| BackgroundsOsteoarthritis(OA)is a chronic joint disease characterized by degeneration of articular cartilage,degenerative joint inflammation and secondary bone hyperplasia.This disease often occurs in the knee,hip,elbow and other small joints and involves the subchondral bone,articular cartilage,joint capsule,synovial membrane,ligament and surrounding muscles.It is common in the middle aged and elderly,women more than men.With the development of the elderly and obese,the incidence of OA has been gradually increasing.By the year 2020,it is estimated that the elderly population will account for about a quarter of the total population,and about half of those with chronic diseases at age 65 suffer from OA in developed countries.In China,the number of OA patients over 60 years old is about 130 million,accounting for 30%of the total elderly population.OA has been one of the joint diseases that affect human health,and it seriously threatens human health and quality of life.Interleukin-1(IL-1)is a typical pro-inflammatory cytokine,and exists in most cells.IL-1 family has three types,including IL-1?,IL-1? and IL-1Ra.IL-1? is the main material with biological activity,and can play a role outside the cell membrane.IL-1? is rare in normal synovial fluid,cartilage and synovial tissues.However,a large amount of IL-1? was detected in synovial fluid,cartilage and synovial tissues of OA.Further studies have found that IL-1? is involved in the occurrence of inflammatory responses and immune regulation,and the production and secretion of IL-1? in the human body are also related to the severity of the disease.microRNAs(miRNAs)regulate the target genes at the post-transcriptional level,and can participate in some important processes of biological life,such as cell proliferation,differentiation,apoptosis,metabolism and immune response.In addition,miRNAs are also linked to the occurrence and development of various diseases,such as hepatocellular carcinoma,endometrial cancer,cardiovascular disease,and esophageal cancer.In recent years,multiple lines of evidence suggest that miRNAs play an important role in OA.The study found that the abnormal expression levels of various miRNAs were found in OA patients,and these abnormal expressions were related to the apoptosis of chondrocytes and related genes.We could further study miRNAs with abnormal expression in OA,and it may provide a new direction for the treatment of OA.miR-126 was reported to be associated with many physiological processes,including vascular integrity and angiogenesis,inflammation,and many disease developments such as cancers.miR-126 has been described in several malignancies including lung cancers,prostate cancers,breast cancers,renal cancers,cervical cancers,thyroid cancers and etc.Furthermore,miR-126 has been implicated in regulating processes of tumor development and progression.However,it is still unclear about the miR-126 expression and potential molecular mechanism(s)in the development of OA.ObjectsThe aim of this study was to investigate the roles of miR-126 in the development of OA and the potential molecular mechanisms.Firstly,IL-1? was used to simulate inflammatory lesions in vitro.Then,we studied the role of miR-126 in the inflammatory lesions.Finally,the potential molecular mechanisms of miR-126 in OA were investigated.MethodsPart ?In this study,IL-1? was used to construct the OA model in vitro.Firstly,human chondrocyte cells CHON-001 were treated with different contents of IL-1?(0,0.1,2,5 and 10ng/ml,respectively),and cell viability was dectected by CCK-8 assay;The 5 and 10ng/ml of IL-1? were taken as effective concentrations for further detection of cell migration.The pictures of a specific position on the simulated scratch areas were taken by an inverted microscope at 0,6,12,24 and 48 h,and the relative width were calculated;Furthermore,flow cytometer was utilized to test cell apoptosis after 5 and 10ng/ml IL-1? treatment;Finally,apoptosis-related factors expressions,including cytochrome c(Cyto c),cleaved-caspase 3 and p53 and inflammatory cytokines expressions,including IL-6,IL-8 and tumour necrosis factor-?(TNF-?)were examined by Western blot or quantitative reverse transcriptase/polymerase chain reaction(qRT-PCR).Part ?In order to investigate the effect of miR-126 on OA,qRT-PCR was used to test the expressions of miR-126 in CHON-001 cells after 0,0.1,2,5 and lOng/ml IL-1?treatment;Then,miR-126 inhibitor was transfected into 10ng/ml IL-1? induced-cell,and the transfection efficiency was detected by qRT-PCR;Subsequently,CCK-8 assay,wound healing assay,flow cytometer and Western blot were used to measure cell viability,cell migration,cell apoptosis and the expressions of apoptosis-related factors,respectively.Then,the expressions of inflammatory cytokines,including IL-6,IL-8 and TNF-? in CHON-001 cells after miR-126 inhibitor transfection were monitored by qRT-PCR;To further confirm the effect of miR-126 inhibitor on IL-1?induced inflammatory response in CHON-001 cells,we also employed the different concentrations of IL-1?