CircRNA-MSR Regulates LPS-Induced C28/I2 Chondrocyte Injury Through MiR-643/MAP2K6 Signaling Pathway

Background Osteoarthritis(OA)is a chronic joint disease characterized by articular cartilage degeneration and secondary osteophyte formation,mainly involving the weight-bearing joints.The etiology of OA is related to many risk factors,however,the Pathophysiology mechanism of osteoarthritis is still not completely clear,so early can not effectively prevent the occurrence of osteoarthritis,surgical intervention is mainly targeted at patients with advanced osteoarthritis to give surgical treatment,to the patient to solve the pain,but also to the patient burden.Previous studies have confirmed the role of circRNA in osteoarthritis,but the role of circRNA-msr in osteoarthritis is unclear.Objective 1.By building the OA model of cartilage cells and testing the expressing of circRNA-MSR.2.Prediction of micro RNA that are targeted to circRNA-MSR.3.To explore the mechanism of the role of circRNA-MSR in the chondrocyte model of OA.Method Human chondrocytes C28/I2 were cultured and treated with lipopolysaccharide(LPS)to establish an osteoarthritis chondrocyte model.The m RNA and protein levels were detected by q RT-PCR or Western Blot.Cell viability was measured by MTT assay.Cell apoptosis was detected by Flow cytometry.Tumor necrosis factor(TNF-α)、 IL-1β and IL-6 levels were detected by ELISA.Determination of the correlation between circRNA-MSR and miR-643 by pull-down experiment.The targeting relationship between miR-643 and circRNA-MSR or MAP2K6 was detected by double luciferase reporter gene assay.Location detection of cirna-msr and miR-643 using RNA Fluorescence in situ hybridization.Results 1.In the groups stimulated by different concentrations of LPS,the expression level of circRNA-MSR increased in a dose effect with the concentration of LPS;MTT assay showed that the relative cell activity of C28/I2 cells stimulated by different concentrations of LPS was significantly lower than that before stimulation,and the decrease level was dose-dependent with the concentration of LPS;Flow cytometry showed that the apoptosis level of C28 / I2 cells stimulated by different concentrations of LPS increased significantly,and the increased level was dose-dependent with the concentration of LPS induced apoptosis。2.After knock down circRNA-MSR,LPS induced C28/I2 cells increased in activity and decreased in apoptosis rate,while IL-6,IL-1β decreased.The expression of BAX,cleaved-caspase-3 and MMP13 decreased,but the expression of BCL-2 and aggrecan and collagen II increased.3.The expression level of miR-643 decreased with the increase of LPS-induced concentration,but increased when low circRNA-MSR was knocked down in LPS-induced C28/I2 cells,the expression of circRNA-MSR and miR-643 in C28/i2 cells was up-or down-regulated,and the RNA pull down test with the probes of biotin-labelled circRNA-MSR showed that miR-643 and circRNA-MSR interacted double luciferase report system shows that WTcircRNA-MSR and miR-643 mimic co-expression cells in Luc/RLUC group was significantly lower than that in the control group,which indicated that they formed a complex on the transcription factors,and there was an interaction between circRNA-MSR and miR-643;The co-localization of circRNA-MSR and miR-643 in the subcellular structure(cytoplasm)was found in the RNA Fluorescence in situ hybridization experiment,which directly verified the interaction between the two.4.Overexpression of miR-643,increased activity of C28/I2 cells induced by LPS,decreased apoptosis rate,decreased expression of IL-6,IL-1β,TNF-α,decreased expression of BAX,cleaved-caspase-3,MMP13,the expression of BCL-2 and collagen II,which are important components of articular cartilage,were increased.5.With the increase of LPS concentration,the expression of MAP2K6 m RNA/protein decreased,and the overexpression of miR-643 in C28/I2 cells induced by LPS decreased.The double luciferase system indicated that Luc/Rluc co-expression of WT-MAP2K6 and miR-643 mimic cells was significantly lower than that of the control group.6.Knock down circRNA-MSR/over-expression MAP2K6,knock down circRNA-MSR and inhibition of miR-643 expression in LPS-induced C28/I2 cells were used to detect the change of miR-643 expression level.Knock down circRNA-MSR and miR-643 suppression were used to detect the change of MAP2K6 m RNA expression,the results suggest that the expression level of circRNA-MSR can directly affect miR-643(inhibiting miR-643),that means circRNA is the upstream molecule of miR-643,while MAP2K6 has no obvious effect on miR-643,MAP2K6 is not upstream of miR-643.By inhibiting miR-643 and promoting MAP2K6 expression,circRNA-MSR can inhibit relative cell activity,induce cell inflammation,inhibit proliferation,promote extracellular matrix and promote apoptosis,MAP2K6 is the effector gene(protein)of this pathway.Conclusion Circ RNA-MSR Regulates LPS-Induced C28/I2 Chondrocyte Injury through miR-643/MAP2K6 Signaling Pathway.