Expression changes of circRNA_009396 in peripheral blood of atrial fibrillation patients and the effects of its overexpression on fibrosis of HCFs

LI Weiyan; CHEN Can

doi:10.3969/j.issn.1002-266X.2025.06.003

Abstract：Objective To investigate the expression changes of circular RNA(circRNA)_009396 in the peripheral blood of atrial fibrillation(AF)patients,and to explore the effects of circRNA_009396 overexpression on fibrosis of Hu-man cardiac fibroblasts(HCFs).Methods Forty-four patients with AF(AF group),and 44 healthy volunteers who re-ceived comprehensive physical examinations(non-AF group)were selected.Real-time fluorescence quantitative PCR was employed to measure the levels of circRNA_009396 in peripheral blood samples from both groups.Subsequently,the re-ceiver operating characteristic(ROC)curve for circRNA_009396 in diagnosing AF was constructed,and its diagnostic effi-cacy was systematically evaluated.HCFs of the 3rd to 5th passages were treated with 20 μmol/L angiotensin Ⅱ(Ang Ⅱ)for 48 h to induce fibrosis.Western blotting confirmed that the relative expression levels of type Ⅰ collagen α1 chain(COL1A1),type Ⅲ collagen α1 chain(COL3A1),and α-smooth muscle actin(α-SMA),which were markers of cellular fibrosis,significantly increased,thereby validating the successful establishment of the fibrosis of HCFs.The the HCFs in-duced to fibrosis were subsequently divided into the observation group and control group.In the observation group,cells were transfected with 2.5 μg circRNA_009396 overexpression plasmid using 4 μL of Lipo8000 transfection reagent,whereas the control group remained untreated.Real-time fluorescence quantitative PCR was utilized to assess the relative mRNA expression levels of circRNA_009396,COL1A1,COL3A1,and α-SMA in the cells,while Western blotting was performed to determine the relative protein expression levels of COL1A1,COL3A1,and α-SMA.Results The levels of circRNA_009396 in the peripheral blood of the AF group and the non-AF group were 0.46±0.05 and 1.22±0.10,re-spectively,with statistically significant difference between the two groups(P<0.05).The area under the curve(AUC)for diagnosing AF using peripheral blood circRNA_009396 was 0.902.When the cut-off value was set at 0.542,the sensitiv-ity reached 0.755,and the specificity was 0.930.Additionally,the relative expression level of circRNA_009396 in the observation group was significantly higher than that in the control group(P<0.05).Conversely,the relative expression levels of COL1A1,COL3A1,and α-SMA mRNA in the observation group were all significantly lower than those in the control group(all P<0.05).Similarly,the protein expression levels of COL1A1,COL3A1,and α-SMA in the observa-tion group were significantly lower than those in the control group(all P<0.05).Conclusion The expression level of circRNA_009396 in the peripheral blood of AF patients is significantly reduced and overexpression of circRNA_009396 may inhibit Ang Ⅱ-induced fibrosis of HCFs.