miR-10a/Klotho在TGF-β诱导肾小管上皮细胞脂肪酸β氧化障碍中的作用

Klotho (KL) , a gene of anti-aging, plays a key protective role in renal interstitial fibrosis. Defective fatty acid oxidation of renal tubular epithelial cells induced by TGF-β has a key role in kidney fibrosis development, although the mechanism is not clear. TGF-β could induce miR-10a, which maybe transcriptional regulate KL gene. The current project is aimed at further understanding of roles of miR-10a/KL in defective fatty acid oxidation of renal tubular epithelial cells induced by TGF-β. Firstly, we are going to uncover the influence of TGF-β on miR-10a/KL and defective fatty acid oxidation by plasmid transfection. Secondly, using UUO model built on KLTG mice and KLhe mice, the influence of TGF-β inhibitor on miR-10a、KL and defective fatty acid oxidation will be explored after transfection in vivo. Finally, we will study the correlation of TGF-β、miR-10a、KL and defective fatty acid oxidation of clinical samples from different stage of chronic kidney diseases using DNA-gold nanoparticle probes. This project is critical for the exploring new mechanism of renal interstitial fibrosis induced by TGF-β and the finding of new molecular pathological marker.

抗衰老基因Klotho（KL）在肾间质纤维化进展中起肾保护作用。TGF-β诱导的肾小管上皮细胞脂肪酸β氧化障碍在肾纤维化进展中起关键作用，但其机制尚不清楚。TGF-β可上调miR-10a，我们发现后者可能调控KL表达，因此认为TGF-β可通过miR-10a/KL引起脂肪酸β氧化障碍。本研究拟从细胞水平通过对人肾小管上皮细胞转染，给予TGF-β，研究TGF-β对miR-10a、KL和脂肪酸β氧化相关CPT、ACOX等的影响；从动物水平通过在KLTG、KLhe小鼠建立UUO模型后体内转染，探索抑制TGF-β对miR-10a、KL和脂肪酸β氧化关键分子的影响；最后从临床样本入手，借助DNA-金纳米探针验证TGF-β、miR-10a、KL等的相关性，阐明miR-10a在TGF-β诱导的脂肪酸β氧化障碍中的作用机制。本研究对探索TGF-β诱导肾间质纤维化的新途径，发现新分子标志物具有重要的指导意义。

肾小管萎缩、间质纤维化是各种慢性肾脏病终末期的共同病理表现，研究表明由TGF-β引起的肾小管上皮细胞脂肪酸β氧化障碍是肾小管上皮细胞死亡的主要原因，并在慢性肾脏病肾纤维化进展中起关键作用。近年发现Klotho在肾间质纤维化进程中起关键性肾保护作用，其启动子区域范围较大，表达调控方式复杂，相关研究提示反式作用因子可能是该基因的主要调控途径。本研究从miR-10a入手，通过体外过表达和低表达肾小管上皮细胞内miR-10a的实验证实了miR-10a可通过负向调控Klotho表达，增强TGF-β诱导的肾小管上皮细胞脂肪酸β氧化障碍。体内实验通过在野生型和Klotho基因敲除的UUO模型小鼠运用RNAi技术干预miR-10a水平发现：miR-10a shRNA可减轻野生小鼠UUO模型的肾脏纤维化程度，下调TGF-β，增加Klotho及脂肪酸β氧化进程关键分子表达水平，但不能改善Klotho基因敲除小鼠UUO模型肾脏纤维化及脂肪酸β氧化障碍，证实了沉默miR-10a可改善UUO模型肾组织的脂肪酸β氧化障碍，对UUO 小鼠肾脏纤维化有保护作用，且这一作用与Klotho密切相关，揭示了抑制TGF-β诱导的肾间质纤维化的新途径。