Kras突变协同Pten低表达在肝内大胆管周围腺体内胆道干细胞恶化转化中的作用及分子机制

Recent efforts, including ours, have shown that there exists heterogeneity in intrahepatic cholangiocarcinoma (ICC), bile duct-type of which has many properties similar to extrahepatic cholangiocarcinoma.. Latest research, including ours, has suggested that biliary tree stem/progenitor cells (BTSCs) are present in peribiliary glands (PBGs) of the intrahepatic large bile ducts, which give rise in vitro and in vivo to cholangiocytes in the selection conditions and are responsible for the renewal of adjacent biliary epithelia, and PBGs could develop precancerous lesions and tumors.. Newest study, including ours, has demonstrated that the above PBGs, adjacent biliary epithelia and preneoplastic lesion, and bile duct-type ICC have high frequency of Kras mutations and low Pten expression, and their related PI3K signal pathway are activated. Moreover, combined Kras mutations and low Pten expression could cooperate during carcinogenesis. . Thus, we hypothesized that both the above might cooperate through PI3K pathway to induce malignant transformation of BTSCs within PBGs of the intrahepatic large bile ducts into bile duct-type ICC.. This study will be undertaken to investigate the effect of mutant Kras expression and low Pten expression on biological behavior of the BTSCs using gene transfection and RNA-interference (RNAi) technique, with an emphasis on tumor formation and type. Then, the expression and phosphorylation levels of AKT and ERK are detected by Western blotting with an aim of clarifying the molecular mechanism underlying the synergistic effects on tumorigenesis.. The present study will further promote our previous investigation on “the heterogeneity of original cells” about ICC, and meanwhile provide the experimental basis for a new classification of ICC and even cholangiocarcinomas based on the cell-lineages-of-origin.

近年研究（包括我们）表明，肝内胆管癌身存异质，其胆管型趋同于肝外胆管癌。最近研究（包括我们）提示，肝内大胆管周围腺体内存在能分化为胆管细胞的胆道干细胞，并负责邻近胆管上皮的更新修复，且该腺体能形成癌前病灶及肿瘤；最新研究（包括我们）显示，上述腺体、毗邻胆管黏膜、该部位癌前病变及胆管型肝内胆管癌均存较频繁Kras突变和Pten低表达及其相关PI3K通路被激活，且两者能协同致瘤。因此，我们假设：上述两者联合异常激活PI3K通路、诱导胆道干细胞逐步恶性转化为胆管型肝内胆管癌。本项目采用基因转染和RNAi技术，观察表达突变型Kras和下调Pten表达对胆道干细胞生物学行为的影响，尤其能否致瘤及肿瘤类型；免疫印迹检测AKT和ERK磷酸化水平，初步阐明两者协同致瘤的调控机理。本研究将进一步完善我们既往探索的肝内胆管癌“起源细胞异质性”这一学说，为肝内胆管癌乃至胆管癌“基于起源细胞”新分类提供实验依据。

按原拟定研究计划及研究内容，Kras突变和Pten低表达联合异常激活PI3K通路，可诱导肝内大胆管周围腺体内胆道干细胞逐步恶性转化为胆管型肝内胆管癌。因此，我们原拟定采用基因转染和RNAi技术，观察表达突变型Kras和下调Pten表达对胆道干细胞生物学行为的影响，尤其能否致瘤及肿瘤类型；免疫印迹检测AKT和ERK磷酸化水平，初步阐明两者协同致瘤的调控机理。.然而，因新鲜标本接济不上，成为初衷研究进展的主要限速因素。为此，我们因地制宜，及时、适当调整研究内容，提高研究效率。基于当前抑癌基因miR-181a和Kras之间的关系以及作用于胆管癌细胞的机制尚无定论，相应的研究目标转变为探讨miR-181a在Kras的表达调控中的作用及对胆管癌细胞QBC939增殖、迁移的影响，可为临床胆管癌的靶向治疗提供参考。我们将胆管癌细胞株QBC939按是否转染miR-181a模拟物进行分组并设对照，检测Kras和miR-181a的表达及对其胆管癌细胞系的影响。结果发现，miR-181a的过表达可抑制Kras从而抑制胆管癌细胞的增殖、迁移。另外，由于对剪接体SNRPB在肝细胞癌（HCC）中的生物学功能知之甚少，我们收集154名符合要求的HCC患者术后标本及其预后信息。结果发现，由c-Myc介导上调的SNRPB呈现出致癌活性，促进HCC细胞增殖和迁移，并在TCGA和oncomine数据库的研究中进一步证实SNRPB在HCC中的表达异常和临床价值。因此，我们认为SNRPB可作为HCC的致癌基因及有前景的预后因子。此外，基于当前转录组高通量测序实验广泛开展的现状，我们对离体后的HCC标本中RNA进行研究，确定其在常温下保持稳定性的最小时限。随机选取5例手术切除的肝癌组织，将每例分成5组，分别在离体后五个时间点处置，提取总RNA进行质量检测。结果证实，HCC标本离体后室温25℃环境下、放置时间在30 min内，提取RNA的稳定性质量尚有保障。最后，我们还对长链非编码RNA-p21（lincRNA-p21）和葡萄糖转运蛋白1（Glut-1）在HCC中的表达及其临床意义进行探讨，发现lincRNA-p21和Glut-1在HCC的发生和发展过程中起重要作用。lincRNA- p21的降低和Glut-1的升高提示预后不良，可作为判断HCC恶性程度和预后的潜在生物标志物。