支链氨基酸氨基转移酶1在转化生长因子-β1刺激的NIH-3T3成纤维细胞向肌成纤维细胞表型转变中的作用

    Role of branched-chain amino acid aminotransferase-1 in phenotypic transition of NIH-3T3 fibroblasts towards myofibroblasts stimulated by transforming growth factor-β1

      摘要
    • 摘要:
      目的 探索成纤维细胞向肌成纤维细胞转化中支链氨基酸氨基转移酶1（branched-chain amino acid transaminase-1，BCAT1）对纤维化相关基因表达及表型的调控作用。
      方法 NIH-3T3小鼠胚胎成纤维细胞系分别转染腺病毒为载体的BCAT1敲低和过表达病毒后，给予转化生长因子-β1（transforming growth factor-β1，TGFβ1）诱导NIH-3T3细胞转化为肌成纤维细胞。利用实时定量PCR、Western blot、免疫荧光实验等检测TGFβ1诱导的纤维化相关基因表达变化。同时通过细胞划痕实验、Transwell、胶收缩实验等检测NIH-3T3细胞向肌成纤维细胞转化的迁移、收缩等表型变化。
      结果 TGFβ1刺激诱导NIH-3T3细胞转化为肌成纤维细胞时BCAT1 mRNA及蛋白表达水平显著升高（均P<0.01）。使用短发卡RNA（short hairpin RNA，shRNA）敲低NIH-3T3细胞的BCAT1表达显著抑制TGFβ1诱导的纤维化相关基因Acta2和Postn的mRNA表达（均P<0.01）及与纤维化过程相关的细胞迁移、收缩等肌成纤维细胞表型（均P<0.01）。反之，过表达BCAT1显著促进TGFβ1诱导的纤维化相关基因表达和肌成纤维细胞表型（均P<0.01）。
      结论 BCAT1是调节成纤维细胞纤维化相关基因表达及其向肌成纤维细胞表型转变的关键分子，可能成为治疗和调控器官纤维化的潜在靶点。

       

      Abstract:
      AIM To explore the effect of branched chain amino acid transaminase 1 (BCAT1) on fibrosis related gene expression and phenotype during the transformation of fibroblasts into myofibroblasts.
      METHODS NIH-3T3 mouse embryonic fibroblast cell line was manipulated to knock down or over-express BCAT1 using adenovirus transfection, followed by stimulation with TGFβ1 to induce NIH-3T3 cells transition into myofibroblasts. The expression of fibrosis-related genes was assessed through RT-qPCR, Western blot and immunofluorescence, while fibrosis-related phenotypic changes were evaluated using cell scratch tests, Transwell assays and gel contraction tests.
      RESULTS The results showed a significant increase in both BCAT1 mRNA and protein levels upon TGFβ1 stimulation in NIH-3T3 cells (both P<0.01). Knockdown of BCAT1 using shRNA significantly inhibited the expression of TGFβ1-induced fibrosis-related genes Acta2 and Postn mRNA (both P<0.01) as well as fibrosis-related migration and contraction phenotypes (all P<0.01). Conversely, over-expression of BCAT1 increased the expression of these genes and phenotypes (all P<0.01).
      CONCLUSION BCAT1 plays a crucial role in regulating the expressions of fibrosis-related genes and phenotypes during the transition of NIH-3T3 fibroblasts towards myofibroblasts, suggesting its potential as a target for fibrosis treatment.

       

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