miR374b在促甲状腺激素致肝脂肪变中的作用及机制

Non-alcoholic fatty liver disease (NAFLD) is a risk factor for cardiovascular diseases. Hallmarks of NAFLD are hepatic steatosis. Recently, microRNAs have been considered as a key regulator in the progress of NAFLD. Additionally, subclinical hypothyroidism (SCH), characterized by an elevated thyrotropin (TSH) level and a normal free thyroxine (T4) level, has recently been demonstrated as a risk factor for NAFLD. Emerging evidence revealed that the prevalence of NAFLD increases steadily with increasing TSH levels. As TSH is the only thyroid function component affected in SCH, it raises the question that whether TSH might also plays an important role in the development of NAFLD. microRNAs as crucial regulators of NAFLD pathogenesis and progression.. Our previous study suggested that TSH increases lipid accumulation in liver and ultimately promotes the development of NAFLD. Additionally, our latest reports showed that compared with wild type (Tshr+/+) mice, Tshr-/- mice exhibit a relatively lower degree of liver steatosis and a decreased miR374b expression induced through a high fat diet. Moreover, using microarray technology we demonstrated that TSH regulates the expression of potential targets of miR374b. Whether miR374b takes part in the process of TSH regulation on hepatic steatosis and the exact functional mechanisms are still uncertain. Therefore, using both in vitro (TSH stimulation on liver cells, interference, blocking or activation of the key molecules) and in vivo (conditionally knockout of liver Tshr /miR374b gene mouse model, TSHR-RNAi mouse model) experiments and the relative key targets intervention approaches, we aim to elucidate the key molecules and major signaling networks about the effect of miR374b in TSH-induced steatosis. Our study will explore the pathophysioligical mechanism of TSH-induce NAFLD and further provide newly theoretical evidence for treatment strategy.

非酒精性脂肪肝（NAFLD）患病率持续增长危害严重，其主要特征是肝脂肪变。研究显示：1.microRNAs 是NAFLD发生发展中的关键调控因素； 2.NAFLD患病率随血清促甲状腺激素（TSH）水平升高而增加，机制未完全阐明。课题组前期研究发现：1.TSH诱发肝脂肪变；2.Tshr基因敲除小鼠肝脂肪变减轻，miR374b表达明显下降；3.miR374b的多个潜在靶基因是参与脂代谢调控的关键分子。目前，关于miR374b在TSH致肝脂肪变中的作用未见国内外报道。本项目应用体内（条件性敲除甲状腺或肝脏Tshr基因、条件性敲除肝脏miR374b基因等多种动物模型）与体外（TSH刺激肝细胞、沉默基因表达及阻断传递等技术）实验相结合技术手段，阐明TSH通过miR374b介导诱发肝脂肪变的机制，为揭示TSH在NAFLD发生发展中的作用提供新思路。

非酒精性脂肪性肝病（NAFLD）已成为世界范围内的重要健康问题之一，约20%的NASH患者可进展为肝硬化，其主要特征是肝脂肪变。NAFLD的发病机制极为复杂，其中，microRNAs是NAFLD发生发展中的关键调控因素，microRNAs 在糖脂代谢、氧化应激、细胞凋亡、炎症、内质网应激和胰岛素抵抗等多个方面对 NAFLD 产生广泛的影响。NAFLD 患病率随血清促甲状腺激素（TSH）水平升高而增加，TSH 对于 NAFLD 的发生有重要作用，并且并不依赖于甲状腺激素。课题组前期研究证实TSH诱发肝脂肪变，TSHR基因敲除小鼠肝脂肪变减轻，miR374b表达明显下降以及miR374b的多个潜在靶基因是参与脂代谢调控的关键分子。 故基于此研究背景和前期研究成果，本研究探讨TSH能否通过肝细胞上的 TSHR，上调 miR374b 表达，然后 miR374b 作用于其靶基因，进而调控脂质代谢关键分子，引起肝细胞脂质蓄积，诱发肝脂肪变，从而阐明 miR374b 在 TSH 诱发肝脂肪变中的作用及机制。我们研究发现：1.随着TSH浓度的增加及作用时间的延长，会加重肝脏脂质的沉积，细胞损伤加重，自噬增加,并且miRNA374b的表达量呈现同样趋势；2. miR374b 可以识别靶基因 C/EBPβ及 FoxO1 mRNA 的3’-UTR 区并且对翻译过程起到抑制作用，从而使其下游分子PPARγ、SREBP2、miR33a、SREBP1c的表达增加；3. C57BL/6 小鼠，尾静脉注射 miR374b 的模拟物后，肝脏脂肪变加重。4.沉默肝细胞 TSHR 表达或条件性敲除肝脏 Tshr 基因后，miR374b 的表达降低，肝脂肪变减轻；5. miRNA374b基因敲除鼠的肝脏脂肪变较正常小鼠减轻，给予btsh注射后，脂肝脏质沉积无明显增加；6.SREBP1c 基因敲除鼠给予尾静脉注射 miR374b agomir后，肝脏脂质沉积无明显增加。 研究发现miR374b 能够作用于C/EBPβ，抑制其转录稳定性和翻译，进而抑制 PPARγ信号通路，以及作用于FoxO1，抑制其表达，进而作用于SREBP1c，在脂质代谢与胰岛素抵抗中发挥重要作用，本研究成果有望进一步揭示TSH 调节肝脏脂质代谢的分子机制，并为NAFLD 的的预测及寻找干预靶点提供依据。