Rheumatoid Arthritis Fibroblast-like Synoviocyte Suppression Mediated by PTEN Involves Survivin Gene Silencing

Survivin is a proto-oncogene biomarker known for its anti-apoptotic and cell cycle regulating properties induced by the activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway. In the context of non-cancer pathology, such as rheumatoid arthritis (RA), survivin has emerged as a feature associated with severe joint damage and poor treatment response. Phosphatase and tensin homolog (PTEN) is a phosphatase antagonizing all classes of PI3K. The interplay between survivin oncogenic mechanisms and proliferation suppression networks in RA has remained largely elusive. This study investigated the effect of PTEN on survivin gene expression in rheumatiod arthritis fibroblast-like synoviocyte (RA-FLS). We showed for the first time that the suppression of RA-FLS was mediated by PTEN involving survivin silencing. Considering that survivin suppressants are currently available in clinical trials and clinical use, their effects in RA-FLS support a probably RA therapy to clinical practice.


Results
Survivin is suppressed after inhibition with LY294002 in RA-FLS. Recent reports showed that survivin is a direct target gene of the PI3K/AKT signaling pathway in the ectopic endometrium and invasive epithelium cells in adenomyosis [16][17][18] . In our study, we further confirmed the regulation of survivin expression in RA-FLS. As shown in Fig. 1, the mRNA levels of survivin were significantly down-regulated after inhibition of the PI3K/Akt pathway with LY294002. By contrast, expression of the PTEN gene did not significantly change in the presence of LY294002. We hypothesized that survivin expression is regulated by the PI3K/Akt pathway in RA-FLS. All evaluations were tested by directed qPCR with normalization to the housekeeping gene GAPDH (Fig. 1A). Relative expression was quantitated using densitometry, and the results of triplicate experiments are shown in Fig. 1B. Construction of recombinant pcDNA3.1-PTEN and shRNA-pENTR/U6-survivin. After electrophoresis using 1.5% agarose gel, the PCR product was visualized under a UV transilluminator and purified with the PCR product purification kit (OMEGA). The expected 1212 bp PTEN PCR product fragment was observed ( Fig. 2A). The PCR product was ligated into the pcDNA3.1 expression vector and transformed into the E. coli DH5α strain. The recombinant plasmid was confirmed by restriction enzyme analysis (Fig. 2B) and sequencing analysis.
Three annealed double-stranded shRNAs were individually cloned into the linearized shuttle vector shRNA-pENTR/U6, which was verified by PCR using the U6 and V5 universal primers. Only #1 and #3 were successfully constructed (Fig. 2C). Sequencing was performed to confirm successful cloning of the recombinant shuttle plasmid.
Over-expression of PTEN reduces the survivin expression. The over-expression of PTEN by transient transfection of the PTEN-pcDNA3.1 plasmid in RA-FLS was verified by qPCR (Fig. 3A) and Western blotting (Fig. 3B). Blank and pcDNA3.1 vector transfection were performed as negative controls. Statistical analysis of three parallel experiments showed the efficiency and stability of gene overexpression ( Fig. 3C and D).   To determine whether survivin production is regulated by the PI3K/Akt pathway inhibitor PTEN, RA-FLS cells transfected with a PTEN over-expression plasmid or control were measured the levels of mRNA and protein of survivin after 24 hours transfection. A significant reduction of survivin production was observed by qPCR ( Fig. 3I) and Western blotting (Fig. 3J). This inhibitory effect was not observed in cells transfected with control plasmid or blank. Three parallel experiments showed similar results with significant differences (P < 0.001) ( Fig. 3K and L). All results suggested that the up-regulation of PTEN can decrease survivin gene expression. We hypothesized that the inhibition of PTEN on the PI3K/Akt pathway leading to the down-regulation of survivin. The result was similar to the research in cells in adenomyosis [16][17][18] .

PTEN over-expression and/or silencing of survivin inhibited RA-FLS proliferation.
The regulation of cell division is a prominent function of survivin. The stimulatory function on RA-FLS proliferation by survivin is important in disease development 5 . We verified the significance of survivin on RA-FLS proliferation using CCK8 tests. RA-FLS cells with survivin knock-down showed an inhibition of cell proliferation (Fig. 4). The lipid phosphatase PTEN is a well established inhibitor of cell proliferation. In our study, a similar reduction in cell growth was exhibited in the PTEN over-expression group.
To determine the relationship between PTEN over-expression and survivin knock-down, co-transfection of PTEN-pcDNA3.1 and survivin shRNA was performed simultaneously. The co-transfection did not show any cooperative relationship on RA-FLS cell proliferation inhibition. There was no significant change between the co-transfection group and either transfection group alone (Fig. 4).

Migration is decreased by PTEN over-expression or survivin knock-down. Wound healing assays
are used for analysis of cell migration in vitro. As shown in Fig. 5, the effect of the PTEN over-expression or survivin knock-down on RA-FLS cells migration was detected to be negative compared to the normal control. We propose that PTEN over-expression or survivin knock-down impairs the migration of RA-FLS.

The effect of PTEN and survivin on arthritis inflammation and RA-FLS invasion.
The pro-inflammatory cytokine IL-6 is important for potentiating the inflammatory response in RA 15 . In our study, the expression of IL-6 was reduced by PTEN over-expression or survivin knock-down. The results suggest the important role of PTEN and survivin in RA inflammation (Fig. 6A).
The matrix metalloproteinases (MMPs), such as MMP1, MMP3 and MMP9 may be important in the destructive joint changes of RA 19,20 . PTEN also inhibits RA-FLS invasion. MMPs were significantly down-regulated in the presence of PTEN over-expression and survivin silencing (Fig. 6C). The triplicate results with statistical analysis were shown in Fig. 6B,D,E and F.

