which are the key molecules of WNT signal pathway.5.miR-640 can promote the senescence and apoptosis of NP cells by suppress WNT signal pathway.【Conclusion】This study verified that the inflammatory microenvironment may be existed in the nucleus pulposus of senescence,the proinflammatory factors and miR-640 expression form a positive feedback loop,and which caused a large accumulation of mir-640,inhibited the activity of downstream WNT pathway and promoted the senescence and apoptosis of nucleus pulposus cells.In this regulatory network,miR-640 is located in the key cross node of signal pathway and apoptosis of nucleus pulposus can be reduced by interfering the expression of miR-640.This study illustrates the regulatory networks of miR-640 related molecula during the degeneration of nucleus pulposus cells.Meanwhile,this study provides potential drug targets for various diseases which related to intervertebral disc degeneration treatment.