The Mechanism Of LncRNA OIP5-AS1/miR-410-3p/Wnt7b Signal Axis Promoting Proliferation Of Fibroblast-like Synoviocytes In Rheumatoid Arthritis And Intervention Of Total Saponins Of Radix Clematis

This study includes the following two parts:Part 1: LncRNA OIP5-AS1 Regulates the miR-410-3p/Wnt7 b Signal Axis and Promotes the Proliferation of Fibroblast-like Synoviocytes in Rheumatoid Arthritis.Objectives: To investigate the molecular mechanism of long non-coding RNA OPA interacting protein 5-antisense RNA 1(LncRNA OIP5-AS1)regulating miR-410-3p /Wnt7 b signal axis and Wnt signal pathway promoting the proliferation of fibroblast-like synoviocytes(FLS).Methods: The adjuvant induced arthritis(AA)rats model was established with freund’s complete adjuvant(FCA),and fibroblast-like synoviocytes(FLS)were cultured from the synovium of AA rats.RT-qPCR was used to detect the m RNA expression levels of LncRNA OIP5-AS1 and miR-410-3p in FLS;The subcellular localization of LncRNA OIP5-AS1 in FLS was detected by fluorescence in situ hybridization(FISH);The effect of LncRNA OIP5-AS1 si RNA on the proliferation of FLS cells was detected by MTT;LncRNA OIP5-AS1,miR-410-3p,Wnt7 b and the downstream key factors of Wnt signaling pathway including beta-catenin,C-myc,Cyclin D1,GSK-3beta,SFRP4 m RNA expression levels were detected by RT-qPCR;Wnt7b and the downstream key factors of Wnt signaling pathway protein expression changes were detected by immunofluorescence and Western blotting.Results: LncRNA OIP5-AS1 was up-regulated in FLS,while miR-410-3p was down-regulated;FISH results showed that LncRNA OIP5-AS1 was mainly distributed in the cytoplasm;The proliferation ability of FLS was significantly inhibited by LncRNA OIP5-AS1 si RNA;Compared with the model group,LncRNA OIP5-AS1 si RNA inhibited the expression levels of LncRNA OIP5-AS1,Wnt7 b,beta-catenin,C-myc,Cyclin D1,p-GSK-3beta(Ser9)/GSK-3beta significantly,and increased the expression levels of miR-410-3p and Wnt signaling pathway negative regulator SFRP4.Conclusion: LncRNA OIP5-AS1 is able to play a biological role in inhibiting the proliferation of FLS by regulating the miR-410-3p/Wnt7 b signal axis,regulating the Wnt signaling pathway under interference.Part 2: Intervention Effect of Total Saponins of Radix Clematis on LncRNA OIP5-AS1/miR-410-3p/Wnt7 b Signaling Axis in Fibroblast-like Synoviocytes in Rheumatoid Arthritis.Objective: To elucidate the effect of total saponins of radix clematis(TSC)on the LncRNA OIP5-AS1/miR-410-3p/Wnt7 b signaling axis in FLS;The effects of TSC(0.5,2.5,12.5,62.5,312.5 and 1562.5 g/m L)on the proliferation of FLS were detected by MTT assay at 12 h and 24h;LncRNA OIP5-AS1,miR-410-3p,Wnt7 b and key factors of Wnt signaling pathway including beta-catenin,C-myc,Cyclin D1,GSK-3beta,SFRP4 m RNA expression levels after TSC intervention at the optimal dose were detected by RT-qPCR;Wnt7b and key factors downstream of Wnt signaling pathway protein expression changes after TSC intervention at the optimal dose were detected by immunofluorescence and Western blotting;In recovery experiments,MTT and flow cytometry were used to detect the effect of the optimal dose of TSC on the proliferation ability and cell cycle of FLS under the overexpression of LncRNA OIP5-AS1.Results: TSC can inhibits the proliferation of FLS;Compared with the model group,TSC could significantly reduce the expression levels of LncRNA OIP5-AS1,Wnt7 b,beta-catenin,C-myc,Cyclin D1,p-GSK-3beta(Ser9)/GSK-3beta,and increase the expression levels of miR-410-3p and SFRP4;Compared with the model group,LncRNA OIP5-AS1 overexpression could significantly promote cell proliferation and significantly increase the proportion of cells in G2/M phase;The promotion of cell proliferation by LncRNA OIP5-AS1 overexpression could be partially counteracted by TSC intervention.Conclusion: The inhibitory effect of total saponins of radix clematis on FLS excessive proliferation may be related to down-regulation of LncRNA OIP5-AS1 expression,regulation of the miR-410-3p/Wnt7 b signaling axis and Wnt signaling pathway.