MiR-17-5p Regulates Fibroblast Osteogenic Differentiation Targeted ANKH In Ankylosing Spondylosis

Objective: This research is to investigate the expression of miR-17-5p in articular capsule tissues of ankylosing spondylitis(AS)with normal tissues as control,and then to study the biological mechanism of miR-17-5p regulates Fibroblast osteogenic differentiation Targeted ANKH in AS,which may be the new therapeutic targets of AS and provide a new theoretical basis.Method : Before the start of this experiment,the ethical review was approved by the ethics committee of the First Affiliated Hospital of Guangxi Medical University.According to the Helsinki declaration,after the informed consent of the patients and their families,a total of 20 hip joint capsule tissues were obtained from AS of the Department of orthopedics of the First Affiliated Hospital of Guangxi Medical University,and 18 of the same parts were served as control.All cases in the experimental group were diagnosed according to the 1984 New York Standard.There were no other unusual conditions,such as rheumatism.The patients in the control group were healthy,without ankylosing spondylitis and rheumatoid disorders.The tissues were stained with BCIP/NBT,alizarin red,compared differences of ectopic ossification of two groups of ligament tissue;detect the expression level of mi R-17-5p,ANKH and osteogenic related gene mRNA by real-time fluorescent quantitative PCR,and compared with the control group,to determine the correlation between miR-17-5p and ANKH with ankylosing spondylitis,and the negative correlation of the expression of ANKH,Runx2,Col1a1;protein level differences in ligament tissue were detected by Western-Blot.apply the method of tissue cultured fibroblasts,HE,BCIP/NBT,alizarin red staining,compared with two groups of cell morphology and mineralization of osteoblast differentiation;detected the expression of miR-17-5p,ANKH and osteogenic related gene mRNA in two groups of cells by real-time fluorescence quantitative PCR.The differences of protein expressions of two groups of cells in ANKH,Runx2 Col1a1 were detect by Western-Blot.TargetScan,Miranda and other online prediction software were used to analyze the action sites on miR-17-5p and ANKH 3 'UTR.The ANKH 3 'UTR sequence was cloned by molecular biology method,and the bioinformatics analysis was carried out to determine the binding site of miR-17-5p.The ANKH 3 'UTR sequence inserted into luciferase reporter gene downstream,while the miR-17-5p binding site sequence mutations by dual luciferase reporter assay,mi R-17-5p verification by acting on the ANKH 3' binding sites on the UTR,the expression and regulation of ANKH gene.Using control technology of lentiviral mediated,miR-17-5p mimic,miR-17-5p inhibitor and constructed miR-17-5p control lentiviral expression vector packaging lentivirus infection of primary fibroblasts can long-term in cells stably expressing miR-17-5p mimic,miR-17-5p inhibitor and miR-17-5p control.Then the expressions of mRNA and protein of ANKH,Runx2,COL1a1 detected at regular intervals,and calcified nodules staining was used to detect differences in osteogenic differentiation,further confirmed that mi R-17-5p directly targeted ANKH and participate in the occurrence of calcification.The results were analyzed with SPSS 22 software,and the difference of P < 0.05 was considered statistically significant.Result: BCIP/NBT staining showed that AS group was dark blue,while the control group did not have obvious coloring;Alizarin Red staining of calcified nodules found in AS group,while the control group had no calcified nodules;the mRNA expression of mi R-17-5p,Col1a1,ALP,Runx2,Bmp2,BGP were high in AS tissues significantly increased compared with the control group.And the expression of ANKH mRNA was low in AS;Western blot showed that the protein expression of Col1a1 and Runx2 in AS were higher than the control group,while the expression of ANKH was significantly lower than the control group;And alkaline phosphatase activity in AS group was significantly higher than the control group.We successful isolated and cultured fibroblasts From the control and AS,there was no difference in appearance,but Alizarin Red staining,calcified nodules of AS was higher than control,BCIP/ NBT staining were dark blue in AS,while the control did not stain;The expression of miR-17-5p,Col1a1,ALP,Runx2,Bmp2,BGP mRNA of AS was significantly higher than the control,but the expression of ANKH mRNA were low;The expression of Col1a1,Runx2 protein was significantly higher than control group and the expression of ANKH protein was low;Alkaline phosphatase activity in fibroblasts from AS was significantly higher than the control group.TargetScan,Miranda and other online prediction software are forecast to ANKH is the target gene of miR-17-5p,Luciferase reporter gene vector of wild type ANKH and mutant ANKH 3 'UTR 3' UTR construction and identification of successful co-transfection of miR-17-5p mimics and P MIR/ANKH reporter gene,luciferase activity decreased significantly,while the co-transfection of miR-17-5p inhibitor and P MIR/ANKH reporter gene,luciferase activity changed little,the signal intensity did not affect cotransfection of miR-17-5p mimics or inhibitors P and MIR/ANKH/mut report gene vector.After increased the expression of miR-17-5p in fibroblasts,the expression of mRNA and protein of ANKH decreased significantly,expression of Col1a1 and Runx2 mRNA and protein were increased,and down-regulation of miR-17-5p expression in fiber cells,the expression of mRNA and protein of ANKH were significantly up-regulated,the expression of Col1a1 and Runx2 the mRNA and protein were decreased.Alkaline phosphatase activity was detected with BCIP/NBT and Alizarin Red staining results were consistent with the level of mi R-17-5p,the differences were statistically significant.Conclusion: The ligaments of hip joint capsule of AS has obvious osteogenic tendency compared with the control.miR-17-5p,Col1a1,ALP,Runx2,Bmp2,BGP and mRNA expression were significantly higher in the AS compared with the control.Col1a1 and Runx2 protein expression significantly higher in the AS ligament as well as the normal fibroblasts compared with the control.ANKH mRNA and protein expression in AS was lower than the control;miR-17-5p down-regulated the expression of ANKH in osteogenic differentiation of fibroblasts.Up-regulated expression of mi R-17-5p could promote fibroblast osteogenic differentiation,and down-regulated expression of miR-17-5p could inhibited fibroblast osteogenic differentiation.This because of heterotopic osteogenesis of fibroblasts around the articular ligament of ankylosing spondylitis.ANKH may become a potential target for treatment of ankylosing spondylitis.