LncRNA cardiac autophagy inhibitory factor is downregulated in rheumatoid arthritis and suppresses the apoptosis of fibroblast-like synoviocytes by promoting the maturation of miRNA-20a

Figure 1. Patient data. (a) Demographic and clinical characteristics of the RA patients (n=62) included in the study. (b) The representative image of H&E staining and CD3 immunostaining. RA: Rheumatoid arthritis; SD: Standard deviation; DAS28: Disease Activity Score in 28 Joints; CRP: C-reactive protein; ENA: Extractable nuclear antigen; RF: Rheumatoid factor.

Figure 2. The expression of CAIF, mature miR-20a and miR-20a precursor was altered in RA. The expression of CAIF (a), mature miR-20a (b) and miR-20a precursor (c) in plasma samples from both 62 RA patients and controls were determined by RT-qPCR. Mean values of three technical replicates were used to express gene expression levels. CAIF: Cardiac autophagy inhibitor; RA: Rheumatoid arthritis; RT-qPCR: Reverse transcription-quantitative polymerase chain reaction; *** p<0.001.

Figure 3. CAIF and mature miR-20a were positively correlated. Correlations between CAIF and mature miR-20a (a) or miR-20a precursor (b) across plasma samples from RA patients were analyzed by linear regression. CAIF: Cardiac autophagy inhibitor; RA: Rheumatoid arthritis.

Figure 4. Overexpression of CAIF resulted in the upregulation of mature miR-20a in HFLSs. To test the hypothesis that CAIF may affect the maturation of miR-20a, HFLSs were transfected with CAIF expression vector or miR-20a mimic, and the transfections were confirmed by RT-qPCR (a). The effects of CAIF overexpression on the expression of miR-20a precursor (b) and mature miR-20a (c), and the effects of miR-20a overexpression on CAIF (d) were also analyzed by RT-qPCR. Luciferase assay (e) for luciferase activity of CAIF-reporter with miR-20a binding sites co-transfected with miR-20a mimics. n=3. Mean±SD values were used to express data of multiple transfection groups. C, control cells without transfections; pcDNA3.1, cells transfected with empty pcDNA3.1 vector; NC miRNA, cells transfected with NC miRNA. CAIF: Cardiac autophagy inhibitor; HFLSs: Human fibroblast-like synoviocytes; RT-qPCR: Reverse transcription-quantitative polymerase chain reaction; * p<0.05.

Figure 5. CAIF and miR-20a inhibited the apoptosis of HFLSs induced by LPS. HFLSs were cultivated in medium containing 0, 1, 2, 4, 6, and 8 μg/mL LPS for 48 h, followed by the determination of CAIF (a) and mature miR-20a (b) expression by RT-qPCR. The roles of CAIF and miR-20a overexpression in the apoptosis of HFLSs induced by LPS (8 μg/mL for 48 h) were analyzed by cell apoptosis assay (c). Expression of Bax and BCL2 in each transfected groups was determined by RT-qPCR. Bax/BCL2 ratios of all groups were calculated (d). Mean±SD values were used to express data of multiple transfection groups. CAIF: Cardiac autophagy inhibitor; HFLSs: Human fibroblast-like synoviocytes; LPS: Lipopolysaccharide; RT-qPCR: Reverse transcriptionquantitative polymerase chain reaction; * p<0.05.

Figure 6. CAIF and miR-20a inhibited the autophagy of HFLSs. Autophagy analysis was performed to analyze the role of CAIF and miR-20a in the autophagy of HFLSs. Each experiment was performed using 10,000 ells in a 96-well plate. The fluorescence was analyzed using a Gemini XPS fluorescent plate reader. The normalized data represent the data of three biological replicates. CAIF: Cardiac autophagy inhibitor; HFLSs: Human fibroblast-like synoviocytes; * p<0.05.

Supplemental Figure 1.. The expression of CAIF, mature miR-20a and miR-20a precursor were altered in synovial tissues from RA patients. (a) Expression of CAIF, (b) mature miR-20a and (c) miR-20a precursor in synovial tissue samples from both 62 RA patients and controls was determined by RT-qPCR. Mean values of three technical replicates were used to express gene expression levels. CAIF: Cardiac autophagy inhibitor; RA: Rheumatoid arthritis; RT-qPCR: Reverse transcription-quantitative polymerase chain reaction; * p<0.05.