| Background:Osteoarthritis(OA)is the most common type of arthritis.It is a disability,delayed and degenerative disease characterized by the disappearance of inflammation of articular cartilage and synovium,resulting in stiffness,swelling,pain and loss of mobility.From an epidemiological point of view,osteoarthritis affects about half of the world’s people over 65 years of age and is considered to be one of the leading causes of disability.Although artificial joint replacement can effectively restore joint function and improve the quality of life in patients with advanced osteoarthritis,it is difficult to diagnose early disease due to the lack of specific symptoms or signs.Therefore,new biomarkers are urgently needed to diagnose and treat early osteoarthritis.Osteoarthritis was originally thought to be a wear and tear disease unrelated to inflammation,but now due to the emergence of molecular biology,it is being redefined as a complex,multifactorial disease.During the development of osteoarthritis,cartilage damage is related to the up-regulation of inflammatory genes,which can induce cartilage damage through multiple signaling pathways.In addition,inflammatory factors can also increase the expression of proteases,including MMP and integrin,which can degrade the extracellular matrix of articular cartilage and aggravate the disease.In addition,chondrocyte is an important component of articular cartilage.Normally,the metabolism of chondrocyte is very active,which plays an important role in maintaining the normal results and function of cartilage.When inflammatory factors are transmitted to transcription factors through intracellular signaling pathways such as MAPK and NF-Kappa B,chondrocyte differentiation becomes a fibrocyte-like phenotype,which reduces the number of cells and induces fibrosis of surrounding tissues.In conclusion,the occurrence of osteoarthritis involves many changes in molecular and signal pathways,and the specific mechanism has not yet been elucidated.Therefore,it is necessary to explore the molecular mechanism of the occurrence and progress of osteoarthritis in order to find new effective treatment strategies for patients.Long non-coding RNAs(lncRNAs)are endogenous RNA with a length greater than 200 nucleotides.It has been found that abnormal expression of lncRNA plays an important role in regulating various inflammatory diseases,including osteoarthritis.The expression profile analysis showed that 4067 up-regulated lncRNA and 2231 down-regulated lncRNA in osteoarthritis patients,suggesting that IncRNA may play an important role in the pathogenesis of osteoarthritis and may be a potential biomarker or therapeutic target.Recent studies have shown that IncRNA plays an important regulatory role in cartilage injury and can promote the degradation of extracellular matrix of chondrocyte in osteoarthritis.For example,lncRNA CIR can promote the degradation of extracellular matrix of chondrocytes by binding to microRNA-27b.Another study revealed that lncRNA-UFC1 promotes osteoarthritis cell proliferation and reduces chondrocyte apoptosis by regulating microRNA-34a.HULC is an important lncRNA,which can regulate cell apoptosis and play an important role in carcinogenesis of many cancers.Increased expression of HULC in cells can induce cell proliferation and tumor growth,and lead to the down-regulation of tumor suppressor p18.In addition,new evidence suggests that the down-regulation of HULC may be related to chondrocyte death during osteoarthritis.However,the expression,role and regulatory mechanism of HULC in osteoarthritis are largely unknown.Research purposes:The purpose of this study was to investigate the regulatory effect of HULC on osteoarthritis.ATDC5 is a mouse chondrogenic cell line,which is often used to construct osteoarthritis inflammation injury model.Therefore,we use TNF-alpha to treat ATDC5 cells and construct an inflammation injury model to simulate the process of osteoarthritis.Then the effects of HULC on cell proliferation,apoptosis and pro-inflammatory cytokines were measured in ATDC5 cells treated with TNF-alpha,and the signal pathways of NF-kappa B and p38MAPK were studied to reveal their potential molecular mechanisms.Methods:Twenty osteoarthritis patients were enrolled in this study.The expression levels of pro-inflammatory cytokines IL-6,IL-8,MCP-1 and HULC in cartilage tissues of osteoarthritis patients and normal cartilage tissues were detected by real-time quantitative PCR.To construct an inflammation injury model of ATDC5 cells induced by TNF-a and study the regulatory effect of HULC on osteoarthritis.ATDC5 cells were treated with different concentrations of TNF-alpha(0,10,20 and 40 ng/ml).Cell viability was measured by CCK-8 and IC50 concentration was calculated for subsequent experiments.Then the cells were treated with 20 ng/ml TNF-a.The expression of apoptosis-related factors Caspase-3 and Caspase-9 was detected by flow cytometry and Western blot to evaluate apoptosis.In addition,quantitative PCR,Western blot and enzyme-linked immunosorbent assay were used to detect the expression and secretion of pro-inflammatory cytokines IL-6,IL-8 and MCP-1.In order to further study the effect of HULC on ATDC5 cells damaged by TNF-a,ATDC5 cells were treated with different concentrations of TNF-a,and the relative expression of HULC in ATDC5 cells was determined by quantitative PCR.ATDC5 cells were transfected with pc-HULC and sh-HULC to change the expression of HULC,and then the changes of cell viability,apoptosis and pro-inflammatory cytokines after HULC overexpression and interference were detected.The relative expression of miR-101 in ATDC5 and human primary chondrocyte was detected after HULC overexpression and inhibition.Targetscan tool was used to predict the possible action sites of HULC and miR-101.The 3’-UTR binding sequence of HULC gene was mutated.The mutant and wild-type HULC were cloned into fluorescent vector and co-transfected with miR-101.The direct interaction between HULC and miR-101 was confirmed by double luciferase reporter