The Role Of LATs In Rheumatoid Arthritis Fibroblastlike Synoviocytes

Objective 1. To assess the biological behaviors and mechanism of LAT1 and LAT3 in rheumatoid arthritis fibroblast-like synoviocytes. 2. To assess the expression regulation of LAT1 and LAT3 in rheumatoid arthritis fibroblast-like synoviocytes.Methods 1. To detect the expression of LATs in rheumatoid arthritis Synovial tissues using q RT-PCR method; Small interfering RNA(si RNA) was designed and synthesized; Primary rheumatoid arthritis fibroblast-like synoviocytes were separated and cultured.We used si RNA to silence LAT1/ LAT3 and detected the silencing efficiency with q RT-PCR and Western Blot method. The proliferation and invasion were studied after LAT1/3 knowing down by MTS and Transwell method.The uptake of amino acids after si RNA knowing down LAT1/3 and BCH inhibited LATs was detected by [~3H] L-leucine uptake measurement. The expression of MMPs in RAFLS was detected using q RT-PCR method. Western Blot was used to detecte the activity of m TOR and p70S6 K after BCH inhibiting LATs. 2 The expression of LAT1 and LAT3 were detected using q RT-PCR method after rheumatoid arthritis fibroblast-like synoviocytes treated with IL-17, TNF-? and LPS. The effect of IL-17 on invasion of RA FLS was preformed using Transwell method and the same method was used to detecte the effect of IL-17 on invasion after si RNA downregulating LAT1/ LAT3 and BCH inhibiting LATs. We used signal pawthway inhibitor to detect the role of signal pawthway on IL-17 upregulating LAT1 and LAT3.Results 1.Three LATs isoforms, LAT1-3, have been found in rheumatoid arthritis synovial tissues(RAST). Expression level of LAT1 and LAT3 was higher in RA ST than in osteoarthritis synovial tissues(OAST, *** P<0.001, * P<0.05). Expression of LAT1 and LAT3 decreased significantly at both m RNA(** P <0.01, *P <0.05) and protein levels after si RNA knowing down LAT1/ LAT3. MTS method showed no affection on RA FLS proliferation after si RNA knocking down LA1/3 compared to control(*P >0.05). Transwell methods showed less FLS invasiveness after si RNA knocking down(*** P <0.001, *** P <0.001). The uptake of Leucine in RA FLS was significantly reduced after si RNA knowing down LAT1/ LAT3 and BCH blocking LATs(***P<0.001, ***P<0.001). Transwell methods also showed less FLS invasiveness with the presense of BCH(*P <0.05). Phosphorylation of m TOR was decreased in the presence of BCH. 2. Cells were treated with IL-17?LPS and TNF-?. Only IL-17 consistently and significantly upreguate the expression of LAT1 and LAT3(*** P<0.001, *** P<0.001). Whatmore, IL-17 stimulate the expression of LAT1 and LAT3 with dose-dependent manner(* P<0.05, ** P<0.01, *** P<0.001) and time-dependent manner(* P<0.05, ** P<0.01, *** P<0.001). RA FLS invasiveness was significantly increased by stimulation with IL-17(***P<0.001) and this effect was neutralized by BCH and si RNA of LAT1/3 respectively(**P<0.01, ***P<0.001). IL-17 upregulated expression of LAT1 and LAT3 through the m TOR and NF-?B pathways respectively(***P<0.001, ***P<0.001).Conclusion 1 Three LATs isoforms, LAT1-3, have been found in rheumatoid arthritis synovial tissues(RAST). Expression level of LAT1 and LAT3 was higher in RA ST than in OAST. LAT1/LAT3 regulated cell invasion via m TOR pathway in RA FLS. 2. IL-17 upregulated expression of LAT1 and LAT3 with dose-dependent manner and time-dependent manner through the m TOR and NF-?B pathways respectively. LAT1/LAT3 may be involved in IL-17-induced invasiveness in RA FLS.