Mechanism Of MiR-106b-5p On Function Of Nucleus Pulposus Cells In Intervertebral Disc By Targeting MAPK1 Mediated ERK1/2 Signaling Pathway

Objective The molecular mechanism of the occurrence and development of Intervertebral Disc Degeneration(IDD)still keep magic.At present,there is no effective treatment plan for degenerative diseases of spine such as Lumbar disc degeneration(LDD),which is based on the pathological manifestations of IDD.T The pathological changes could not be reversed with the aim of alleviating symptoms and improving quality of life.Therefore,we need to understand the molecular mechanism of IDD at the gene level.In recent years,a large number of studies have shown that micro RNA molecules are involved in the pathological process of IDD.What is the relationship between miR-106b-5p and IDD? What are the changes in the function of Nucleus Pulposus cells(NP cells)caused by miR106b-5p? These problems need to be studied and explored.According to the prediction of bioinformatics analysis,we speculated that miR106b-5p might interfere with the expression of ERK1/2 by targeting MAPK1.It affects the signal transduction function of ERK1/2 signal pathway and changes the expression level of ADAMTs-4 and ADAMTs-5.We transfected the lentivirus containing has-premiR-106b-5p and has-miR-106b-5p into the NP cells.We successfully constructed the NP cell model with high and low expression of miR-106b-5p.The purpose of this study is to explore the molecular mechanism of IDD through miR-106b-5p targeting MAPK1 mediated ERK1/2 signaling pathway.This regulatory mechanism provides a new research direction for the treatment of IDD.Methods(1)Analysis of micro RNA chip by Bioinformatics(GSE19943,GSE45856,GSE63492).(2)The data of 212 cases of disc degeneration and 80 cases of lumbar fracture were collected.We collected and preserved the nucleus pulposus tissue of these patients during the operation.Pfirrmann classification based on MRI to evaluate the severity of IDD.Using PCR to verify the prediction of bioinformatics in intervertebral disc tissue.(3)We verified whether MAPK1 is the target gene of miR-106b-5p by bioinformatics analysis and Dual-Luciferase Reporter Assay System.(4)We cultured human NP cells in vitro.(5)We transfected the lentivirus packed with has-pre-miR-106b-5p and has-miR-106b-5p into the NP cells.The experiment was divided into four groups: miR-106b-5p mimics,miR-106b-5p inhibitor,Negative control,Blank.Mi R-106b-5p was up-regulated and downregulated in group-miR-106b-5p mimics and group-miR-106b-5p inhibitor respectively.Group-Negative control only transfected with empty lentivirus,Group-Blank did not transfect virus.(6)The transfected cells were screened by puromycin.RT-PCR was used to detect the expression level of mir-106b-5p to verify whether the transfection was successful.(7)We used RT-PCR to detect the expression level of MAPK1,ADAMTs-4 and ADAMTs-5.(8)We used Western blotting to detect the protein expression level of ERK1/2,ADAMTs-4 and ADAMTs-5.(9)We measured the scratch healing ability,migration ability and cell activity of NP cells by cell scratch test and Trans-well migration test.(10)We used MTT proliferation test to detect the change of proliferation ability of NP cells.(11)Detection of cell cycle and apoptosis rate of nucleus pulposus cells by flow cytometry.Results(1)The results of GEO CHIP(GSE19943,GSE45856,GSE63492)analysis showed that the expression level of miR-106b-5p was up-regulated 3.82 times compared with the control group(P<0.05).RT-PCR results showed that the expression level of mir-106b-5p was up-regulated by 4.26 times(P<0.05).The expression level of miR-106b-5p was positively correlated with the severity grade of IDD(Pfirrmann grade)(P<0.05).(2)The 445-452 base of MAPK1 3’UTR and miR-106b-5p can be directly combined.MAPK1 is the target of miR-106b-5p.(3)Overexpression of miR-106b-5p inhibited the expression level of MAPK1 and ERK1/2 protein(P< 0.05).On the contrary,the down-regulation of miR-106b-5p can increase the expression level of MAPK1 and ERK1/2 protein(P < 0.05).(4)Overexpression of miR-106b-5p can cause up-regulation of ADAMTs-4,ADAMTs-5 and their corresponding proteins and vice versa(P < 0.05).(5)We transfected NP cells with lentivirus and overexpressed miR-106b-5p.MTT assay showed that the proliferation of NP cells decreased(P=0.041).Down-regulating the expression level of miR-106b-5p can improve the proliferation of NP cells(P=0.02).(6)Overexpression of miR-106b-5p can decrease the ability of wound healing and migration of nucleus pulposus cells,and decrease the cell activity(P<0.05).Down-regulating the expression level of mir-106b-5p can improve the expression ability of NP cells,such as scratch healing ability and migration ability(P<0.05).(7)Inhibition of miR-106b-5p can increase the DNA synthesis and promote the proliferation of NP cells.up-regulating the expression level of miR-106b-5p can improve the proliferation of NP cells(P<0.05).upregulating the expression level of miR-106b-5p can decreased the DNA synthesis and inhibited the proliferation of NP cells,and can cause S-phase arrest of cell cycle of NP cells.(8)Overexpression and inhibition of miR-106b-5p can increase the apoptosis rate of NP cells.However,Overexpression expression of miR-106b-5p can induce more apoptosisConclusion(1)This study confirmed that miR-106b-5p interferes with the expression level of ERK1/2 protein and the ERK1/2 signal pathway by targeting MAPK1,thus changing the expression level of ADAMTs-4 and ADAMTs-5,and destroying destroys the balance of synthesis and degradation of extracellular matrix of NP cells.(2)By regulating miR-106b-5p,the wound healing ability,migration ability,proliferation ability,apoptosis and cell activity of NP cells can be affected.(3)The molecular mechanism of miR-106b-5p regulate MAPK1-ERK1/2 signaling pathway has a high influence weight in the process of IDD.This study provides a candidate target with high efficacy in the treatment of IDD.