【作者】 房林；

【导师】 杨广顺；

【作者基本信息】 第二军医大学 ， 外科学， 2010， 博士

【摘要】 原发性肝癌是高度恶性并严重危害人类健康的疾病,但其发病机制仍不十分清楚。虽然现在有很多的方法用于治疗原发性肝癌,如化疗、放疗、外科手术、生物治疗等,但是都不能明显的提高原发性肝癌患者的生存率。因此寻找有效的治疗手段有非常重要的意义。根据肿瘤干细胞理论,在原发性肝癌中也必然有肝癌干细胞存在,与正常组织是由少数正常组织干细胞通过自我更新和不断的分化产生正常的组织细胞来维持正常功能相似,肿瘤组织也是由少数肿瘤干细胞不断自我更新和分化来触发和维持肿瘤生长的。考虑到目前传统的化疗和放疗只能使肿瘤组织缩小,而不能从根本上消除肿瘤,原因可能是这些传统的治疗方法不能有效的杀灭肿瘤组织中的肿瘤干细胞。因此,深入研究肝癌干细胞特有的生物学特性对于我们深入理解肝癌的发病机制及设计有效的治疗策略都有重要的意义。miRNA是一类内源性非蛋白编码的单链小分子RNA,长度为20-25nt,广泛存在于真核生物中。成熟miRNA与靶mRNA 3非翻译区互补结合,诱导靶mRNA降解或抑制靶mRNA的翻译,从而实现转录后基因调控作用。miRNA对细胞的增殖、分化和凋亡有重要的调节作用。miRNA参与了机体的免疫调控,并在肿瘤发生中发挥重要的调节作用。本研究对肝癌miRNA进行检测,观察肝癌组织中miRNA变化情况。同时根据检测结果,应用miRNA转染,观察转染后肝癌细胞NF-κB信号通路、β-catenin、抑癌基因P53基因和Tg737基因的变化情况,及凋亡相关基因caspase-3、caspase-8的变化情况。同时以CD90阳性细胞为肝癌干细胞标记物,分离肝癌干细胞样细胞,检测肝癌干细胞样细胞上述基因和部分蛋白的变化情况,观察肝癌细胞的生物学特性。对揭示肝癌发生发展的病理机制,从而为研究设计肝癌特异性的诊断和治疗方法提供可能的新思路。研究方法：1.经病理组织学确诊的肝癌组织及癌旁组织标本,TRIzol法提取细胞总RNA和按照miRNA提取分离试剂盒提取miRNA,用realtime-PCR检测miR-155、miR-198、miR-373、miR-520b、miR-548c、miR-1289、miR-1301等miRNA及NF-kB、β-catenin、p53、Tg737基因mRNA表达情况。2.人肝癌细胞株HepG2,根据上述结果选择miR-1301 mimics进行转染,提取细胞总RNA,用realtime-PCR检测转染后p65、β-catenin、p53、Tg737、bcl-2、bcl-XL、caspase-3、caspase-8等基因的变化情况；Western Blot检测相关蛋白表达情况；划痕实验观察细胞迁移能力：transwell小室检测细胞侵袭能力；MTT法观察细胞生长抑制情况。3.人肝癌细胞株HepG2,以CD90作为肝癌干细胞标记物,免疫磁珠法筛选CD90阳性细胞,提取细胞总RNA,用realtime-PCR检测miR-155、198、373、548c-5p、1289、145、375、874、1183、1324等水平,检测p65、β-catenin、p53、Tg737、caspase-3、caspase-8、bcl-2、bcl-XL等基因转录情况。选择miR-548c-5p顺式转染,用realtime-PCR检测转染后p65、β-catenin、p53、Tg737、caspase-3、caspase-8、bcl-2、bcl-XL等基因mRNA表达情况。Western Blot检测相关蛋白表达情况。结果：1.在肝癌组织中,miR-155、miR-198、miR-373、miR-520b、miR-1301表达下降。miR-548c、miR-1289在肝癌组织表达增高。β-catenin、Tg737基因表达下降。NF-kB、p53基因无明显变化。2.转染miR-1301后p53基因水平上调、P53蛋白增加,bcl-2、bcl-XL基因水平下调,Bcl-2、Bcl-XL蛋白减少。转染miR-1301后,划痕实验观察到转染后细胞迁移能力,(?)answell小室观察到细胞侵袭力下降,细胞生长受到抑制。3.肝癌干细胞样细胞中miR-548c-5p.miR-145、miR-375、miR-874水平下降。miR-155、miR-198、miR-1289升高。miR-373、miR-1183、miR-1324无明显变化。肝癌干细胞样细胞中caspase-3、bcl-2基因水平下调,caspase-8基因水平上调,Bcl-XL无明显变化,p65、β-catenin、p53、Tg737等表达无明显变化。转染miR-548c-5p后肝癌干细胞样细胞中P-catenin、Tg737、bcl-2、bcl-XL、Caspase-3基因水平下调,Bcl-2、Bcl-XL、Caspase-3蛋白水平下降,p65、p53、Caspase-8基因水平变化没有统计学意义。转染后transwell小室观察到细胞侵袭力下降。结论肝癌组织中miRNA表达水平有变化。转染miR-155、198、373、520b等可引起P65、β-catenin、P53、Tg737、Caspase-3、Caspase-8基因的变化,推测miR-155、198、373、520b等可miRNA对肝癌细胞的生物学特性产生重要影响。miR-1301可上调p53基因、下调bcl-2、bcl-XL基因。推测miR-1301总体上对肿瘤发生发展可能起抑制作用,但需要进一步研究。肝癌干细胞样细胞中,转染miR-548c-5p mimics后对Bcl-2、Bcl-XL、Caspase-3等多个基因产生影响,这些说明miR-548c-5p可能在肝癌干细胞样细胞的凋亡-抗凋亡过程中起一定作用。肝癌干细胞样细胞中miR-145、miR-375、miR-874、miR-155、miR-198、miR-1289等水平发生改变,这些说明miRNAs可能在肝癌干细胞的发生发展中起重要作用。肝癌干细胞样细胞caspase-3、bcl-2基因水平下调,caspase-8基因水平上调,推测肝癌干细胞样细胞可能存在凋亡-抗凋亡功能紊乱,从而影响肝癌细胞的生物学特性。更多还原