(0.1,2 and 5ng/ml)to stimulate CHON-001 cells and assessed the expression levels of inflammatory factors with or without the presence of miR-126 inhibitor by Western blot and qRT-PCR.Part ?To assess the potential mechanisms that miR-126 affects OA,Western blot was performed to assess the B-cell lymphoma-2(Bcl-2)expressions in CHON-001 cells after 5ng/ml and 10ng/ml IL-1? administration.Then,miR-126 inhibitor was transfected into CHON-001 cell after 10ng/ml IL-1? treatment,and the expression of miR-126 was detected by Western blot;In order to study the effect of miR-126 on mitogen activated protein kinase(MAPK)/c-Jun N terminal kinases(JNK)signaling pathway,si-Bcl-2 and miR-126 inhibitor were co-transfected into CHON-001 cell after 10ng/ml IL-1? treatment,and the expressions of Bcl-2,p-p38 MAPK,p38 MAPK,p-JNK and JNK were also detected by Western blot.ResultsPart ?Cell viability assay results suggested that after treatment with 0.1 and 2ng/ml IL-1?,there are no significant differences compared with control group.However,after treatment with 5 and 10ng/ml IL-1?,cell viability was decreased(P<0.01).Meanwhile,at the concentration of lOng/ml IL-1?,the cell viability was the lowest;The results of wound healing analysis showed that with the time increases,the wound width was gradually decreased.The relative wound width of 5 and 10ng/ml IL-1? administrated groups were higher than control group that without IL-1?treatment.Besides,The relative wound width of 10ng/ml IL-1? administrated group was higher than 5ng/ml IL-1? administrated group;The apoptosis assay results showed that IL-1? treatment remarkably increased relative numbers of apoptotic cells(P<0.01 or P<0.001),and the numbers of apoptotic cells after 10ng/ml IL-1?treatment were higher than 5ng/ml IL-1? treatment;Western blot analysis results also showed that Cyto c,cleaved-Caspase 3 and p53 expressions were both increased in IL-1? administrated groups compared with control group.The protein expressions of IL-6,IL-8 and TNF-? were all increased after been administrated with IL-1?,and the qRT-PCR results were the same as the Western blot(P<0.01).Both the expression levels of apoptosis-related factors and inflammatory,cytokines in 10ng/ml IL-1?treatment cells were higher than 5ng/ml IL-1? treatment cells.Part ?The qPCR analysis results showed that the expression of miR-126 was increased in CHON-001 cells after 5ng/ml and 10ng/ml IL-1? treatment(P<0.01 or P<0.001),and at the concentration of 10ng/ml IL-1?,the expression of miR-126 was the highest.In the further study,miR-126 inhibitor was transfected into CHON-001 cell after 10ng/ml IL-1? treatment,and the expression of miR-126 was obviously decreased after miR-126 inhibitor transfection compared with negative control group even if with the presence of 10ng/ml IL-1?(P<0.001).For cell viability,the results showed that after IL-1? inhibited cell viability of CHON-001,miR-126 inhibitor transfection promoted cell viability compared with IL-1?administration alone(P<0.05).The wound healing assay results also showed that wound width was decreased by miR-126 inhibitor transfection compared with IL-1?administration alone.The relative apoptotic cells were decreased by miR-126 inhibition transfection after IL-1? administration(P<0.01).Meanwhile,the expressions of Cyto c,cleaved-Caspase 3 and p53 were all decreased by miR-126 inhibition transfection.We also measured the effect of miR-126 inhibitor on inflammatory cytokines,and Western blot results showed that miR-126 inhibitor transfection suppressed IL-1?-induced increasing effect on expressions of IL-6,IL-8 and TNF-a.qRT-PCR results were the same as Western blot results(P<0.05).Finally,we also employed the different concentrations of IL-1?(0.1,2,5ng/ml)to stimulate CHON-001 cells,and the expression levels of inflammatory factors were assessed by Western blot and qRT-PCR.The results showed that the expressions of pro-inflammatory cytokines were up-regulated by the treatment of IL-1? at the dosage of 0.1ng/mL(P<0.05),However,the up-regulated pro-inflammatory factors were inhibited by the transfection of miR-126 inhibitor(P<0.05).Similar results were observed in the treatment of IL-1? with the dosage of 2ng/ml and 5ng/ml(P<0.05 or P<0.01).Part ?Western blot was performed to assess the Bcl-2 expression in CHON-001 cells after IL-1? administration.Results showed that IL-1? induction decreased Bcl-2 expressions(P<0.05 or P<0.01),and the expression of Bcl-2 was the lowest by the treatment of IL-1? at the dosage of 10ng/ml.Whereas,miR-126 inhibitor transfection in CHON-001 cells reversed the effect of IL-1? on Bcl-2 expression,as significantly increased Bcl-2 expression levels compared with IL-1? alone.Moreover,IL-1?increased expressions of phosphorylated(p-)MAPK and p-JNK,miR-126 inhibitor transfection decreased these expressions.In IL-1(3-induced CHON-001 cells,miR-126 inhibitor + si-Bcl-2 transfection increased expressions of p-p38 MAPK and p-JNK.ConclusionsIn conclusion,in this study,we simulated human inflammatory chondrocytes by using IL-1? in vitro,and found that the up-regulated miR-126 might be involved in the regulation of inflammatory response.Suppression of miR-126 could inhibit MAPK/JNK signaling pathway activation via increasing Bcl-2 expression.It hits that more animal or clinical researches in vivo are still necessary for the better understanding of pathogenesis and improving clinical therapy of OA. |
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