Deoxycytidine Kinase Gene Silencing By Lentiviral-mediated RNA Interference Inhibits The Migration And Invasion Of Fibroblast-like Synoviocytes From Rheumatoid Arthritis Patients

INTRODUCTIONIn the broad spectrum of inflammatory joint diseases, rheumatoidarthritis (RA) has a prominent position. By causing symmetricalinflammation in the affected joints, RA leads to pain and joint destruction,eventually resulting in disfiguration and disability. Fibroblast-like synovialcell (FLS) plays an important role in each link of the pathogenesis of RA,erosion of the extracellular matrix and cartilage are its importantcharacteristics. The characteristics are similar to tumor cells. Kinases playan important role in migration and invasion of various types of cells. Thepresent study of Kinases focuses on the metastasis of tumor and nervoussystem diseases. Deoxycytidine kinase (DCK) is a key enzyme in DNAbiosynthesis in the salvage pathways, which is high expression in thymus and bone marrow. In virus infection and autoimmune diseases,phosphorylated DCK actived nucleoside analogues to drug precursor. ForDCK and rheumatoid arthritis fibroblast-like synovial cells (RA FLS)relationship, there are no research reports.OBJECTIVETo investigate the effect of deoxycytidine kinase (DCK) gene silencingby lentiviral-mediated RNA interference on regulating migration andinvasion in rheumatoid arthritis fibroblast-like synoviol cells (FLS).MATERIALS AND METHODSTo suppress DCK expression, FLS cells were infected with a lentivirusexpressing DCK-shRNA. The silencing efficacy of DCK shRNA lentivirus atmRNA and protein levels in FLS cells was determined by quantitativereal-time PCR analysis and Western blot. Wound healing, transwell migrationand invasion assays were performed and analyzed to investigate the effect ofDCK shRNA knockdown on migration and invasion of FLS cells. F-actinstained with phalloidin reorganization was performed to further confirm it.MTS assay was performed to evaluate the FLS cell viability. qRT-PCR andBio-plex were used to measure the production of MMPs.RESULTSOur data revealed that DCK silencing had a strong inhibitory ability to migration and invasion of FLS cells, which not due to toxic effect of virus.DCK shRNA knockdown decreased production of MMP-1and MMP-3, andprevented cytoskeletal reorganization of RA FLS cells. AKT played anessential role in cell motility and affect phosphorylation of FAK, whichresults in activation of FAK, focal adhesion disassembly, and change cellmotility in the DCK-mediated FLS migration and invasion. Morever,DCK-mediated AKT pathway also affect NF-К B activation throughinteracting with IKKa in FLS, which results in induced level of MMPexpression.CONCLUSIONSDCK plays an important role in migration, invasion, MMPs expressionand cytoskeletal reorganization in RA FLS. The mechanism may throughAKT activation affected phosphorylation of FAK and NF-κB signalingpathways. These findings suggest that DCK may be a novel target for controljoint destruction of RA.