Bmi1通过调控DKK1激活Wnt/β-catenin信号通路诱导成体肝干细胞转化为肝细胞癌的机制的研究

At the present stage, we think that hepatocellular carcinoma may originate from liver progenitor cells. In our experiment, Bmi1 were conducted in rat liver progenitor cells (OC3) through gene transfection technique. It showed that forced expression of Bmi1 accelerated the carcinogenesis of OC3 in nude mice. And at the same time, we found that Wnt signal pathway activated and DKK1 expression of OC3 cells increased because of the enhanced Bmi1 expression. DKK1 exert important modulatory effect on Wnt signal pathways. To be specific, loss-of-function assay of DKK1 in high-expression Bmi1 of OC3 showed that down-regulation of DKK1 has a positive correlation with the proliferation in vitro, colony-forming efficiency, invasive ability and migration ability of OC3. Meanwhile, the expressions of Wnt1 and β-catenin are down-regulated. Therefore, we presume that Bmi1 can activate Wnt signal pathway and promote the carcinogenesis of OC3 by regulating DKK1 expression.In this research, we use gene transfection technique to down-regulate or up-regulate the expression of DKK1 in high expression and normal expression of OC3 respectively. Then we will detect the following two aspects: 1 proliferation, colony formation and migration of OC3 in vitro; 2 Molecular mechanisms in Wnt signal pathway induced by Bmi1 on DKK1 expression.This research reveals the effect of the key gene and signal pathway on carcinogenesis of liver progenitor cells. So it lays a theoretical basis for the specific targeted therapy of hepatocellular carcinoma.

现阶段认为原发性肝癌很可能起源于癌变的成体肝干细胞。我们通过上调Bmi1的表达诱导大鼠成体肝干细胞（卵圆细胞）在裸鼠体内形成肝细胞癌，并发现在此过程中Wnt信号通路激活，并且DKK1表达升高。DKK1是调控Wnt信号通路的关键蛋白。下调高表达Bmi1的卵圆细胞中DKK1表达后，其体外增殖、克隆形成、迁移和侵袭能力下降显著，并且Wnt1和β-catenin表达下降。在以上基础上我们假设：Bmi1通过调控DKK1激活 Wnt通路诱导大鼠成体肝干细胞转化为肝细胞癌。本研究采用基因转染技术，分别下调或上调高表达Bmi1卵圆细胞和正常卵圆细胞中DKK1的表达，然后检测：1 .卵圆细胞在体外增殖、克隆形成、迁移/侵袭、耐受化疗药物能力以及在裸鼠体内成瘤能力；2. Bmi1调控DKK1激活Wnt通路的分子机制。本研究揭示关键基因/信号通路在成体肝干细胞癌变中的作用，为肝细胞癌特异靶向治疗提供理论依据。

肝癌可能起源于成体肝干细胞。我们前期研究发现Bmi1基因促进成体肝干细胞转化为肝细胞癌；进一步研究发现Bmi1可能通过调控Dkk1基因促进成体肝干细胞发生癌变。然而，DKK1在肝脏肿瘤发生发展中的作用目前尚不清楚。因此，我们首先探讨了DKK1基因对人肝细胞癌和胆管细胞癌生物学特性的影响。免疫组织化学技术研究发现，DKK1在人肝细胞癌组织中表达升高，DKK1的表达水平与肿瘤直径相关；shRNA下调DKK1的表达后，人肝癌细胞株体外增殖、克隆形成、侵袭能力和裸鼠体内成瘤能力显著下降；上调DKK1的表达后，人肝癌细胞株体外增殖、克隆形成、侵袭能力和裸鼠体内成瘤能力显著增强。.与此同时，我们研究发现，DKK1在人肝门部胆管癌组织中表达显著升高，DKK1的表达水平与肿瘤淋巴转移相关；下调DKK1的表达后，人胆管细胞癌细胞株体外增殖、克隆形成、侵袭能力和裸鼠体内成瘤能力显著下降；进一步研究发现。DKK1通过调控β-catenin/MMP-7信号通路促进促进人肝门部胆管细胞癌发生淋巴转移。在以上研究基础上我们认为，DKK1在肝癌发生发展中发挥重要的作用。在本项目的支持下我们研究发现Bmi1基因和DKK1基因在乙肝转基因小鼠恶性转化过程中发挥关键作用。更重要的是，我们发现Bmi1基因通过调控DKK1基因促进大鼠成体肝干细胞转化为肝细胞癌，上调Bmi1基因的表达后大鼠成体肝干细胞可以在裸鼠体内直接转换为肝细胞癌。综上所述，BMI1和DKK1在肝脏肿瘤发生发展过程中发挥重要的作用，BMI1通过调控DKK1促进成体肝干细胞发生癌变转化为肝细胞癌。