| Objective To assess the effect of silence of PCSK6 by si RNA on biological behaviors in collagen-induced arthritis(CIA) fibroblast-like synoviocytes(FLS). To assess the effect of exogenous PCSK6(human) recombinant protein on biological behaviors and molecular mechanisms in rheumatoid arthritis(RA) fibroblast-like synoviocytes(FLS).Methods CIA models of rat were induced with bovine collagen type Ⅱ. Cultured FLS from CIA models were treated with anti-PCSK6 si RNA. A negative si RNA was used as the negative control. The inhibiting effects were detected with RT-q PCR after 24 h and 36 h. The proliferation, invasion, migration capacity, secretion of inflammation factors and cell cycle were studied following by MTT, transwell, wound healing, ELISA and flow cytometry. Expression of genes related with proliferation, invasion, migration and inflammation were detected by RT-q PCR. Different concentrations of PCSK6(human) recombinant protein were used to induce FLS of RA. The proliferation, invasion, migration, secretion of inflammation factors, cell cycle and apoptosis were studied following by MTT, transwell, wound healing, ELISA and flow cytometry. RT-q PCR was used to detect the expression of functional genes. ERK, STAT3, Wnt3 a, Nodal, m TOR, NF-κB p65 and NF-κB p105/p50 signaling transduction pathways were tested by western blot, as well as MMP9 protein. PD98059, Stattic, PDTC inhibitors were used to induce FLS of RA, which has been induced PCSK6(human) recombinant protein. The proliferation, invasion, migration, secretion of inflammation factors,cell cycle and apoptosis were studied following by MTT, transwell, wound healing, ELISA and flow cytometry.Results The inhibiting effect was remarkable after anti-PCSK6 si RNA was transfected into FLS at 24 h in the level of m RNA compared with negative control(P<0.001). PCSK6-si RNA treatment significantly decreased cell proliferation(P<0.001), invasion(P<0.001) and migration(P<0.001) in FLS of CIA, as well as secretion of TNF-a and IL-1β(P<0.001). Flow cytometry revealed a G0/G1 arrest of FLS(P<0.05). Expression of genes, including CXCL9, MMP-2, MMP-9, NOSTRIN, HIF-1α, MPZL2, IGF-2 and PADI4 were all decreased obviously(P<0.05). The promoting effect was the most remarkable after 150ng/m L PCSK6(human) recombinant protein was induced into FLS of RA compared with blank control. PCSK6(human) recombinant protein treatment significantly increased cell proliferation(P<0.01), invasion(P<0.001) and migration(P<0.001) in FLS of RA, as well as secretion of IL-1α, IL-1β, IL-17, IL-6 and TNF-α(P<0.05). Flow cytomety revealed the S and G2/M phase gather of FLS(P<0.01) and the late stage apoptosis inhibition of FLS(P<0.01). Expression of genes, including C10ORF116, CXCL9, CXCL10, HIF-1α, IL-6, IL-10, IL-26, MMP9, NOSTRIN and TNF-α were all increased obviously(P<0.001). Expression of pre-MMP9 and active-MMP9 proteins were also increased. Along with the added PCSK6(human) recombinant protein, ERK, STAT3 and NF-κB p65 signaling transduction pathways were activated at 6h. PD98059, Stattic and PDTC inhibitors treatment significantly decreased cell proliferation(P<0.05), invasion(P<0.001) and migration(P<0.001) in FLS of RA, as well as secretion of IL-1α, IL-1β, IL-17, IL-6 and TNF-α(P<0.05) compared with 150ng/m L PCSK6(human) recombinant protein control. Flow cytometry revealed a G0/G1 phase arrest of FLS following Stattic and PDTC inhibitors treatment(P<0.05). Only added PDTC inhibitor could promote FLS apoptosis(P<0.05).Conclusion Inhibition of PCSK6 might play a protective role in CIA FLS on biological behavior, and laid the foundation for further study the pathogenesis of CIA. Exogenous PCSK6(human) recombinant protein might play a destructive role of RA FLS on biological behavior. Also, PCSK6 might activate ERK, STAT3 and NF-κB p65 signaling transduction pathways to regulate biological behavior of RA FLS. It could become a new target to studyRA pathogenesis, drug screening and immunotherapy, and so on. |
PDF Full Text Request