DISCUSSION
So far, the pathogenesis of RA has not been completely clarified, therefore no therapeutic specific has been found clinically. The current treatment mainly focuses on drug therapy and surgical treatment. Current clinically used drugs include nonsteroidal anti-inflammatory drugs, glucocorticoid, disease modifying antirheumatic drugs, biologics, traditional Chinese medicine, etc. However, various therapies have their own limitations in the clinical treatment of RA. Recent studies have suggested genetic approach may offer new therapeutic methods, several genetics have been identified as genetic factors that contribute to RA, or have potential role in regulating the pathogenesis of RA, such as microRNAs[22,23], lncRNAs[24], etc. There is growing evidence that FLS of active RA play a major role in the onset and progression of RA. In addition, FLS obtained from synovia of patients with RA exhibit tumour-like behaviours, including aggressive proliferation, increased migration, invasion, and reduced apoptosis. Emerging evidence indicate that inhibition of RA-FLS effector molecules might be beneficial for RA therapy. In this study, we expolred the role of overexpression of EBP50 in FLS proliferation, migration/invasion, and cell apoptosis.
There have many studies reported that EBP50 expression was down-regulated in breast cancer, gastric cancer and prostate cancer, and overexpression of EBP50 could effectively inhibit the proliferation of tumor cells and promote the apoptosis of tumor cells. Here in our study, to explore the potential effects of EBP50 on the proliferation of FLS, FLS were left untreated (CON) or transfected with control lentivirus (LV-Plvx) or LV-EBP50 for 72 h, the expression of EBP50 were detected by WB and qRT-PCR firstly. The results indicated that the expression of EBP50 was significantly up-regulated in LV-EBP50 transfected cells compared with untransfected cells and LV-Plvx transfected cells. Then, we found that EBP50 overexpression in FLS significantly inhibited cell proliferation by MTT assay, consistent with previous reports showing EBP50 as an inhibitor against cell proliferation in certain proliferative diseases such as cancer[25-27]. At the same time, EBP50 can stimulate autophagy and silencing EBP50 restrains it in cytoplasm[28]. EBP50, an activator of autophagy, causes lysosomal degradation of c-Myc to suppress the growth of MCF-7 breast cancer cells overexpressing[29]. Recent studies also have shown that in RA-FLS from RA patients or from animal models of RA, the autophagy pathway is persistently stimulated and apoptosis is downregulated, leading to synovial fibroblast proliferation and activation. So, we investigated the effect of EBP50 overexpression on autophagy in FLS. We observed that the autophagy-related proteins LC3 remain unchanged. Our results thus suggest that EBP50 overexpression had no effect on the autophagy of FLS, which might inhibit FLS proliferation through other pathways.
Apoptosis is another important target for anticancer therapy. Related literature reports that overexpression of EBP50 significantly promoted the tumor cells apotosis, and we found that overexpression of EBP50 upregulates the apoptosis ratio of macrophage by increasing the expression of Bax and caspase-3 in an NO-dependent mechanism in a previous study[30]. Thus, we investigated whether EBP50 can perform the same function in FLS. It is well known that the Bcl-2 and caspase families play a vital role in cell survival/death. The Bcl-2 family can be functionally divided into antiapoptotic members (Bcl-2, Bcl-XL, Bcl-w, and CED 9), and proapoptotic members (Bax, Bad, and Bid)[31,32]. Among the members of the caspase family, caspases-3, -6, and -7 are the effector caspases involved in the execution of cells in response to apoptotic stimuli, and caspase-3, the most important terminal executive molecule in the process of apoptosis, activation will lead to irreversible apoptosis[33]. In this study, after transduced with LV-EBP50, the apoptosis of FLS were increased when compared with LV-Plvx transduced FLS (Fig. 4B). Furthermore, we evaluated the expression of the apoptosis-related proteins Caspase3 (Fig. 4B). The results showed that cleaved caspase-3 were significantly increased in LV-EBP50-transduced FLS, which revealed that EBP50 overexpression promote apotosis of FLS through activating caspase-3. Our results demonstrated EBP50 transient overexpression significantly increased RA-FLS apoptosis, and therefore, may offer novel approaches against synovial hyperplasia.
FLS is the major cell population involved in the development of RA in synovial tissues. Previous studies have shown that FLS migrate and invade the cartilage and bone, contributing to pannus formation and tissue damage during RA progression. Regulation of FLS migration and invasion may be a new therapeutic strategy to prevent the destructive progress of RA. Prior studies indicated that the suppression of FLS migration and invasion might protect against joint destruction in RA. Therefore, we determined the effect of overexpression of EBP50 on the migration and invasion of FLS in this study. As a result, transfection of FLS with the LV-EBP50 over-expression vector had a profound inhibitory effect on their migration and invasion. It is well documented that membrane-associated proteins formed by the combination of E-cadherin and β-catenin participate in the regulation of cell adhesion, and cell movement, including metastasis and invasion, is also regulated by membrane-associated adhesion proteins. EBP50 stabilizes β-catenin on the cell surface by interacting with β-catenin and promotes the binding of β-catenin to E-cadherin complex, inhibiting cell migration and thus tumor cell metastasis. In addition, Matrix metalloproteinases (MMPs) are the main proteases responsible for the invasion and degradation of basement membranes and extracellular matrix. MMP-1, MMP-3, MMP-9, and MMP-13 are markedly elevated in synovial fluid from RA patients and play a significant role in collagen degradation, causing disease progression and bone erosion[34]. Among these proteins, endogenous MMP2 and MMP9 contribute to synovial fibroblast survival, proliferation, migration, and invasion in RA [35]. Studies have shown that EBP50 can down-regulate the expression of MMPs in breast cancer cells, especially MMP-2 and MMP-9, thus inhibiting the invasion ability of breast cancer cells, indicating that EBP50 can inhibit the invasion of tumor cells by inhibiting the expression of MMPs. To determine the effect of EBP50 on E-cadherin, β-catenin, MMP2 and MMP9 expression of FLS, we used western blot to measure the expression of E-cadherin, β-catenin, MMP2 and MMP9. In the present study, we observed that EBP50 overexpression markedly down-regulated the expression of MMP-9, but had no effect on MMP-2, E-cadherin and β-catenin, suggesting that one of the mechanisms for EBP50-regulated aggressive behavior of FLS is reducing MMP-9 production.