| Purpose : The biological function of LncRNA NEAT1 in tendinopathy was systematically investigated to verify the regulatory role of LncRNA NEAT1 in tendinopathy by affecting mi R-148a-3p,and to reveal the mechanism of NEAT1 in the occurrence and development of tendinopathy.To explore and verify the mechanism of LncRNA NEAT1/mi R-148a-3p/KLF6 signaling axis on the expression and secretion of TSP-4 and the regulation of angiogenesis in tendon cells by cell and in vivo experiments.Methods: LncRNA microarray technology was used to quantitatively analyze the expression profiles of LncRNA and m RNA in the sample tissues,which were obtained from tendinopathy patients and normal people without tendinopathy.The expression results were screened to identify the differential expression of these two genes in tendinopathy patients.At the same time,RT-q PCR technology was used to verify and analyze the chip detection results.Then GO analysis and Pathway analysis were carried out to predict the specific functions and possible signaling pathways of LncRNA target genes.At the same time,RT-q PCR was used to verify the expression of selected lncrnas and mrnas in tissues,and their correlation was detected.Starbase ce RNA prediction,dual luciferase reporter gene technology,RT-q PCR and Western blot were used to predict and verify the downstream signaling pathway of NEAT1.The integrity of the studied signaling pathway was further verified by NEAT1 gene overexpression and silencing.To establish a rat model of tendinopathy to verify the signaling pathway regulated by NEAT1 and its regulation on angiogenesis.Results: LncRNA and m RNA expression profile data showed that a total of 17 LncRNAs were found,of which 10 were up-regulated and the rest were down-regulated.Four differentially expressed LncRNAs and four m RNAs were randomly selected,and their expression trends were consistent with the results of LncRNA microarray detection.Through lncRNA-m RNA co-expression prediction analysis,GO analysis and Pathway analysis,it was found that LncRNA NEAT1 and TSP-4 m RNA were coexpressed and played a role in cell adhesion and angiogenesis.By RT-q PCR,it was found that LncRNA NEAT1 screened by LncRNA chip was abnormally up-regulated in tendinopathy tissues,while TSP-4 m RNA was abnormally high in tendinopathy tissues.Pearson test showed that NEAT1 and TSP-4 were positively correlated.The potential target gene of NEAT1 was predicted to be mi R-148a-3p by Starbase software.Abstract The detection of dual luciferase reporter gene showed that NEAT1 regulated mi R-148a-3p.Subsequently,by RT-q PCR,we found that the content of mi R-148a-3p in tendinopathy samples was significantly higher than that in the control group(P<0.01).Pearson test showed that NEAT1 was positively correlated with mi R-148a-3p(P<0.001).KLF6 was identified as a potential target of mi R-148a-3p by Target Scan prediction and confirmed by luciferase reporter detection.After transfection of mi R-148a-3p mimics and inhibitors in tendon cells,RT-q PCR was used to detect KLF6 m RNA expression,and it was found that KLF6 m RNA expression was significantly inhibited in mi R-148a-3p mimics group(P<0.01),while KLF6 m RNA expression was significantly increased in the mi R-148a-3p inhibitors group(P <0.01).In addition,RTq PCR was used to detect KLF6 expression in human tendon tissue samples,and it was found that KLF6 expression was significantly decreased in tendon tissue(P<0.01),and Pearson correlation analysis showed that the expression levels of mi R-148a-3p and KLF6 were negatively correlated in tendinopathy(P<0.001).Combined with literature reports,KLF6 can target the promoter binding TSP-4 gene.We transfected tendon cells with adenovirus to overexpress and silence NEAT1 gene and divided them into 6groups(NEAT1 overexpression group: NC group,NEAT1 group,NEAT1+mi Rinhibitor group;NEAT1 silencing group: NC group,NEAT1-shr NA group,NEAT1-shr NA +mi R-mimic group).RT-q PCR results showed that compared with the NC group,the m RNA expression level of KLF6 in the tendon cells of