gene analysis.This is the case.Subsequently,we used biotin-avidin Pull-down system to study the interaction between the two:biotin-labeled wild-type miR-101 gene was transfected into ATDC5 cells,and avidin mediator was incubated with cell lysate to specifically adsorb miR-101 and the genes interacting with it.Quantitative PCR was used to determine the enrichment and expression of HULC.The reverse Pull-down experiment was carried out by using the biolabeled HALC probe.Next,we use anti-Ago2 antibody to carry out RNA immunoprecipitation experiment,and further explore the interaction between HULC and miR-101.miR-101 mimics and pcHULC were simultaneously transfected into ATDC5 cells to overexpress both miR-101 and HULC.RT-qPCR was used to detect the transfection efficiency.The ATDC5 cells were treated with TNF-a to detect whether the overexpression of miR-101 could affect the cell viability,apoptosis and the expression of IL-6,IL-8 and MCP-1 induced by HULC.miR-101 inhibitor was used to transfect cells to detect the changes of cell function,including cell viability,apoptosis,expression of active Caspase-3 and active Caspase-9,and expression of pro-inflammatory cytokines.Finally,we detected the phosphorylation levels of the important components of the NF-kappa B and p38/MAPK signaling pathways by Western blot,and evaluated the effects of HULC and miR-101 on the NF-kappa B and p38/MAPK signaling pathways in TNF-alpha-treated ATDC5 cells.Results:Quantitative PCR results showed that the expression of IL-6,IL-8 and MCP-1 in OA cartilage tissues of patients with arthritis was significantly higher than that in normal cartilage tissues(P<0.01 or P<0.001),and the expression of HULC in cartilage tissues of patients with arthritis was significantly lower(p<0.001).TNF-a treatment of ATDC5 cells significantly reduced HULC expression and cell viability,and IC50 concentration was about 20 ng/ml.20 ng/ml TNF-a was used to treat ATDC5 cells for subsequent experiments.It was found that the apoptotic level was significantly increased,the expression of active Caspase-3 and Caspase-9 was increased,and the expression and secretion of IL-6,IL-8 and MCP-1 were significantly increased at the levels of gene and protein.These results indicated that TNF-a-induced inflammation injury model of ATDC5 cells.Successful construction.Overexpression in HULC can significantly increase the viability of ATDC5 cells treated with TNF-alpha and inhibit cell apoptosis,and induce the down-regulation of Caspase-3/-9,and inhibit the expression of IL-6,IL-8 and MCP-1 at the levels of gene and protein,as well as the extracellular secretion level;interfere with the expression of HULC on TNF-alpha-induced cell damage The effect on cells was contrary to that of overexpression(p<0.01 or P<0.001).Similarly,HULC has similar results in primary chondrocytes,suggesting that HULC can alleviate inflammatory damage of chondrocytes induced by TNF-a.Compared with the control group,HULC overexpression significantly decreased the expression level of microRNA-101(p<0.05),while inhibiting HULC expression significantly increased the expression level of microRNA-101(P<0.001).We also found that pc-HULC and sh-HULC transfection could also significantly alter the expression of miR-101 in human primary chondrocytes.It was predicted that HULC could bind to miR-101 directly.Dule luciferase reporter gene analysis showed that miR-101 could inhibit the activity of luciferase in wild-type HULC transfection group,but there was no significant change in mutant HULC group.Pull-down experiment showed that the concentration of HULC increased after adsorbing avidin(p<0.001),but the mutant miR-101 could not change the amount of HULC,suggesting a direct interaction between HULC and mir-101,and the reverse Pull-down experiment also obtained similar results.In addition,in ATDC5 cells with Ago2 expression,HULC and miR-101 enriched with anti-Ago2 antibody were significantly up-regulated(p<0.01).These results suggest that HULC plays a role in ATDC5 cells by binding to miR-101,and there may be mutual inhibition between HULC and miR-101.In ATDC5 cells treated with TNF-alpha,over-expression of microRNA-101 significantly reversed the effects of HULC on cell viability,apoptosis and expression of IL-6,IL-8 and MCP-1(p<0.05,P<0.01 or P<0.001).These data suggest that over-expression of microRNA-101 inhibits the protective effect of HULC on ATDC5 cells damaged by TNF-alpha.However,when the expression of ATDC5 cells was inhibited by miR-101 inhibitor,the activity of ATDC5 cells treated with TNF-alpha increased significantly,apoptosis decreased,and the expression of active Caspase-3 and active Caspase-9 decreased(P<0.01).In addition,the expression of IL-6,IL-8 and MCP-1 was inhibited by miR-101 inhibitor(p<0.05 or P<0.01).These results suggest that miR-101 inhibitor has protective effect on TNF-α-treated ATDC5 cells.After TNF-α treatment,the phosphorylation levels of I-kappaB-α,p65,p38MAPK,ERK1/2 and JNK1/2 were significantly increased(P<0.001).Overexpression of HULC can down-regulate the phosphorylation of these proteins after TNF-alpha treatment.However,over-expression of microRNA-101 reversed the inhibitory effects of HULC on phosphorylation of I-kappa Ba,p65,p38MAPK,ERK1/2 and JNK1/2(p<0.01 or P<0.001).There were no significant changes in t-I-kappa B alpha,t-p65,t-p38MAPK,t-ERK1/2 and t-JNK1/2 in the non-phosphorylated state in different groups.These data suggest that HULC can protect cells from inflammatory damage by down-regulating the signal transduction pathways of NF-kappa B and p38MAPK in TNF-alpha-treated ATDC5 cells.Conclusion:In conclusion,our results suggest that lncRNA HULC can protect ATDC5 cells from TNF-alpha-induced inflammation by interacting with and inhibiting the expression of microRNA-101,inhibiting the activation of NF-kappa B and MAPK signaling pathways.These results suggest that lncRNA HULC may be an important regulator in the pathogenesis of osteoarthritis and a new biomarker for the diagnosis and treatment of osteoarthritis. |
PDF Full Text Request