MiRNA-mediated GAS1 Inhibition Regulates The Proliferation And Apoptosis Of Fibroblast-like Synoviocytes In Osteoarthritis

BackgroundsOsteoarthritis(OA)is a common,non-septic,chronic arthritis mostly involved in knee joints.OA is characterized by high morbidity,lack of efficacious treatment,unendurable pain and prognostic disability by patients.It could be a huge economic burden to whole society.The research on OA pathogenesis is of great significance.Recent studies indicated that inflamed and hyperplastic Fibroblast-like synoviocytes(FLSs)were widely presented in OA joints and might play an important role in OA progress.Growth arrest-specific gene 1(GAS1),regarded as a cell growth repressor and apoptotic inducer,was found downregulated in OAFLSs according to our preliminary data.The protein product of GAS1 gene could regulate the cell proliferation and apoptosis via a PI3K-Akt pathway.Based on these preliminary findings,we hypothesized that the knockdown of GAS1 contributed to the over-proliferation of FLSs in OA developments.miRNA is known as a branch of small non-coding RNA(approximately 20 to 26 nucleotides in length)that can down-regulate gene expression at a post-transcriptional level.Evidence shows that multiple miRNAs were aberrantly expressed between non-OA and OAFLSs.We further postulate that some miRNA candidates could probably regulate the expression of GAS1 in FLSs under OA inflammatory circumstances.ObjectsTo acquire human non-OAFLSs or OAFLSs and to detect the cell viability of both cells;To detect the mRNA and protein level of GAS1 in non-OAFLSs,OAFLSs and IL-1β-stimulated FLSs and to detect the protein intensity of non-OA and OA synovial tissues;To establish and verify the GAS1 up-/down-regulated FLSs;To explore the possible molecular mechanism of the effect of GAS1 on FLSs proliferation and apoptosis;To further investigate the potential miRNA candidates that can directly down-regulate the GAS1 expression in FLSs under OA inflammatory circumstances.Methods1.Synovial fibroblasts were separated and primary cultured from synovial tissues for further experiments.2.GAS1 expression were detected by qPCR and Westernblot assay among non-OAFLSs,OAFLSs,and FLSs treated with various dosage of IL-1β;GAS1 expression intensity was detected by ICH staining among non-OA and OA synovial tissues;3.Reconstructed pcDNA3.1(+)-GAS1 or siRNA-GAS1 were transfected into non-OAFLSs separately for establishing up-/down-regulated FLSs.GAS1 expression of each group were tested by qPCR and cellular immunofluorescence staining;4.Cell viability and proliferation curve were measured by Cell Counting-Kit 8 assay;For cell cycling and apoptosis,FLSs were stained with PI and Annexin V-FITC,then performed through flow cytometry.5.Proteins involved in PI3K-Akt pathway like PI3 K,pAkt,CDK2 and BAX,were tested by westernblot assay;LY294002,a PI3 K inhibitor,was also used for further testing.6.MiRNA candidates were predicted by Targetscan 7.2 and verified by qPCR,the mechanism of the miRNA candidates was tested by westernblot and Luciferase assay.Results1.Primary-cultured FLSs were obtained and verified for further cellular experiments;2.The expression of GAS1 was down-regulated in primary cultured OAFLSs and IL-1β-stimulated FLSs compared to non-OAFLSs and GAS1 expression was negatively related to the exposed dosage of IL-1β;GAS1 intensity was lower in OA synovial tissues compared to non-OA synovial tissues;3.The expression of GAS1 in pcDNA-GAS1 FLSs were up-regulated,while the expression of GAS1 in siRNA GAS1 group were down-regulated detected by qPCR and cellular immunofluorescence staining assay.4.GAS1 down-regulated FLSs showed higher cell proliferation rate and lower apoptosis rate.On the contrary,GAS1 up-regulated FLSs presented lower cell viability,obvious cell cycle arrest and higher apoptosis rate compared to control groups;5.Growth inhibitory effects of GAS1 in FLSs by inactivating PI3K/Akt pathway were verified by testing the protein level of PI3 K,pAkt,cell cycle related proteins,CDK2,and apoptotic protein,BAX;6.MiR-34a-5p and miR-181a-5p were verified to down-regulate the GAS1 expression in OA or IL-1β-stimulated FLSs by directly targeting the GAS1 3’UTR proved by westernblot and Luciferase assay.ConclusionDown-regulation of GAS1,mediated by miR-34a-5p and miR-181a-5p,leads to over-proliferation of FLSs in OA pathogenesis through the PI3K-Akt pathway.Therefore,targeting GAS1 holds great promise for anti-hyperplasia of FLSs in OA treatment.