the NEAT1 overexpression group decreased.However,TSP-4 m RNA showed a higher expression level;In contrast,in the co-transfected mi R-148a-3p inhibitor group,compared with the single overexpression group,the expression level of KLF6 gene was increased,while the expression level of TSP-4 was decreased.Compared with the NC group,the expression level of KLF6 m RNA was higher in the NEAT1 group,but the expression level of TSP-4 m RNA decreased significantly.In contrast to the single silencing group,the expression level of KLF6 gene was decreased and the expression level of TSP-4was increased in the co-transfected mi R-148a-3p mimic group.Western blot results showed a similar trend.It was further verified that the LncRNA NEAT1/mi R-148a-3p/KLF6 regulatory axis mediates the signaling pathway of TSP-4 expression in tendon cells.An animal model of tendinopathy in rats was established and divided into two groups: NEAT1 overexpression group: normal saline group;Injection of NEAT1 overexpression virus group;Injection of NEAT1 overexpressing virus group and antiTSP-4 protein;NEAT1 silencing group: normal saline group;Injection of NEAT1-sh RNA virus group;Injection of NEAT1-sh RNA virus group + rh TSP-4 protein.RTq PCR results showed that the gene expression levels of mi R-148a-3p and TSP-4 in the overexpression of NEAT1 group and the overexpression of NEAT1+anti-TSP-4 group were significantly increased,and the expression level of KLF6 was decreased.However,in the expression of CD31 gene,the two experimental groups showed opposite trend.The expression trend of KLF6 and CD31 in the Western blot was similar to that of their genes,while the expression trend of TSP-4 was opposite to the protein level in the NEAT1+anti-TSP-4 overexpression group.However,the gene expression levels of mi R-148a-3p and TSP-4 were decreased and KLF6 was increased in both NEAT1 silencing group and NEAT1 silencing group +rh TSP-4 group.In terms of CD31 gene expression,the two experimental groups also showed opposite trend.Western blot results were similar.However,the expression of TSP-4 gene was opposite to the protein level due to the addition of rh TSP-4.The results of immunohistochemistry showed that KLF6 was significantly concentrated in both nuclei and cytoplasm.The expression of KLF6 in NEAT1 overexpression group and NEAT1 overexpression group and NEAT1 overexpression group +anti-TSP-4 was decreased compared with that in the control group,while the expression of KLF6 in NEAT1 silencing group and NEAT1 silencing group +rh TSP-4 group was opposite.TSP-4 was found to be distributed in cytosol and extracellular cells in immunohistochemical results,and the positive rate of overexpression of NEAT1 was higher than that of control group,while the opposite was true in NEAT1 silencing group.The results of H&E staining showed that the tendon tissue cells in the normal saline group had disordered arrangement,increased number of cells,non-uniform nucleoplasm,decreased extracellular matrix content,and increased angiogenesis.Compared with the control group,the content of extracellular matrix was decreased and angiogenesis was increased in NEAT1 overexpression group.The overexpression of NEAT1+anti-TSP-4 showed a significant decrease in angiogenesis.In NEAT1 silencing group,the cell arrangement was more orderly and angiogenesis was reduced.However,NEAT1 silencing group +rh TSP-4 group showed a relative increase in angiogenesis.By immunohistochemistry,we found that the positive rate of CD31 in NEAT1 overexpression group was higher than that in the control group,and the positive rate of CD31 was significantly decreased under the influence of anti-TSP-4.The positive rate of CD31 in NEAT1 silencing group was lower than that in control group,while the positive rate of CD31 in NEAT1 silencing+rhTSP-4 group was relatively higher.Conculsions: Axis of Lnc NEAT1/mi R-148a-3p/KLF6 could regulate TSP-4 from tenocyte to promote angiogenesis,which provided a new sight into the treatment of tendinopathy. |
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