Abstract：
BACKGROUND: Excessive fatty acids in the liver lead to the accumulation of lipotoxic lipids and then cellular stress to further evoke the related disease, like non-alcoholic fatty liver disease (NAFLD). As reported, fatty acid stimulation can cause some specific miRNA dysregulation, which caused us to investigate the relationship between miRNA biogenesis and fatty acid overload.
METHODS: Gene expression omnibus (GEO) dataset analysis, miRNA-seq, miRNA cleavage assay, RT-qPCR, western blotting, immunofluorescence and co-immunoprecipitation (co-IP) were used to reveal the change of miRNAs under pathological status and explore the relevant mechanism. High fat, high fructose, high cholesterol (HFHFrHC) diet-fed mice transfected with AAV2/8-shDrosha or AAV2/8-shPRMT5 were established to investigate the in vivo effects of Drosha or PRMT5 on NAFLD phenotype.
RESULTS: We discovered that the cleavage of miRNAs was inhibited by analysing miRNA contents and detecting some representative pri-miRNAs in multiple mouse and cell models, which was further verified by the reduction of the Microprocessor activity in the presence of palmitic acid (PA). In vitro, PA could induce Drosha, the core RNase III in the Microprocessor complex, degrading through the proteasome-mediated pathway, while in vivo, knockdown of Drosha significantly promoted NAFLD to develop to a more serious stage. Mechanistically, our results demonstrated that PA can increase the methyltransferase activity of PRMT5 to degrade Drosha through MDM2, a ubiquitin E3 ligase for Drosha. The above results indicated that PRMT5 may be a critical regulator in lipid metabolism during NAFLD, which was confirmed by the knocking down of PRMT5 improved aberrant lipid metabolism in vitro and in vivo.
CONCLUSIONS: We first demonstrated the relationship between miRNA dosage and NAFLD and proved that PA can activate the PRMT5-MDM2-Drosha signalling pathway to regulate miRNA biogenesis.
METHODS: Gene expression omnibus (GEO) dataset analysis, miRNA-seq, miRNA cleavage assay, RT-qPCR, western blotting, immunofluorescence and co-immunoprecipitation (co-IP) were used to reveal the change of miRNAs under pathological status and explore the relevant mechanism. High fat, high fructose, high cholesterol (HFHFrHC) diet-fed mice transfected with AAV2/8-shDrosha or AAV2/8-shPRMT5 were established to investigate the in vivo effects of Drosha or PRMT5 on NAFLD phenotype.
RESULTS: We discovered that the cleavage of miRNAs was inhibited by analysing miRNA contents and detecting some representative pri-miRNAs in multiple mouse and cell models, which was further verified by the reduction of the Microprocessor activity in the presence of palmitic acid (PA). In vitro, PA could induce Drosha, the core RNase III in the Microprocessor complex, degrading through the proteasome-mediated pathway, while in vivo, knockdown of Drosha significantly promoted NAFLD to develop to a more serious stage. Mechanistically, our results demonstrated that PA can increase the methyltransferase activity of PRMT5 to degrade Drosha through MDM2, a ubiquitin E3 ligase for Drosha. The above results indicated that PRMT5 may be a critical regulator in lipid metabolism during NAFLD, which was confirmed by the knocking down of PRMT5 improved aberrant lipid metabolism in vitro and in vivo.
CONCLUSIONS: We first demonstrated the relationship between miRNA dosage and NAFLD and proved that PA can activate the PRMT5-MDM2-Drosha signalling pathway to regulate miRNA biogenesis.
摘要：
背景：肝脏中脂肪酸过多导致脂毒脂质的积累，然后导致细胞应激，从而进一步引起相关疾病，如非酒精性脂肪性肝病（NAFLD）。据报道，脂肪酸刺激可以引起一些特定的miRNA失调，这促使我们研究miRNA生物发生与脂肪酸过载之间的关系。
方法：基因表达综合(GEO)数据集分析，miRNA-seq,miRNA裂解试验,RT-qPCR,西方印迹,免疫荧光和免疫共沉淀（co-IP）用于揭示病理状态下miRNAs的变化并探讨相关机制。高脂肪,高果糖,建立了转染AAV2/8-shDrosha或AAV2/8-shPRMT5的高胆固醇饮食（HFHFrHC）小鼠，以研究Drosha或PRMT5对NAFLD表型的体内影响。
结果：我们发现通过分析miRNA含量并检测多种小鼠和细胞模型中的一些代表性pri-miRNAs来抑制miRNA的裂解，这通过在棕榈酸(PA)存在下微处理器活性的降低进一步证实。体外，PA可以诱导Drosha，微处理器复合体中的核心核糖核酸酶III，通过蛋白酶体介导的途径降解，而在体内，Drosha的击倒显着促进NAFLD发展到更严重的阶段。机械上,我们的结果表明，PA可以增加PRMT5的甲基转移酶活性，从而通过Drosha的泛素E3连接酶MDM2降解Drosha。以上结果表明，PRMT5可能是NAFLD过程中脂质代谢的关键调节因子。这通过PRMT5的敲低在体外和体内改善异常的脂质代谢得到证实。
结论：我们首先证明了miRNA剂量与NAFLD之间的关系，并证明PA可以激活PRMT5-MDM2-Drosha信号通路来调节miRNA的生物发生。
方法：基因表达综合(GEO)数据集分析，miRNA-seq,miRNA裂解试验,RT-qPCR,西方印迹,免疫荧光和免疫共沉淀（co-IP）用于揭示病理状态下miRNAs的变化并探讨相关机制。高脂肪,高果糖,建立了转染AAV2/8-shDrosha或AAV2/8-shPRMT5的高胆固醇饮食（HFHFrHC）小鼠，以研究Drosha或PRMT5对NAFLD表型的体内影响。
结果：我们发现通过分析miRNA含量并检测多种小鼠和细胞模型中的一些代表性pri-miRNAs来抑制miRNA的裂解，这通过在棕榈酸(PA)存在下微处理器活性的降低进一步证实。体外，PA可以诱导Drosha，微处理器复合体中的核心核糖核酸酶III，通过蛋白酶体介导的途径降解，而在体内，Drosha的击倒显着促进NAFLD发展到更严重的阶段。机械上,我们的结果表明，PA可以增加PRMT5的甲基转移酶活性，从而通过Drosha的泛素E3连接酶MDM2降解Drosha。以上结果表明，PRMT5可能是NAFLD过程中脂质代谢的关键调节因子。这通过PRMT5的敲低在体外和体内改善异常的脂质代谢得到证实。
结论：我们首先证明了miRNA剂量与NAFLD之间的关系，并证明PA可以激活PRMT5-MDM2-Drosha信号通路来调节miRNA的生物发生。
