miR-375调控肝癌细胞线粒体能量代谢的作用及分子机制研究

Abnormal energy metabolism is an extremely important indicator of malignant transformation. As important regulatory factors, miRNAs have become a major focus in research on the regulation of energy metabolism in tumor cells. miR-375, as a member of the miRNA family, has been reported to be related to glucose metabolism. Our previous screening study of miRNAs showed that the expression of miR-375 was significantly reduced in hepatocellular carcinoma and that miR-375 had four potential targets that might play a regulatory role in the core steps of mitochondrial energy metabolism. Therefore, we speculate that miR-375 may have a significant effect on the regulation of abnormal energy metabolism in the liver, which has not yet been studied worldwide. Based on the above assumption, this project is aimed to: ① demonstrate the impact of the miR-375 expression level on energy metabolism in hepatocellular carcinoma by using a variety of hepatocellular carcinoma cell lines and their models in nude mice; ② identify the regulatory relationship between miR-375 and the four target genes ( PDHK1, LDHB, ACSL4 and OCNT1 ) with the help of the technologies such as the gene reporter, the regulation of the miRNA expression, and the RNA and protein detection techniques; ③ explore the influence of miRNA on the pathway of mitochondrial energy metabolism by further regulating its expression level. This project is expected to lay a theoretical foundation for the mechanism by which hepatic mitochondrial energy metabolism is regulated, and to provide new directions in the targeted therapy for hepatocellular carcinoma.

能量代谢异常是肿瘤细胞恶性转变的重要生物学特征。miRNAs作为重要的调控因子，在肿瘤细胞能量代谢过程中如何发挥调控作用是当前的研究热点。本项目组前期筛选发现，miR-375作为近期报道的血糖调控相关miRNA，在肝癌细胞中表达显著降低，并有4个潜在靶点可调控线粒体能量代谢的关键步骤。由此我们推测,miR-375极有可能通过调控线粒体功能进而影响肝癌细胞能量代谢水平。基于上述设想，本项目拟：①利用多种肝癌细胞株及裸鼠荷瘤模型，明确miR-375表达水平对肝癌细胞能量代谢的影响；②采用报告基因、miRNA表达调控、RNA及蛋白检测技术验证miR-375与4个靶基因（PDHK1、LDHB、ACSL4和OCNT1）的调控关系；③进一步通过调控miR-375表达水平检测其对线粒体内能量代谢通路的影响。本项目的完成将为探究肝癌线粒体能量代谢的调控机制奠定新的理论基础，亦为肝癌靶向治疗提供新的思路。

能量代谢异常是肿瘤细胞恶性转变的重要生物学标志。以有氧条件下糖酵解水平增强为主要表现的能量代谢异常不仅适应肿瘤细胞恶性增殖的需求，同时也与肿瘤细胞恶性程度密切相关。miRNAs作为重要的调控因子，通过降解靶基因mRNA或抑制靶基因mRNA翻译，参与细胞增殖、分化及凋亡等多种生物学过程。作为细胞恶变的能量基础，miRNA参与调控肿瘤细胞异常能量代谢的观点逐渐受到人们关注。然而，目前报道的肿瘤能量代谢相关miRNAs很少。能量代谢作为一个巨大的调控网络，现有报道能否充分解释其复杂的调控机制？尤其是在线粒体能量代谢的调控过程中，是否需要更多的miRNA参与？基于这一思路，本课题拟通过生物信息学筛选和分子生物学实验，探讨线粒体相关miRNA对于肝癌代谢的影响。.本项目研究内容包括：（1）利用多种肝癌细胞株及裸鼠荷瘤模型，明确miR-375表达水平对肝癌细胞能量代谢的影响；（2）采用报告基因、miRNA表达调控、RNA及蛋白检测技术验证miR-375与4个靶基因（PDHK1、LDHB、ACSL4和OCNT1）的调控关系；（3）进一步通过调控miR-375表达水平检测其对线粒体内能量代谢通路的影响。.结果表明：（1）生物信息学预测及分子生物学检测证实，miR-375在肝癌组织中显著低表达，且与肝癌细胞能量代谢密切相关。（2）肝癌细胞中过表达miR-375促进了肝癌细胞的乳酸生成及葡萄糖摄取，并抑制了线粒体数目以及线粒体DNA拷贝数，最终抑制了肝癌细胞活性。（3）通过microRNA靶基因预测及肝癌组织标本进行相关性分析，筛选出PDHK1、LDHB、ACSL4以及OCTN1作为miR-375的潜在靶点。（4）基因芯片数据表明，过表达miR-375的Hep3B以及HepG2细胞中，有67个基因下调4倍以上，并且和多条通路相关，导致细胞内乳酸的堆积和线粒体功能紊乱受损。.综上所述，我们发现，miR-375可能靶向抑制LDHB、LDHC，进而导致糖酵解水平增强以及线粒体氧化磷酸化水平降低，造成肿瘤细胞中的乳酸堆积。筛选出的67个差异表达基因中4个基因（TIMM10，CHCHD7，C6orf136，RPS15A）属于线粒体核编码基因。这些基因的受损或者抑制都可能导致线粒体功能的紊乱或异常，因此miR-375很有可能是抑制了线粒体核编码基因的表达，从而使得肝癌细胞线粒体的数目以及DNA拷贝