| Objectives:To explore the differential expression profile of lncRNAs in the course of cartilage degeneration mediated by the SDF-1/CXCR4 axis,and to establish of lncRNA-miRNA-mRNA that played a regulatory role in ceRNA and identify candidate lncRNAs;to clarify the regulatory mechanism of SDF-1/CXCR4 axis-mediated OA articular cartilage degeneration.Methods:(1)The analysis of differential expression profiles of lncRNA in osteoarthritis chondrocytes induced by SDF-1. The cartilage tissue was remaining in total knee arthroplasty collect from 10 patients with knee OA for chondrocyte culture.The chondrocytes were randomly divided into an experimental group and a control group.The cell culture medium of the two groups was 10%fetal bovine serum and penicillin.Double antibody in high glucose DMEM medium.In the experimental group 100 μg/L SDF-1 was added to the culture medium,while the control group was not given any treatment.The chondrocytes of the two groups were routinely cultured for 48 h,and the lncRNA chip was screened and analyzed,verified by real-time quantitative PCR;RNA central software was used to predict miRNA to lncRNA binding,and the lncRNA which could target miR-146a-5p was obtained.(2)Key lncRNA screening from lncRNA/miRNA-146a-5p/mRNA co-expression network in OA chondrocytes.The human OA chondrocytes were cultured from in method 1,and the lncRNAs and miRNAs in OA chondrocytes induced by SDF-1 were screened based on high-throughput sequencing;The lncRNAs and miRNAs significantly related to the serious cartilage degeneration mediated by SDF-1/CXCR4 axis process were screend,the lncRNA/miR-146a-5p/CXCR4 co-expression network was established,and the key lncRNAs and miRNAs which were most likely to play a role in the regulation of ceRNAs were explored.The binding activity of miR-146a-5p and lncRNA was detected by dual luciferase reporter,and the mRNA expression of lncRNA,miR-146a-5p and CXCR4 in OA chondrocytes was detected by qRT-PCR,and the expression levels of key lncRNAs in OA chondrocytes were explored.The key lncRNAs which played the ceRNA regulatory role on miR-146a-5p in the SDF-1/CXCR4 axis-mediated OA process were identified.(3)NONHSAT248596.1 regulated miR-146a-5p/CXCR4 axis to mediate cartilage degeneration in OA Human knee joint primary OA chondrocytes cultured according to method 1 were randomly divided into seven groups,Group A was the blank control group;Group B was chondrocytes+lncRNA overexpression control plasmid+10Ong/mL and SDF-1 treated for 48 h;Group C chondrocytes+lncRNA overexpression plasmid+100ng/mL and SDF-1 treated for 48h;Group D:chondrocytes+lncRNA overexpression plasmid+miR-146a-5p mimic+100ng/mL and SDF-1 treated for 48h;Group E:chondrocytes+lncRNA interference control plasmid+100ng/mL and SDF-1 treated for 4 8h;Group F was chondrocytes+lncRNA interference plasmid+100ng/mL and SDF-1 treated for 48h;Group G was chondrocytes+lncRNA interference plasmid+miR-146a-5p inhibitor+100ng/mL and SDF-1 was treated for 48h;after routine culture in each group,qRT-PCR was performed to detect the mRNA expression of target lncRNA,miR-146a-5p,CXCR4,qRT-PCR was used to detect MMP-3,9,13 in chondrocytes.The mRNA expression of aggrecan and type Ⅱcollagen,the protein levels of aggrecan and type Ⅱ collagen were detected by Western blot;the dual luciferase experiment was used to verify the targeting binding relationship between miR-146a-5p and key lncRNA and CXCR4;MTS tested the proliferation of chondrocytes,and the apoptosis of chondrocytes was detected by flow cytometry.Results:1.① HE staining and immunofluorescence combined with laser confocal showed that the chondrocytes induced by SDF-1 had degenerative changes and the expression of CXCR4 increased.② High-throughput sequencing analysis showed differential changes in lncRNAs in OA chondrocytes added with SDF-1,a total of 52,741 lncRNAs had expression changes,of which 186 lncRNAs were significantly changed,88 lncRNAs were up-regulated,and 98 lncRNAs were down-regulated.The molecular functions of differential lncRNAs and their target genes were analyzed by GO enrichment.The Most molecular functions were enriched in receptor regulation and calcium ion transmembrane translocation signaling pathways;biological processes were enriched in type I interferon signaling pathway and ion transmembrane transporter activity.regulation;cellular composition is enriched in single organism metabolic processes,cellular intermolecular connections.KEGG is enriched in NF-kB signaling pathway,TGF-β signaling pathway,cytokine-cytokine receptor function,osteoclast differentiation,and ion signaling pathway.