DLK1-DIO3印迹区基因在肝癌干祖细胞分化过程中的作用及表观遗传调控机制研究

As known, the expression status of DLK1-DIO3 imprinted region genes are related to the status of pluripotent stem cell，iPS cells included，and regulated by complex epigenetic mechanism. Our study showed that the expression of paternally allele expressed DLK1 increased in human and mice HCC specimen, its expression was limited to only a few cells, but there is no imprinting loss of DLK1-DIO3 imprinted region. DLK1+ cells showed stronger ability of proliferation. RNAi targeting to DLK1 could suppress the ability of proliferation. The expression of DLK1 decreased along with differentiation of HCC cells. All these data suggested that: 1) DLK1+ cells are potential HCC stem/progenitor cells. 2) The expression status of genes in DLK1-DIO3 imprinted region was critical to maintain the charater of HCC stem/progenitor cells. 3) Regulation expression of DLK1 or DLK1-DIO3 imprinted region gene maybe promote differentiation of HCC cells.. The aim of our research is to study the expression profile of DLK1-DIO3 imprinted region genes of HCC stem/progenitor cells, the related epigenetic mechanism and its effection to biological character of HCC stem/progenitor cells; regulate the gene expression of DLK1 or DLK1-DIO3 region through genetics and epigenetics method; and found its effection to the biological character of HCC stem/progenitor cells, and build the foundation of differentiation therapy of HCC.

已知基因组DLK1-DIO3印迹区的基因表达状态与干细胞包括诱导的多能干细胞分化阶段有关且受复杂的表观遗传机制调节。本课题组前期研究表明，父系等位基因表达的DLK1在人和鼠肝癌样本中均升高且局限在少量细胞表达，但DLK1-DIO3印迹区未发生印迹丧失；DLK1+细胞呈高增殖性，RNA干扰DLK1则减弱其增殖；DLK1随肝癌细胞分化而降低。提示1）DLK1+细胞是潜在肝癌干/祖细胞；2）DLK1-DIO3印迹区基因表达状态可能对维持肝癌干/祖细胞特征起关键作用；3）调控DLK1或DLK1-DIO3区基因表达可能促进肝癌细胞分化。本课题拟研究基因组DLK1-DIO3印迹区基因在肝癌干/祖细胞的表达谱特征、表观遗传学机制以及对其生物学特征的影响；通过遗传学或表观遗传学手段调控DLK1或DLK1-DIO3区基因表达对肝癌干/祖细胞生物学特征尤其分化的作用，为肝癌分化治疗奠定理论基础。

项目的背景:已知基因组DLK1-DIO3 印迹区的基因表达状态与干细胞包括诱导的多能干细胞分化阶段有关且受复杂的表观遗传机制调节。本课题组前期研究表明，父系等位基因表达的DLK1 在人和鼠肝癌样本中均升高且局限在少量细胞表达，但DLK1-DIO3 印迹区未发生印迹丧失；DLK1+细胞呈高增殖性，RNA 干扰DLK1 则减弱其增殖；DLK1 随肝癌细胞分化而降低。本课题拟研究基因组DLK1-DIO3 印迹区基因在肝癌干/祖细胞的表达谱特征、表观遗传学机制以及对其生物学特征的影响；通过遗传学或表观遗传学手段调控DLK1 或DLK1-DIO3 区基因表达对肝癌干/祖细胞生物学特征尤其分化的作用，为肝癌分化治疗奠定理论基础。主要研究内容:A. DLK1 阳性肝癌细胞基本生物学和DLK1-DIO3 印迹区基因表达谱特征研究B. DLK1-DIO3 印迹区基因表达状态和表观遗传学机制对维持肝癌干/祖细胞特征的作用C. DLK1 或DLK1-DIO3 区基因作为治疗靶标的潜力研究。重要结果:干扰DLK1基因的表达可以影响肿瘤细胞的体内外生长和成瘤能力和成球能力。转染miR-495-3p的模拟物，可以促进肝癌细胞Hep3B的生长，而转染miR-495-3p的抑制物，则抑制肝癌细胞Hep3B的生长。miR323-3p可以影响肝癌细胞Hep3B的生长和克隆形成。动物实验研究发现DLK1的条件沉默可以抑制异种移植小鼠的肿瘤生长。腺病毒介导的DLK1表达下调可抑制小鼠肝癌模型中的肿瘤生长。RNAi介导的DLK1表达下调可在原位移植模型中抑制肿瘤的生长。DLK1表达下调抑制细胞周期由G1进入S期，并下调细胞周期素E1的表达。DLK1表达下调还可以促进p27kip1蛋白的降解和诱导HCC的分化。关键数据及其科学意义:我们首先利用一个四环素诱导的shRNA系统表明DLK1的诱导下调可以抑制Hep3B和Huh7肝癌细胞的克隆形成，成球形成，和异种移植肿瘤生长的能力。随后，尾静脉注射腺病毒介导DLK1表达下降可以减少二乙基亚硝胺（DEN）诱导的小鼠肝癌模型中肝癌的早期发生，并且降低晚期的最大肿瘤直径的大小。此外，我们在体内原位移植模型中，通过活体荧光成像证明DLK1的靶向对肿瘤生长的抑制作用。最后，我们研究了抗肿瘤机理。一个干细胞相关基因，p27kip1蛋白，随DLK1的表达下调从而下降其蛋白表达水