| Objective:In this study,we investigated the effect of overexpression of lnc RNA NONHSAT042241 on the biological behavior of RA-FLS,and explored the effect of LLDT-8 on RA-FLS by regulating the expression of lnc RNA NONHSAT042241 and the mechanism of lnc RNA NONHSAT042241.These results provide theoretical basis and new research ideas for further understanding of the pathogenesis of RA and the clinical application of LLDT-8 in the treatment of RA.Methods:1)The expression of NONHSAT042241 in RA synovial tissue and OA synovial tissue was detected by q RT-PCR,as well as the expression of NONHSAT042241 of primary cultured RA-FLS cells and OA-FLS cells.2)We constructed the overexpression recombinant lentiviral vector of NONHSAT042241,and transfected the RA-FLS cell line with the lentiviral vector.The cell proliferation ability was observed by the CCK8 method and the cloning formation experiment.The cell apoptosis was observed by the flow cytometry experiment.Western Blot experiment was used to observe the expression of apoptosis-related proteins.Scratch experiment was used to observe the cell migration and cell invasion ability was detected by Transwell invasion experiment.m RNA and protein expression of inflammatory factors and metalloproteinase was detected q RT-PCR and ELISA.3)We used CCK8、cloning formation experiment、flow cytometry、scratch、Transwell invasion、q RT-PCR、 ELISA and Western Blot to detect the cell proliferation,apoptosis,invasion,migration and the secretion of inflammatory factors and metal matrix proteases of overexpressing and knocking down NONHSAT042241 in RA-FLS after treating with LLDT-8.4)The cell location of NONHSAT042241 was determined by FISH experiment.RNA pull-down experiment combined with mass spectrometry was used to detect the protein which interacted with NONHSAT042241 and the RIP experiment was used to verify whether the target protein is directly bound to NONHSAT042241.Western Blot were used to observe the effect of lnc RNA NONHSAT042241 on Wnt/β-catenin signaling pathway and target genes,as well as the changes of Wnt/β-catenin signaling pathway and target genes after treating with LLDT-8.Restults:1)The expression of NONHSAT042241 in RA synovial tissue and RA-FLS was significantly lower than that in OA synovial tissue(P<0.01)and OA-FLS(P=0.0281).2)Overexpression of NONHSAT042241 in RA-FLS can inhibit the proliferation(P<0.01),clone formation(P<0.01)and can also promote apoptosis of RA-FLS(P<0.01),at the same time increasing the expression of pro-apoptotic proteins[Bax(P<0.01)and Cleaved-caspase 3(P<0.05)].The overexpression of NONHSAT042241 can also inhibit invasion of RA-FLS(P<0.01),and inhibit the secretion of inflammatory factors IL-1(P<0.01),IL-6(P<0.01)and metal matrix protease MMP-1(P<0.05),MMP-3(P<0.01),and increase the expression of IL-10(P<0.01).3)LLDT-8 can inhibit cell proliferation(P<0.01),clone formation(P<0.01)enhance cell apoptosis(P<0.01)and further enhance the expression of pro-apoptotic proteins Bax(P<0.01)and Cleaved-caspase 3(P<0.05)by increasing NONHSAT042241,while reducing the expression of IL-1(P<0.01),IL-6(P<0.01)and MMP-1(P<0.01)、MMP-3(P<0.01).4)NONHSAT042241 can directly bind to RNA binding proteins hn RNP D and PBAP1 and affect the Wnt/β-catenin signaling pathway.NONHSAT042241-protein complex can affect the expression of β-catenin and affect the Wnt/β-catenin signaling pathway.Overexpression of NONHSAT042241 can reduce the expression levels ofβ-catenin(P<0.01)and p-GSK-3β(P<0.01),and inhibit the expression of downstream target genes Cyclin D1(P<0.01),while reducing the expression of cell proliferation marker protein PCNA(P<0.01).After treatment with LLDT-8,it can increase the expression of GSK-3β(P<0.01),and further inhibit the expression of β-catenin,p-GSK-3β and downstream target genes C-myc(P<0.01),Cyclin D1(P<0.01)and PCNA(P<0.01).Concolusion:1)NONHSAT042241 is low expressed in RA tissues and cells.Overexpression of NONHSAT042241 can inhibit the proliferation of RA-FLS cells,promote cell apoptosis,and inhibit their invasion and secretion of inflammatory factors and MMPs.LLDT-8 can further promote the inhibition of the proliferation by increasing NONHSAT042241,and further reduce the secretion of inflammatory factors and MMPs,and promote apoptosis.2)NONHSAT042241 can directly bind to RNA binding proteins hn RNP D and PBAP1,and affect the Wnt/β-catenin signaling pathway.Overexpression of NONHSAT042241 can down-regulate the expression of β-catenin and p-GSK-3β,inhibit Wnt/β-catenin pathway activation,and down-regulates the expression of,Cyclin D1,and PCNA,thereby affecting cell proliferation,etc.While LLDT-8 can further down-regulate their expression and play a synergistic effect. |
