miR-130a作用于LDLR抑制HCV复制的分子机制研究

Hepatitis C is an infectious disease caused by hepatitis C virus (HCV) infection. Despite the presence of DAA treatment，HCV infection is becoming an increased threat to our health system due to viral resistance and lack of vaccine. It has been shown that microRNA plays an important role in anti-viral process, but the mechanism of different microRNA is also different. Our previous study demonstrated that microRNA 130a (miR-130a) inhibits HCV replication in Huh7.5.1 cells, but the molecular mechanism remains unclear. Further bioinformatics analysis, fluorescence quantitative PCR and Western blot found that LDLR was a potential target of miR-130a, and the target was preliminary validated by double luciferase reporter gene experiments (wild type). LDLR is not only the receptor of HCV, but also the key molecule of lipid metabolism. This study proposed to construct the 3 'UTR region of the LDLR genes (mutant) to the vector which contains luciferase gene, then confirm the target of miR-130a by luciferase reporter gene experiments. Subsequently, construct the expression vector of the LDLR gene, then, study the function of the LDLR gene for the HCV replication (endocytosis, replication and secretion), as well as the key molecules related to lipid metabolism and HCV replication (such as CD36, FAS etc.) by overexpression and gene silencing experiments, finally, the correlation between miR-130a expression and HCV titer was verified at the clinical level, so as to reveal the molecular mechanism of miR-130a inhibit the HCV replication. Results from these studies will shed light not only on the anti-viral mechanism of microRNA but also on the new drug screening and new method for HCV therapy.

丙型肝炎是由丙型肝炎病毒引起的传染性疾病，尽管DAA已广泛应用于临床，但由于病毒耐药及疫苗缺乏等原因，其危害仍然严重。已有研究证实小分子RNA在抗病毒过程中具有极为重要的作用，但不同小分子的作用机制也不同。我们前期研究发现：过表达miR-130a能显著抑制HCV的复制，进一步实验提示LDLR为miR-130a的潜在靶点，后经双荧光素酶报告基因（野生型）实验得到初步验证。LDLR既是HCV的入胞受体，又是脂代谢关键分子。因此，本研究拟通过突变型实验确认miR-130a的靶点；在此基础上，构建LDLR的表达载体，通过过表达及基因敲除等实验研究其对HCV复制（入胞、复制及分泌）以及与HCV复制相关的脂代谢相关分子（如CD36、FAS等）的影响；最后在临床水平验证miR-130a表达与HCV滴度的关联，从而揭示miR-130a抑制HCV复制的分子机制。研究成果将为丙型肝炎的治疗提供潜在的药物靶点。

丙型肝炎是由丙型肝炎病毒引起的传染性疾病，尽管直接抗病毒药物已广泛应用于临床，但由于病毒耐药及疫苗缺乏等原因，其危害仍然严重。已有研究证实小分子RNA在抗病毒过程中具有极为重要的作用，但不同小分子的作用机制也不同。本项目研究在人体内广泛存在的小分子RNA130a（miR-130a）对HCV复制的影响，发现了miR-130a可以显著抑制HCV的复制，进一步研究发现，miR-130a通过作用于靶点PKLR来抑制HCV的复制。为了证实这个研究结果的可靠性及广谱性，我们在HBV中也证实了这一结论。表明miR-130a可以通过作用于靶点PKLR，从而影响病毒的复制。本研究成果不仅可以为小分子RNA 的抗病毒机理积累数据，也为丙型肝炎治疗的药物靶点筛选及潜在新方法研究提供广阔的探索空间。