③46 lncRNAs that could target miR-146a-5p were predicted by RNA central software.2.① In the miRNA expression profile,miR-146a-5p was predicted to have a targeting relationship with CXCR4 through target gene prediction,and inhibited the effect of SDF-1;②The top 15 lncRNA target genes with up-regulated fold and down-regulated fold of differential expression were predicted among the lncRNAs of miR-146a-5p,a total of 4 lncRNAs were mutually targeted genes;qRT-PCR verification found that ENST0000055945,NONHSAT203408.1,NONHSAT248596.1,NONHSAT060379.2 had the same trend as the gene chip results,NONHSAT248596.1 and NONHSAT060379.2 were significantly up-regulated,which were involved in the regulation of cartilage degeneration in OA;③Dual luciferase reporter assay showed that:after co-transfection of NONHSAT060379.2 mimic,in wild-type NONHSAT060379.2 plasmid+miR-146a-5p mimic group,the relative luciferase activity in OA chondrocytes was inhibited,and the fluorescence intensity decreased,but the activity of the mutant plasmid did not change;while the fluorescence intensity of the other experimental groups did not change significantly;After co-transfection of NONHSAT248596.1,the relative luciferase activity in OA chondrocytes in the plasmid+miR-146a-5p mimic group was also inhibited,and the fluorescence intensity decreased,but the activity of the mutant plasmid did not change;while the fluorescence intensity of the other experimental groups did not change significantly.④ Through the tool database DIANA tools predicted the CXCR4 targeting interaction miRNAs,a total of 30 miRNAs were analyzed;the target lncRNAs of 30 miRNAs were re-predicted,of which 23 miRNA molecules had corresponding lncRNA interaction molecules,with a total of 491,Among them,there are 37 lncRNA molecules targeting hsa-miR-146a-5p molecules.⑤ The ceRNA network was constructed based on CXCR4,miRNA,and lncRNA molecules,and constructed the interaction network by Cytoscape.3.①CCK8 detection of chondrocyte proliferation and flow cytometry detection of chondrocyte apoptosis:lncRNA overexpression could regulate the SDF-1/CXCR4 axis by adsorbing miR-146a-5p,inhibited SDF-1-induced OA chondrocyte viability and proliferation,and promoted SDF-1 induced apoptosis of OA chondrocytes;②miR-146a-5p was the target of lncRNA,and CXCR4 was the direct target of miR-146a-5p.qRT-PCR validation found that lncRNA increased the expression of CXCR4 by sponge adsorption of miR-146a-5p,and observed low expression of miR-146a-5p and high expression of CXCR4.After overexpression of lncRNA NONHSAT248596.1,the expression level of miR-146a-5p was significantly decreased,the mRNA expression of MMPs was significantly increased,and the mRNA expression of COL Ⅱ and ACAN was inhibited;after inhibited the expression level of lncRNA NONHSAT248596.1,the result was the opposite.③Western blot detected the protein secretion levels of Aggrecan and Collagen Ⅱ in chondrocytes of each group,and the expression levels of Aggrecan and Collagen Ⅱ in the experimental group overexpressing lncRNA were significantly decreased;the expression levels of Aggrecan and Collagen Ⅱ in the experimental group overexpressing miR-146a-5p significantly increased.Conclusions:①The regulatory role of lncRNAs in OA cartilage degeneration induced by SDF-1 was confirmed obtained lncRNAs that may be related to OA,successfully constructed differential expression profiles of lncRNAs,and predicted 46 lncRNAs that are targeting related to miR-146a-5p;②The lncRNA-miRNA-mRNA network most likely to play a ceRNA regulatory role in the pathogenesis of OA,was explored and identified candidate lncRNAs NONHSAT248596.1 and miR-146a-5p were identified;③In SDF-1-induced OA chondrocytes,lncRNAs NONHSAT248596.1 and CXCR4 were up-regulated,while miR-146a-5p was down-regulated;the level of miR-146a-5p was negatively correlated with the level of lncRNA NONHSAT248596.1 or CXCR4;④lncRNA NONHS AT248596.1 acted as a molecular sponge for miR-146a-5p;lncRNA NONHSAT248596.1 was an endogenous sponge RNA that could directly bind to miR-146a-5p and inhibited its expression and activity;lncRNA NONHSAT248596.1 adsorbsed miR-146a-5p and regulated the SDF-1/CXCR4 axis which induced the expression of MMPs,COL II,ACAN and the degradation of ECM in OA chondrocytes. |
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