Overexpression of EBP50 Suppresses the proliferation of FLS
Inhibiting abnormal proliferation and activation of FLS may be a potentially therapeutic strategy to block or delay the progression of RA. To investigate the potential effects of EBP50 on the proliferation of FLS, fls were transfected with LV-EBP50 or LV-Plvx for 72 h, then analyzed for the EBP50 expression and the proliferation of FLS. The results showed that LV-EBP50 transfection significantly increased the expression of EBP50 in both mRNA levels (Fig. 1A) and protein levels (Fig. 1B-1C), and MTT assay demonstrated that overexpression of EBP50 can suppresse the proliferation of FLS significantly, when compared with untransfected cells and LV-Plvx transfected cells (Fig. 1D). It has been reported that EBP50 can inhibit the proliferation of breast cancer cells by stimulating the autophagic lysosomal degradation, so we speculate whether EBP50 inhibits FLS cell proliferation by inhibiting autophagy. Then, the expression of LC3 was measured by western blotting in LV-EBP50 and LV-plvx transfected cells and untreated cells.As shown in (Fig. 1E-1F), the expressions of LC3 in FLS remain unchanged after transfected with LV-EBP50.Collectively, these results suggest that overexpression of EBP50 suppresses the proliferation of FLS and has no influence on autophagy.
Overexpression of EBP50 promote apotosis of FLS By activating caspase-3
FLS can exhibit tumor-like behavior, such as significant inhibition of apoptosis. As the major tumor suppressor genes, related literature reports have indicated that overexpression of EBP50 promoted significantly the tumor cells apotosis, and our previous results have showed that the overexpression of EBP50 upregulates the apoptosis ratio of macrophage by increasing the expression of Bax and caspase-3 in an NO-dependent mechanism. To investigated whether EBP50 can perform the same function in FLS, FLS were transfected with LV-EBP50 and LV-Plvx at the MOIs of 10 for 48 h, 72 h, 96 h, and the cell apoptosis of FLS was analyzed by FCM. Results showed that EBP50 overexpression increased distinctly the apoptosis of FLS when compared with LV-Plvx transduced FLS (P< 0.001) (Fig. 2A); The expression of apoptosis-related protein caspase-3 in FLS were measured by western blotting. As shown in (Fig. 2B), when FLS were transduced with LV-EBP50 and LV-Plvx for 96h ,the protein expressions level of cleaved caspase-3 were significantly increased .
overexpression of EBP50Suppresses the Migration and Invasion of FLS