MicroRNA675靶向异染色质蛋白1异构体激活丙酮酸激酶M2影响肝癌干细胞的表观调控机制研究

The hepatocarcingenesis is a gradual process and if the liver cancer stem cells can be blocked in the early stage, the development of liver cancer is to be prevented. To explore the mechanism of the occurrence and development of liver cancer stem cells from the epigenetic views is the current research hotspot. MicroRNA675 is located in the first exon in long noncoding RNA H19, which can promote the growth of embryo and also enhance the cell malignant proliferation. Using the separated or induced liver cancer stem cells, we try to investigate the influence of microRNA675 on liver cancer stem cells phenotypes in vitro and in vivo, and on the expression and dynamic changes on the chromosome of three molecular subtypes (HP1 alpha, HP1 beta, HP1 gamma) of heterochromatin senior structure regulating protein HP1. Next, based on the effect of miceoRNA675 on HP1, histone and DNA epigenetic modification alternation should be studied in the liver cancer stem cells , and thus the abnormal expression and function alternation of H19 caused by function change of the early growth response gene 1 (EGR1) triggered by histone and DNA epigenetic modification alternation will be examined. Moreover, the functions of Hub protein pyruvate kinase PKM2 which links micro-glucose metabolism, environmental cell growth signaling and oxidative stress in cancer cells may further be explored in the liver cancer cells, involving in the abnormal cell metabolism ability, abnormal DNA damage repair and chromatin reprogramming. We will try to reveal the important functions of miR675 in the regulation and control of liver cancer stem cells’ fate and provide with a theoretical basis for studying the mechanism of reversal of liver cancer stem cells and liver cancer gene therapy.

肝癌的发生是一个渐进过程，在早期阻断肝癌干细胞向肝癌发展能预防肝癌的发生，从表观遗传学的角度去探讨肝癌干细胞的发生发展机制是目前的研究热点。microRNA675位于长链非编码RNA H19的第一个外显子，其既能促进胚胎生长发育，又能增强细胞的恶性增殖。我们将研究miR675对肝癌干细胞体内外表型的影响，并对miR675调控异染色质蛋白HP1三个亚型分子（HP1α、HP1β、HP1γ）的表达及其在染色体上的动态变化进行解析。基于miR675对HP1的作用，研究miR675对肝癌干细胞中的组蛋白和DNA表观修饰的影响，以及可能由此引发的早期生长应答基因1(EGR1)功能改变而介导的H19的表达和功能的异常改变。进一步对可能导致的癌细胞中的枢纽蛋白丙酮酸激酶M2功能改变而促发的异常的细胞物质能量代谢、DNA损伤修复、染色质重编程进行分析，旨在揭示miR675在调控肝癌干细胞命运上的重要功能。

本研究利用胚胎干细胞、诱导的肝干细胞、分离的肝癌干细胞，通过构建基因定点特异性插入过表达或RNA干扰的方法，建立稳定细胞系和动物模型，从表观遗传学的角度去探讨了miR-675及其相关分子在干细胞恶变过程中的作用机制。结果显示：1）miR-675与丙酮酸激酶M2(PKM2)协同促进肝癌干细胞生长。miR675协同 PKM2增强了甲基化转移酶SUV39h2的活性，增加H3K9me3 与 HP1α相互作用而上调了癌基因C-myc 的表达。2）miR675通过阻碍DNA错配修复加速间充质干细胞的恶性转化。miR675增强了SETD2 与P62间的相互作用,减少了HBMMSCs中H3K36me3修饰和hMSH6-H3k36me3-Skp2 四复合物的形成，阻碍了DNA错配修复，促发了DNA 微卫星不稳定性。3）miR675 依赖RARP1激活Rad51 ，并增强癌基因H-Ras的表达及其功能促进了人类肝癌干细胞的生长。 4）长链非编码RNA HULC通过miR-675促发肝癌干细胞细胞自噬而促进丙酮酸激酶M2和细胞周期蛋白的表达。5）miR-24-2依赖miR-675在表观遗传水平上促进肝癌干细胞恶性生长。 miR24-2通过miR675促进了人类肝癌干细胞中Histone H4第16位赖氨酸的乙酰化修饰和抑制了Histone H4第20位赖氨酸的甲基化修饰，从而增强了磷脂酰肌醇3-激酶（PI3K）的表达，并导致了细胞自噬的发生。6）a.发现长链非编码RNA HULC通过促发细胞自噬促进肝癌发生的分子机制。b.发现 miR372-PTEN-CTCF-pYB1-β-catenin-PKM2-H3K9Ac-erbB2信号通路促进了人类肝癌的恶性进展。c.发现炎症相关基因 P62促发了小鼠炎性肝脏中间充质干细胞的恶性转化.d.发现炎症因子Toll样受体4（TLR4）促进了组蛋白甲基化转移酶SUV39h2的功能，进而增强了肝癌干细胞中端粒维持正相关蛋白pTOP1,TOP1, pExo1, Exo1, pSNM1b, NM1b与端粒DNA的结合能力。e.长链非编码RNA　MEG3减少了PKM2的表达和核定位。总之，本研究结果提示：miR675及其相关分子在调控肝癌干细胞命运上发挥重要作用，这些结果为人类肿瘤防治提供了理论依据。