Discussion
This study shows a close relationship between PTEN and survivin for nearly all RA-FLS functional processes that induce the pathogenesis of RA, such as cell proliferation, migration, invasion and inflammation.
Survivin is a direct target gene of the PI3K/AKT signaling pathway in the ectopic endometrium and invasive epithelium cells in adenomyosis [16][17][18] . In our study, we showed the regulation of this pathway in RA-FLS. The PI3K inhibitor LY294002, significantly decreased the expression of survivin. Then, we explored the relationships between PTEN, the antagonizing phosphatase of PI3K/AKT pathway, and survivin by the construction and transfection of plasmids that over-express PTEN and knock-down survivin in RA-FLS. Consistent with the results of the LY294002 inhibitor, PTEN overexpression reduced survivin gene-expression. The results provide direct evidence of the survivin regulation by the PTEN/PI3K/AKT pathway in RA-FLS. The hyporhesis was similar to the research in adenomyosis [16][17][18] . So we got a general idea that the PTEN/PI3K/AKT pathway on survivin expression seemed to be the molecular mechanism underlined in the epithelium cell biological malignant behavior.
The close associations between survivin and RA disease activity, production of autoantibodies, and the predicted development of joint damage 3, 21 were previously reported. In our study, we also found that survivin is important in RA. The results showed that survivin helps the tumor-like proliferation of RA-FLS and is involved in the secretion of the proinflammatory cytokine IL-6 and MMPs. IL-6 is one of the most abundantly expressed cytokines identified in the sera of RA patients. It promotes B cell growth and differentiation, Th17 cell generation, and osteoclast formation [22][23][24][25] . The levels of MMP-1 and MMP-3 were proved to correlated with the degree of articular surface erosion 26 . MMP-9 was showed to involve in the angiogenesis and the pannus formation in RA synovium tissues, which has been considered as essential events in the development of RA 27 . All the previous studies, as well as our results elucidated the molecules play pivotal roles in the development of synovial hyperplasia, sustained inflammation and joint destruction in arthritic joints.
In our study, we showed the important role of PTEN in RA. We provide an integrated explanation for the down-regulation of PTEN initially shown by the Thomas Pap group 13 . The loss of PTEN promotes RA-FLS in cell proliferation, migration, invasion and inflammation.
Notably, the simultaneous overexpression of PTEN and silencing of survivin did not have a greater effect on inhibition of cell proliferation. We hypothesize that PTEN may play the inhibitory roles by targeting survivin, and that survivin can only be reduced by the PTEN/PI3K/AKT pathway in RA-FLS cell proliferation.

Methods
Ethics approval. This experimental protocols and consent procedure was approved by the ethics committee of the Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangdong, China. In addition, all the participants were given written informed consent in the study. All procedures and methods involving human samples were in accordance with approved guidelines.

RNA isolation and reverse transcription polymerase chain Reaction. Total RNA was isolated by
TRIzol reagent (Life Technologies, Basel, Switzerland) according to the manufacturer's protocol. First strand cDNA was synthesized by oligo(dT) primers and Moloney murine leukemia virus (M-MuLV) reverse transcriptase (Boehringer-Mannheim, La Jolla, California, USA).
Tissue preparation and cell cultures. Synovial tissue specimens were obtained from five RA patients (F = 3, M = 2, average age = 55.8 ± 9.7) undergoing synovectomy or joint replacement in the Second Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine and the Third Affiliated Hospital of Sun Yat-sen University. All the patients were diagnosed by the 1987 American College of Rheumatology criteria for RA 28 and had actively inflamed knee joints. Immediately after surgery, the tissues were brought to our laboratory and enzymatically digested. The released cells were grown in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) in a humidified incubator maintained at 37 °C in the presence of 5% CO 2 29 . Cells from passages 4-6 were used in the experiments. The results have been collected in all five patients, and the results were similar in all five patients.
In experiments assessing the role of PI3K inhibition on survivin, 100 μM LY294002 (Cell Signaling Technology, Beverly, MA, USA) was added to culture media and DMSO as a control. Western blotting analysis. Cells were lysed at 4 °C with RIPA lysis buffer containing 1 μmol/L PMSF.
Equal amounts of protein were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene fluoride membrane. The membrane was sealed with PBS containing 5% Skimmed milk powder for 1 h and incubated with primary antibodies (survivin, PTEN and GAPDH) at 4 °C overnight. The membrane was washed with PBST (PBS, pH 7.5, containing 0.1% Tween-20) and incubated with a secondary antibody for 1 h. Anti-mouse or anti-rabbit antibodies against IgG conjugated with HRP were adopted as the secondary antibodies. Immobilon western HRP substrate was used to detect peroxidase activity.
Wound healing assay. In wound healing assay, the cells were cultured to 80-90% confluence in six-well plates. A 200 μl sterile pipet tip was used to make a central linear wound, and the plates were washed with PBS to remove the floating cells. The speed of wound closure was analyzed by measuring the distance of the wound edge to the original wound site after 24 h. Each experiment was performed in triplicate.
Cell proliferation assays. Cell proliferation was determined using the CCK-8 Kit (Dojindo Laboratories, Kumamoto, Japan) according to manufacturer protocols.