Centre for Liver Research, School of Digestive and Liver Diseases, Institute of Post Graduate Medical Education and Research , Kolkata, India.
Human Genetics Unit, Indian Statistical Institute , Kolkata, Human Genetics Unit, India.
RNA Biol. 2020 Sep;17(9):1352-1363. doi: 10.1080/15476286.2020.1768353. Epub 2020 Jun 7.
Imbalance in lipid metabolism induces steatosis in liver during Chronic hepatitis C (CHC). Contribution of microRNAs in regulating lipid homoeostasis and liver disease progression is well established using small RNA-transcriptome data. Owing to the complexity in the development of liver diseases, the existence and functional importance of yet undiscovered regulatory miRNAs in disease pathogenesis was explored in this study using the unmapped sequences of the transcriptome data of HCV-HCC liver tissues following miRDeep2.pl pipeline. MicroRNA-c12 derived from the first intron of LGR5 of chromosome 12 was identified as one of the miRNA like sequences retrieved in this analysis that showed human specific origin. Northern blot hybridization has proved its existence in the hepatic cell line. Enrichment of premiR-c12 in dicer-deficient cells and miR-c12 in Ago2-RISC complex clearly suggested that it followed canonical miRNA biogenesis pathway and accomplished its regulatory function. Expression of this miRNA was quite low in CHC tissues than normal liver implying HCV-proteins might be regulating its biogenesis. Promoter scanning and ChIP analysis further revealed that under expression of p53 and hyper-methylation of STAT3 binding site upon HCV infection restricted its expression in CHC tissues. Centrosomal protein 350 (CEP350), which sequestered PPARα, was identified as one of the targets of miR-c12 using Miranda and validated by luciferase assay/western blot analysis. Furthermore, reduced triglyceride accumulation and enhanced PPARα mediated transcription of β-oxidation genes upon restoration of miR-c12 in liver cells suggested its role in lipid catabolism. Thus this study is reporting miR-c12 for the first time and showed its' protective role during chronic HCV infection.
脂质代谢失衡会导致慢性丙型肝炎（CHC）患者肝脏脂肪变性。利用小 RNA 转录组数据，已经证实 microRNA 在调节脂质稳态和肝病进展方面发挥着重要作用。由于肝脏疾病的发展较为复杂，本研究利用 HCV-HCC 肝组织转录组数据中未映射序列，通过 miRDeep2.pl 管道，探索了疾病发病机制中尚未发现的调控 microRNA 的存在和功能重要性。microRNA-c12 来源于染色体 12 上 LGR5 的第一内含子，是本分析中检索到的具有人类特异性起源的 microRNA 类似序列之一。Northern blot 杂交证明了其在肝细胞系中的存在。pre-miR-c12 在 dicer 缺陷细胞中的富集和 miR-c12 在 Ago2-RISC 复合物中的富集清楚地表明，它遵循典型的 microRNA 生物发生途径，并发挥其调节功能。与正常肝脏组织相比，该 microRNA 在 CHC 组织中的表达水平较低，这表明 HCV 蛋白可能调控其生物发生。启动子扫描和 ChIP 分析进一步表明，p53 表达下调和 STAT3 结合位点的高甲基化限制了其在 CHC 组织中的表达。微管相关蛋白 350（CEP350），它能隔离 PPARα，被鉴定为 miR-c12 的一个靶点，通过 Miranda 进行验证，并通过荧光素酶测定/western blot 分析进行验证。此外，在恢复肝细胞中 miR-c12 表达后，甘油三酯积累减少，PPARα 介导的β氧化基因转录增强，提示其在脂质分解代谢中的作用。因此，本研究首次报道了 miR-c12，并显示其在慢性 HCV 感染期间具有保护作用。
