microRNA-200b靶向调控Bmi1基因促进成体肝干细胞恶性转化的作用和机制研究

Cancer stem cells probably derived from malignant transformation of adult stem cells, while oncogene Bmi1 plays pivotal roles in the self-renewal and "stemness" maintenance of stem cells. Previouly, we have found that rat oval cells (adult liver stem cells) with high expression of Bmi1 gene produced tumors in nude mice with histological features of poorly differentiated hepatocellular carcinoma. Further, our array analysis revealed that miRNA-200b correlated negatively with Bmi1 gene. Bmi1 has also been predicted to be a potential target gene of miRNA-200b by the use of miRNA-target software prediction algorithms. Therefore, we hypothesised that miRNA-200b can promote malignant transformation of liver stem cells to liver cancer stem cells by targeted regulation of Bmi1 in oval cells. In this study, first real time PCR, Western blot and Dual-Luciferase assay were employed to validate whether Bmi1 was a target gene of miRNA-200b. Next, the effect of miRNA-200b on oval cells self-renew, multipotent differentiation, chemoresistance as well as tumorigenecity was studied in vitro and in vivo. At last, the effects of miRNA-200b/Bmi1 on Wnt, SHH and Notch signaling pathways were investigated. This study aimd to explore the key genes, signaling pathways, upstream and downstream regulatory mechanisms in malignant transformation of stem cells.

癌干细胞很可能来自癌变的成体干细胞，而原癌基因Bmi1在干细胞自我更新和"干性"维持中起关键作用。我们前期研究发现，高表达Bmi1基因的大鼠卵圆细胞(成体肝干细胞)可在裸鼠体内形成低分化肝癌；进一步的芯片结果提示miRNA-200b与Bmi1的表达显著负相关，miRNA靶基因预测软件也提示Bmi1可能是miRNA-200b的下游靶基因。在此基础上我们提出假设：miRNA-200b可通过靶向调控Bmi1基因进而促进成体肝干细胞向肝癌干细胞转分化。本项目拟首先通过qPCR、Western和双荧光素酶报告系统验证miRNA-200b能否靶向调控Bmi1；然后检测miRNA-200b对卵圆细胞体外自我更新、多向分化、耐受化疗药及在裸鼠体内成瘤能力的影响；最后检测miRNA-200b/Bmi1对Wnt、SHH和Notch信号通路的影响；以探明在干细胞癌变中起关键作用的基因、信号通路及上下游调控机制。

癌干细胞很可能来自癌变的成体干细胞，而原癌基因Bmi1在干细胞自我更新和“干性”维持中起关键作用。本项目是在前期Bmi1促进肝成体干细胞恶性转化的研究基础上，进一步探索miRNA-200b能否通过靶向调控Bmi1基因进而促进成体肝干细胞向肝癌干细胞转分化。我们首先通过real time PCR、Western blot和双荧光素酶报告系统证实miRNA-200b在人肝癌细胞系和大鼠成体肝干细胞中均可靶向沉默Bmi1；然后通过体内外实验证实miRNA-200b对大鼠成体肝干细胞的克隆形成能力、侵袭能力及裸鼠体内成瘤能力等均有一定的影响；最后通过iTRAQ定量蛋白组学技术证实miRNA-200b/Bmi1对Wnt、SHH和Notch信号通路均有一定的调控作用。此外我们通过MSP（甲基化特异性PCR）和BSP（亚硫酸氢盐测序PCR）等实验方法还发现，miRNA-200b甲基化沉默可通过靶向上调Bmi1基因促进人肝细胞癌进展和化疗耐药。上述结果表明，miRNA-200b不但可通过靶向上调Bmi1基因促进人肝细胞癌的发生、而且在人肝细胞癌的进展及化疗耐药中也发挥重要作用。上述成果利于我们更好的理解肝癌的细胞起源和肝癌发生、发展、化疗耐药中起关键作用的基因、信号通路及上下游调控机制，为肝癌的早期诊断和分子靶向治疗提供实验依据。