| 摘要: |
| 目的 探讨lncRNA成纤维细胞活化抑制因子1（FAIF1）调控晚期糖基化终产物（AGE）诱导的人心肌成纤维细胞增殖、活化及纤维化的机制。方法 将人心肌成纤维细胞分为对照组、AGE组、FAIF1重组慢病毒（Lv-FAIF1）＋AGE组和对照慢病毒（Lv-control）＋AGE组。通过qPCR和蛋白质印迹法检测AGE诱导下人心肌成纤维细胞中miRNA-424-5p、FAIF1及Smad7的表达水平。通过生物信息学分析预测miRNA-424-5p、FAIF1和Smad7的相互关系，并用萤光素酶报告基因实验验证。利用CCK-8检测细胞增殖活性，免疫荧光染色观察Ⅰ/Ⅲ型胶原蛋白的表达分泌情况，qPCR评估Lv-FAIF1对AGE诱导细胞活化标志物α-平滑肌肌动蛋白（α-SMA）和基质金属蛋白酶9（MMP9）表达的影响。结果 qPCR及蛋白质印迹法检测结果显示，AGE诱导后人心肌成纤维细胞中FAIF1和Smad7表达降低，miRNA-424-5p表达升高（与对照组比较，均P＜0.05）。生物信息学分析显示，Smad7 mRNA 3'-非翻译区存在miRNA-424-5p结合位点（序列为UGCUGCU），而FAIF1序列中存在3个相同的结合位点。萤光素酶报告基因实验显示，miRNA-424-5p抑制Smad7的表达，而FAIF1通过竞争性结合miRNA-424-5p解除miRNA-424-5p对Smad7 mRNA的抑制作用。功能实验显示，Lv-FAIF1能够抑制AGE诱导的细胞增殖、Ⅰ/Ⅲ型胶原蛋白表达分泌及α-SMA和MMP9表达（与AGE组比较，均P＜0.01），促进Smad7的表达（与AGE组比较，P＜0.01）。结论 miRNA-424-5p抑制Smad7的表达，FAIF1通过调控miRNA-424-5p/Smad7轴有效抑制AGE诱导的人心肌成纤维细胞过度活化。本研究为糖尿病心肌病的防治提供了新的分子靶点。 |
| 关键词: 长链非编码RNA 微RNA-424-5p Smad7 心肌成纤维细胞 晚期糖基化终产物 |
| DOI：10.16781/j.CN31-2187/R.20250045 |
| 投稿时间：2025-01-20修订日期：2025-03-05 |
| 基金项目: |
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| lncRNA FAIF1 regulates the inhibitory effect of miRNA-424-5p/Smad7 axis against cardiac fibroblast dysfunction induced by advanced glycation end products |
| YUE Wenheng,HUANG Kun,WU Yue,WEN Jiayu,LIANG Chun* |
(Department of Cardiovasology, The Second Affiliated Hospital of Naval Medical University (Second Military Medical University), Shanghai 200003, China
*Corresponding author) |
| Abstract: |
| Objective To explore the mechanism of long non-coding RNA fibroblast activation inhibitory factor 1 (FAIF1) regulates the proliferation, activation, and fibrosis of human cardiac fibroblasts induced by advanced glycation end products (AGEs). Methods Human cardiac fibroblasts were assigned to control group, AGE group, FAIF1 recombinant lentivirus (Lv-FAIF1)＋AGE group or control lentivirus (Lv control)＋AGE group. The expression levels of miRNA-424-5p, FAIF1, and Smad7 in myocardial fibroblasts induced by AGEs were detected by quantitative polymerase chain reaction (qPCR) and Western blotting. Bioinformatics analysis was used to predict the interactions between miRNA-424-5p, FAIF1, and Smad7; and luciferase reporter assays were used for verification. Cell proliferation activity was measured by cell counting kit 8 assay, the expression and secretion of collagen Ⅰ/Ⅲ were observed by immunofluorescence staining, and the effect of Lv-FAIF1 on cell activation markers α-smooth muscle actin (α-SMA) and migration proteins matrix metalloproteinase 9 (MMP9) induced by AGEs was evaluated by qPCR. Results qPCR and Western blotting results showed that AGEs significantly reduced the expression of FAIF1 and Smad7 in myocardial fibroblasts and upregulated the level of miRNA-424-5p (compared with the control group, all P＜0.05). Bioinformatics analysis revealed that the 3'-untranslated region of Smad7 mRNA contained a binding site for the miRNA-424-5p seed sequence “UGCUGCU”, and FAIF1 sequence contained 3 identical binding sites. Luciferase assays showed that miRNA-424-5p inhibited the expression of Smad7, while FAIF1 competed with miRNA-424-5p for binding, thereby relieving the inhibitory effect of miRNA-424-5p on Smad7 mRNA. Functional experiments showed that Lv-FAIF1 significantly inhibited AGEs-induced cell proliferation, collagenⅠ/Ⅲ expression and secretion, as well as α-SMA and MMP9 expression (compared with AGE group, all P＜0.01); and it promoted the expression of Smad7 (compared with AGE group, P＜0.01). Conclusion miRNA-424-5p can inhibit the expression of Smad7, and FAIF1 effectively suppresses AGEs-induced over-activation of cardiac fibroblasts by regulating the miRNA-424-5p/Smad7 axis, which provides a new molecular target for the prevention and treatment of diabetic cardiomyopathy. |
| Key words: long non-coding RNA microRNA-424-5p Smad7 cardiac fibroblasts advanced glycation end products |
