Inhibition of lncRNA NEAT1 induces dysfunction of fibroblast-like synoviocytes in rheumatoid arthritis via miRNA-338-3p-mediated regulation of glutamine metabolism

Figures

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  Fig. 1. correlation of NEAT1 and miR-338-3p with cellular dysfunction and glutamine metabolism in FLSs-RA. A Expression of NEAT1 in FLSs from healthy donors and RA patients. B FLSs-RA cells without or with NEAT1 silencing by NEAT1 siRNA transfection were treated with the indicated concentrations of H2O2 and cell viability was determined by MTT assay, C Annexin V assay. D Glutamine uptake and E glutaminase activity were examined in FLSs-RA cells without and with NEAT1 silencing. F Predicted NEAT1-miR-338-3p interaction from StarBase. G Expression of miR-338-3p in FLS of healthy donors and RA patients. H FLSs-RA cells that did or did not overexpress miR-338-3p were treated with the indicated concentrations of H2O2, and cell viability was determined by MTT assay and I Annexin V assay. J Glutamine uptake and K glutaminase activity were examined in FLSs-RA cells without or with overexpression of miR-338-3p. Significant differences between the two groups were identified using the Mann–Whitney U test. Values are means (± SD, n = 3). *p < 0.05, **p < 0.01

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  Fig. 2. NEAT1 downregulates miR-338-3p by sponging as ceRNA. A FLSs-RA was transfected with control siRNA or NEAT1 siRNA and miR-338-3p expression was detected by qRT-PCR. B FLSs-RA cell lysates were incubated with biotin-labeled control, sense or antisense NEAT1 probes for pull-down assays. miR-338-3p enrichment was assessed by qRT-PCR. C Luciferase reporter assays were performed by transfection of FLSs-RA with luciferase vectors containing WT- or Mut-NEAT1 and control miRNA or miR-338-3p. Luciferase activity was examined; significant differences between the two groups were identified using the Mann–Whitney U test. Values are means (± SD, n = 3). *p < 0.05, **p < 0.01

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  Fig. 3. The rate of glutamine metabolism is increased in FLSs-RA. A Glutamine uptake and B glutaminase activity were examined in FLSs from normal subjects and RA patients. C FLS of normal and RA patients were cultured under normal or low glutamine conditions. Cell viability was examined by clonogenic and MTT assays. D Protein expression of GLS in FLS of normal and RA patients was detected by Western blotting. β-actin served as an internal control. The significant differences between the two groups were identified using the Mann–Whitney U test. Values are means (± SD, n = 3). *p < 0.05, **p < 0.01

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  Fig. 4. miR-338-3p directly targets the 3' UTR of the GLS of FLSs. A miR-338-3p binding sites on the 3' UTR of GLSs were predicted from StarBase. B The expression of GLS mRNA in normal FLSs and FLSs-RA was detected by qRT-PCR. C FLSs-RA cells were transfected with control siRNA or GLS siRNA for 48 h, and cells were treated with the indicated concentration of H2O2. Cell viability was examined by MTT assay. D FLSs-RA cells were transfected with control miRNA or miR-338-3p, and protein expression of GLS was detected by Western blot. E WT- or Mut-3'UTR of GLS was transfected into normal FLSs and F FLSs-RA with control miRNA or miR-338-3p, and a luciferase reporter assay was performed. Luciferase activity was examined; significant differences between the two groups were identified using the Mann–Whitney U test. Values are means (± SD, n = 3). *p < 0.05, **p < 0.01

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  Fig. 5. Restoration of GLS restored miR-338-3p-mediated glutamine metabolism and cellular dysfunction in FLSs-RA. A FLSs-RA were transfected with control, miR-338-3p alone, or GLS plus, and GLS expression was examined by Western blot. B Glutamine uptake and C glutaminase activity were detected from the above-transfected cells. D FLSs-RA cells transfected with control, miR-338-3p alone, or plus GLS were treated with H2O2 at the indicated concentrations for 8 h, and cell viability was determined by MTT assay. Significant differences between the two groups were identified using the Mann–Whitney U test. Values are means (± SD, n = 3). *p < 0.05, **p < 0.01, ***p < 0.001

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  Fig. 6. Role of the NEAT1-miR-338-3p-GLS axis in FLSs-RA. A FLSs-RA was transfected with control, NEAT1 alone, or in combination with miR-338-3p, B protein expression of GLS was examined by qRT-PCR and Western blot, respectively. C Glutamine uptake assay and D glutaminase activity were measured from the above-transfected cells. E FLSs-RA were transfected with control, NEAT1 alone or in combination with miR-338-3p, cells were treated with H2O2 at the indicated concentrations for 48 h and cell viability was determined by MTT assay and F Annexin V apoptosis assay. Significant differences between the two groups were identified using the Mann–Whitney U test. Values are means (± SD, n = 3). *p < 0.05, **p < 0.01