Majumdar Swagata, Chowdhury Deeya Roy, Chakraborty Bidhan Chandra, Chowdhury Abhijit, Datta Simanti, Banerjee Soma
Centre for Liver Research, School of Digestive and Liver Diseases, Institute of Post Graduate Medical Education and Research, Kolkata, West Bengal, India.
Multi-disciplinary Research Unit, Institute of Post Graduate Medical Education and Research, Kolkata, West Bengal, India.
J Transl Med. 2025 Mar 13;23(1):322. doi: 10.1186/s12967-025-06286-9.
Lipotoxicity is one of the causes for the progression of fatty liver in chronic hepatitis (CH) towards end-stage liver diseases. The role of miRNAs in the signalling pathways of lipid metabolism has been studied, but their direct targets in this pathway have not been identified yet. Here, we have characterized a downregulated miRNA in CH namely miR-451a, which has a direct impact on the lipid metabolism pathway.
Liver tissue samples and blood were collected from CHC/CHB patients and normal individuals. Huh7 and SNU449 cell lines were used for in vitro assays. Expressions of miRNA/mRNAs and proteins were confirmed by qRT-PCR and immuno-blot analysis. Oil Red O staining, Colorimetric, and Fluorometric assay kit were used to quantify triglyceride (TG) and cholesterol from tissue and serum, respectively. Target prediction and pathway analysis were performed using Targetscan, miRWalk, and DAVID respectively. 3'UTR-Luciferase assay and Co-immuno-precipitation were conducted to determine direct interaction between miRNA-mRNA and protein-protein, respectively. Unpaired two-tailed Student's t-test and Mann-Whitney test were employed as required using GraphPad prism. P < 0.05 was considered as significant.
The miRNA, miR-451a was selected as one of the downregulated miRNAs in progressive liver disease stages of CHC and CHB. Target identification and pathway analysis of this miRNA revealed that lipid metabolism pathway gene, glycerol kinase (GK), could be the target of this miRNA. Subsequent 3'UTR Luciferase assay and immuno-blot analysis confirmed the binding of miR-451a to GK. Though both hepatitis viruses, HCV and HBV, could alter the lipid metabolism pathways, intracellular TG and cholesterol content were observed to be significantly higher upon HCV infection only. It also suppressed the expression of miR-451a, resulting in overshooting of GK expression. GK interacted positively with the transcription factor SREBP1, which led to overexpression of Fatty acid synthase, Acetyl- CoA Carboxylase, and Stearoyl-CoA desaturase. As a result, intracellular fatty acids, TG, and cholesterol synthesis and accumulation heightened but trafficking dropped, resulting in hypo-cholesterolemia in blood. While, restoration of miR-451a impeded lipid accumulation, reduced steatohepatitis and suppressed HCV replication as well.
These findings suggest that the alteration in the hepatic lipid profile upon HCV/HBV infection is attributed to the downregulation of miR-451a, which has the potential to restrict the expression of GK and SREBP1 in the TG biosynthesis pathway, implying that supplementation of miR-451a may be a potential therapeutic strategy for impeding CHC.
脂毒性是慢性肝炎（CH）中脂肪肝向终末期肝病进展的原因之一。miRNA在脂质代谢信号通路中的作用已得到研究，但其在该通路中的直接靶点尚未确定。在此，我们鉴定了一种在CH中表达下调的miRNA，即miR-451a，它对脂质代谢通路有直接影响。
收集慢性丙型肝炎/慢性乙型肝炎（CHC/CHB）患者和正常个体的肝组织样本及血液。使用Huh7和SNU449细胞系进行体外实验。通过qRT-PCR和免疫印迹分析确认miRNA/mRNA和蛋白质的表达。分别使用油红O染色、比色法和荧光测定试剂盒对组织和血清中的甘油三酯（TG）和胆固醇进行定量。分别使用Targetscan、miRWalk和DAVID进行靶标预测和通路分析。进行3'UTR荧光素酶测定和免疫共沉淀以分别确定miRNA与mRNA之间以及蛋白质与蛋白质之间的直接相互作用。根据需要使用GraphPad prism进行非配对双尾学生t检验和曼-惠特尼检验。P < 0.05被认为具有统计学意义。
miRNA miR-451a被选为CHC和CHB进展性肝病阶段中表达下调的miRNA之一。对该miRNA的靶标鉴定和通路分析表明，脂质代谢通路基因甘油激酶（GK）可能是该miRNA的靶标。随后的3'UTR荧光素酶测定和免疫印迹分析证实了miR-451a与GK的结合。虽然丙型肝炎病毒（HCV）和乙型肝炎病毒（HBV）都可改变脂质代谢通路，但仅在HCV感染时观察到细胞内TG和胆固醇含量显著升高。它还抑制了miR-451a的表达，导致GK表达过度。GK与转录因子固醇调节元件结合蛋白1（SREBP1）正向相互作用，导致脂肪酸合酶、乙酰辅酶A羧化酶和硬脂酰辅酶A去饱和酶过表达。结果，细胞内脂肪酸、TG和胆固醇的合成与积累增加，但转运下降，导致血液中胆固醇过低。而miR-451a的恢复可阻止脂质积累，减轻脂肪性肝炎并抑制HCV复制。
这些发现表明，HCV/HBV感染后肝脏脂质谱的改变归因于miR-451a的下调，它有可能限制TG生物合成途径中GK和SREBP1的表达，这意味着补充miR-451a可能是阻碍CHC的一种潜在治疗策略。
