Knockdown Of YAP And Knockdown Of TAZ Inhibit The Migration And Invasion Of RA-FLS By Regulating Autophagy

Objective:In the present study,we aimed to explore the effects of YAP/TAZ,the effectors of Hippo signaling pathway,on the migration and invasion of fibroblast-like synoviocytes(FLS)in rheumatoid arthritis(RA),and to preliminarily clarify knockdown of YAP and knockdown of TAZ may affect the migration and invasion of RA-by regulating autophagy.Methods:1.Rheumatoid arthritis fibroblast-like synoviocytes(RA-FLS)with good growth condition were selected and were verified by flow cytometry after incubating with FITC labeled m anti-human CD90 antibody and PE labeled mouse anti-human CD55 antibody.2.Western Blot was used to detect the expressions of YAP and TAZ in RA-FLS(RA-FLS group)and normal FLS(NC-FLS group),and the differences between two groups analyzed.3.Lentivirus vectors were constructed for knockdown of YAP and knockdown of TAZ,lentivirus infected cells after lentivirus packaging.Puromycin was further used to screen and obtain cell models of stable knockdown of YAP(sh-YAP group),stable knockdown of TAZ(sh-TAZ group)and empty virus control group(sn-C group),and RT-qPCR and Western Blot were performed for verification.4.Wound healing assay,transwell cell migration and invasion assays were conducted to explore the effects of knockdown of YAP and knockdown of TAZ on the migration and invasion of RA-FLS.5.Western Blot was used to detect the effects of knockdown of YAP and knockdown of TAZ on the expressions of LC3B and p62 as the autophagy related proteins in RA-FLS.6.Imrmunofluorescence staining assay was performed to investigate the effects of knockdown of YAP and knockdown of TAZ on the formation of LC3B puncta in RA-FLS.7.Wound healing assay,transwell cell migration and invasion assays were carried out to observe the effects of autophagy inhibitors CQ and 3-MA on the migration and invasion of RA-FLS after respective knockdown of YAP and knockdown of TAZ.8.Western Blot was employed to measure the effects of autophagy inhibitors CQ and 3-MA on the expressions of LC3B and p62 in RA-FLS after respective knockdown of YAP and knockdown of TAZ.Results:The positive rate of MH7A expressing CD90 and CD55 were 96.7±1.18%(n=3)verified by flow cytometry,and the cell line was identified as RA-FLS,which could be used for following experiments.In the present study,Western Blot was first used to detect the expressions of YAP and TAZ in RA-FLS and NC-FLS.The results manifested that the expressions of YAP and TAZ in RA-FLS were significantly increased indicating that YAP and TAZ were highly expressed in RA-FLS.Then,Lentivirus vectors were constructed for knockdown of YAP and knockdown of TAZ,and lentivirus infected cells after lentivirus packaging.Puromycin was further used to screen and obtain cell models of stable knockdown of YAP(sh-YAP group),stable knockdown of TAZ(sh-TAZ group)and empty virus control group(sh-C group),and RT-qPCR and Western Blot were performed for verification.It was showed that the expressions of YAP in RA-FLS of the sh-YAP group and TAZ in RA-FLS of the sh-TAZ group were significantly declined respectively at the mRNA and protein levels.At the same time,RT-qPCR was used to detect the mRNA levels of CTGF and CYR61,which are recognized as downstream target genes regulated by YAP/TAZ transcription currently.The results manifested that the mRNA levels of CTGF and CYR61 in RA-FLS of sh-YAP group and sh-TAZ group were also significantly reduced.It was suggested that cell models were successfully constructed and could be used for further experiments.Wound healing assay and Transwell cell migration and invasion assays were used to observe the effects of stable knockdown of YAP(sh-YAP group)and stable knockdown of TAZ(sh-TAZ group)on the migration and invasion of RA-FLS.The result of wound healing assay showed that the wound healing of the sh-YAP group and the sh-TAZ group slowed down significantly.The result of transwell cell migration assay manifested that the numbers of RA-FLS migration in the sh-YAP group and the sh-TAZ group were significantly reduced,which was consistent with the results of wound healing assay.The result from transwell cell invasion assay indicated that the numbers of RA-FLS migration in the sh-YAP group and the sh-TAZ group were significantly decreased.The results suggested that knockdown of YAP and knockdown of TAZ could inhibit the migration and invasion of RA-FLS.It was further studied whether stable knockdown of YAP(sh-YAP group)and stable knockdown of TAZ(sh-TAZ group)would affect the autophagy of RA-FLS.Western Blot results showed that expression levels of LC3B-II in RA-FLS of the sh-YAP group and the sh-TAZ group were markedly increased,but expressions of p62 were obviously decreased.The results of immunofluorescence staining indicated that formations of LC3B puncta were increased in RA-FLS of the sh-YAP group and the sh-TAZ group.The results implied that knockdown of YAP and knockdown of TAZ could promote the autophagy of RA-FLS.To clarify whether the effects of stable knockdown of YAP(sh-YAP group)and stable knockdown of TAZ(sh-TAZ group)on the migration and invasion of RA-FLS could be achieved by regulating autophagy,the effects of autophagy inhibitors CQ and 3-MA on RA-FLS in sh-YAP group and sh-TAZ group were further investigated.Wound healing assay,transwell cell migration and invasion assays results demonstrated that,compared with the sh-YAP group,the migration and invasion of RA-FLS from the sh-YAP+CQ group and the sh-YAP+3-MA group were obviously enhanced;compared with the sh-TAZ group,the migration and invasion of RA-FLS from the sh-TAZ+CQ group and the sh-TAZ+3-MA group were also significantly enhanced.Western Blot results manifested that,compared with the sh-YAP group,the expressions of LC3B-II and p62 in RA-FLS of the sh-YAP+CQ group increased significantly;compared with the sh-TAZ group,the expressions of LC3B-II and p62 in RA-FLS of the sh-TAZ+CQ group enhanced obviously;compared to the sh-YAP group,the LC3B-II expression in RA-FLS of sh-YAP+3-MA group decreased slightly,but the expression of p62 increased significantly;compared with sh-TAZ group,the LC3B-II expression in RA-FLS of sh-TAZ+3-MA group declined slightly,but the expression of p62 elevated obviously.It was suggested that inhibiting the RA-FLS autophagy could reverse the effects of knockdown of YAP and knockdown of TAZ on the migration and invasion of RA-FLS.Conclusion:YAP and TAZ were highly expressed in RA-FLS,and knockdown of YAP and knockdown of TAZ could significantly inhibit the migration and invasion of RA-FLS.Meanwhile,knockdown of YAP and knockdown of TAZ could promote the autophagy of RA-FLS,and inhibiting the autophagy of RA-FLS could reverse the effects of knockdown of YAP and knockdown of TAZ on the migration and invasion of RA-FLS.The results revealed that knockdown of YAP and knockdown of TAZ could inhibit the migration and invasion of RA-FLS by regulating autophagy.