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.