厌氧诱导的长非编码RNA在肝细胞癌恶性进展中的作用及其在线粒体代谢重构中的分子功能和网络调控机制的研究

It has been demonstrated that the development of Hepatocellular Carcinoma (HCC) has been accompanied by the multiple-molecular changes and the multiple-step complicated process. And it has been recognized for a long time that a unique metabolic phenotype referred to as the Warburg effect, characterized by enhanced glycolysis and reduced oxidative phosphorylation, even in the presence of oxygen. Recently, we found the new long non-coding RNA in the cytoplasm and mitochondria of LO2 and LM9 under the condition of normoxia and hypoxia (0.2%O2), the expression of which is higher in the cytoplasm and mitochondria after hypoxia. And the knockdown of AC093642 in LM9 with normoxia and hypoxia leads to a strong decrease in glucose uptake and secreted lactate production. And the silence of AC093642 inhibits the invasion and reverses EMT in LM9 cell with normoxia. So maybe the high expression of AC093642 has associated with the mitochondrial metabolic reprogramming and glycolysis, which may result in HCC aggressive progression. However, it has been unknown for the molecular carcinogenic character and relative regulating network mechanisms of AC093642 disregulation in the mitochondrial metabolic reprogramming and glycolysis. .We will examine the expression of AC093642 in carcinoma and adjacent tissue by the use of associated molecular oncological study methods and high-output detection technology and analyze the correlation between the expression levels of AC093642 and HCC clinical pathological parameters. In addition, we will explore the effect of the overexpression of AC093642 in HCC cell lines on the glycolysis and mitochondrial reprogramming and epithelial-mesenchymal transition, migration, invasion and in vivo tumor metastasis. All the more, we will study the mechanisms on lincRNA mitochondrial shuttle and the important down-stream associated-metastasis gene. Then we will find that the clinical oncological significance of AC093642 and the related target gene in HCC metabolic abnormity. And we will explore that the potential therapeutical effect of the AC093642 knockdown on the tumor metastasis, life expectancy and the formation of the ascite in the HCC orthopedic pulmonary metastatic model. We will further explore the molecular mechanisms which result in the high expression of AC093642, and find a new signaling network pathway which regulates the expression of AC093642. So we will devote ourselves to supplying the experimental scientific support for the deep recognization of the tumor metabolism in HCC aggressive progression.

通过在常氧和厌氧培养的细胞浆和线粒体的基因芯片中发现一个厌氧后均高表达的长非编码RNA, 在常氧或厌氧条件下AC093642的沉默明显地抑制了糖酵解，逆转了EMT，减少了侵袭，但AC093642在线粒体代谢重构和糖酵解中的分子功能和网络调控机制的研究还不清楚。.本项目拟应用分子肿瘤学方法和高通量检测技术，检测HCC病人癌和癌旁组织中AC093642的表达，分析AC093642的表达水平和HCC转移的相关性；分别探讨AC093642对糖酵解、线粒体代谢重构、EMT、动物体内肿瘤转移等生物学行为的影响；确定AC093642穿梭线粒体内外的机制和重要的下游转移相关基因；明确AC093642作用的靶基因异常表达的信号转导通路及其在肝细胞癌恶性进展中的临床肿瘤学意义。深入研究AC093642通过调控肿瘤代谢促进HCC恶性进展的分子网络机制，致力于为认识肿瘤代谢在HCC恶性进展的分子遗传学作用提供基础

我们研究了非编码RNA在不同肿瘤发生及恶性进展的机制。探讨和确证了长链非编码RNAAC093642促进了肝细胞癌细胞系糖酵解和线粒体的发生，通过和LDHA 及G6PD结合影响了了线粒体的代谢重构。进一步研究发现染色体乙酰化酶HDAC4促进转录因子STAT4对AC093642转录，从而促进HCC的发生和恶性进展。另一肝细胞长非编码RNALINC01137也通过稳定 NFYB 蛋白水平募集 SMYD3 上调 IL-1β、CXCL2 和 CCL20 来扩大 MDSC 以促进 HCC 转移，从而减轻抗肿瘤 T 细胞反应。机制上发现LINC01137 招募 SMYD3 与 NFYB 相互作用以增强 H3K4me3 在 IL-1β、CXCL2 和 CCL20 启动子处的结合，进而上调 IL-1β、CXCL2 和 CCL20。同时我们在MVI肿瘤中circSEC62 作为 miR-625-5p 的竞争性内源 RNA，通过 SNRPA/NOTCH1/Snail 途径促进迁移、侵袭和 EMT。乳腺癌中我们发现lncRNA HIFAL对维持和增强 HIF-1α 介导的反式激活和糖酵解至关重要。机制上，HIFAL募集PKM2和PHD3且诱导PKM2羟基化，结合 hnRNPF引导而PKM2/PHD3复合物入核，并促进HIF-1α的反式激活。相互地，HIF-1α可以调控HIFAL转录，从而形成正反馈回路而维持HIF-1α的反式激活。临床意义上，我们发现乳腺癌中HIFAL的高表达和乳腺癌的恶性表型密切相关，HIFAL的高表达也促进了乳腺癌的增殖，而靶向HIF-1α和HIFAL的联合治疗明显地阻止了乳腺癌的增殖。在乳腺癌的PDX治疗中我们也发现ATRA（Pin1的抑制剂）和奥拉帕尼（PARP抑制剂）的共同使用使得肿瘤明显减小，并阐明了Pin1通过阻止BRCA1的Lysine1037位点泛素化而抑制BRCA1蛋白的降解的机制。在鼻咽癌中我们通过lncRNA 表达谱微阵列等技术发现长链非编码RNA NKILA在 NPC 组织中降低，NKILA 的低表达是NPC预后不良的独立预后因素。进一步在体内外实验中过表达NKILA可以通过调节NF-KB通路从而抑制鼻咽癌的侵袭和转移。因此这些结果为肿瘤的治疗提供了有用的靶标。