【Abstract】 Background Hepatocellular carcinoma(HCC) is a common malignant tumor in China. Researchers have long been searching for the cellular origin and causes of HCC to try to find effective preventive and treatment measures under the hints of the pathogenesis of HCC. Recent researches had discovered that there were some stem-like subpopulations in tumor tissues which had the potential to proliferate infinitely and played much crucial role in initiating carcinogenesis and development, while the other cells would die after transitory differentiation. It was thought that the tumor growth resulted from the proliferation of some tumor stem cells with special surface markers. For this reason, the tumor treatment must aim directly at the tumor stem cells but not at the bulk of tumor cells with little ability to proliferate and differentiate. This theory might bring about some revolutionary in tumor therapeutics.The key point of the research on tumor stem cells was how to establish the special surface markers of them. However, it was hope to achieve an efficient sorting scheme of tumor stem cells by detecting the surface markers with some directing antigens, which would save a lots of time and research funds. But the following findings published in recent years are exciting:the marker molecule ABCG2 of liver SP cells, which have the biological characteristics of liver cancer stem cells, acts as a marker of liver cancer stem cells; CD 133 is also considered as a marker of liver cancer stem cells; and CD90 is another specific marker of liver cancer stem cells.MicroRNAs(miRNAs), a new class of endogenous, noncoding and single-stranded RNAs, were recently discovered in both animals and plants. They trigger translational repression and/or mRNA degradation mostly through complementary binding to the 3’-untranslated regions of target mRNAs. Studies have shown that miRNAs can regulate a wide array of biological processes such as cell proliferation, differentiation, and aberrant expression. Metabolism of miRNAs are associated with human diseases including malignancy. Alterations of miRNAs expression may play various roles in the pathogenesis of many human cancers. Some miRNAs have been shown to possess oncogenic or tumor suppressor activity, influencing cell transformation, tumor progression or metastasis. Accumulating evidences suggest that miRNAs could potentially be widely used in the diagnosis, prognosis and therapy of human cancer.Objective In this study, we screened differentially miRNAs expressed between hepatocellular carcinoma tissue and adjacent non-tumor tissue through realtime PCR technology, and hope to find that miRNAs changes in tumor tissue. Than we transfected these miRNAS into cells. We colledted the total RNA. Realtime PCR were used to examine the different expression of the potential target mRNA in test groups and the control groups. We hope our study could provide convincing evidences on miRNAs association with hepatocellular carcinoma tumorigenesis, and facilitate the search of suitable candidates for cancer targeting therapy.Using CD90 as specific marker of liver cancer stem cells,we isolate, culture and identify the stem-like subpopulations from HepG2 cells in vitro, to investigate the basic characteristics of stem-like liver cancer cells.MethodsTissue samples were obtained from 20 patients undergoing hepatectomy for hepatocellular carcinoma. Tumor samples were resected at surgery. Non-tumor samples from the macroscopic tumor margin were isolated at the same patients and used as the matched adjacent non-neoplastic tissue. Real-time PCR technology was applied to screen differentially expressed miR-155, miR-198, miR-373, miR-520b, miR-548c, miR-1289, miR-1301 between tumor tissue and adjacent non-tumor tissue.We transfected miR-1301 mimics into HepG2 cells. Real-time PCR technique was applied to screen p65,β-catenin, p53, Tg737, bcl-2, bcl-XL, caspase-3, caspase-8 mRNA between transfected groups and contral groups. Western Blot technique was applied to detect their protein. Transwell cabin was used to observe cellular invasiveness. The cellular growth activity was assayed by MTT assay.Using CD90 as specific marker of liver cancer stem cells, we isolate, culture and identify the stem-like subpopulations from HepG2 cells in vitro. Real-time PCR technique was applied to screen miR-155, miR-198, miR-373, miR-568c-5p, miR-1289, miR-145, miR-375, miR-874, miR-1183, and miR-1324 between CD90 positive cells and negative cells. P65,β-catenin, P53, Tg737, caspase-3, caspase-8,bcl-2, bcl-XL mRNA between CD90 positive cells and negative cells were also detectd by Real-time PCR method. miR-568c-5p mimics was transfected into CD90 positive cells, and change of P65,β-catenin, P53, Tg737, caspase-3, caspase-8,bcl-2, bcl-XL mRNA were detected. Western Blot technique was applied to detect their protein. Transwell cabin was used to observe cellular invasion capacity. Results 1. Compared with adjacent non-tumor tissues, miR-155, miR-198, miR-373, miR-520b, miR-1301 were down-regulated while miR-548c,miR-1289 were up-regulated in tumor tissue.2. p53 mRNA and protein were up-regulated in miR-1301 transfected groups. bcl-2, bcl-XL mRNA and protein were down-regulated in miR-1301 transfected groups. Cellular invasive capacity was depressed. Cellular inhibition ratio were high in transfected groups.3. In CD90 positive cells, miR-155, miR-198, miR-1289 were up-regulated while miR-548c-5p,miR-145, miR-375, miR-874were down-regulated.Caspase-3,bcl-2 mRNA were down-regulated in CD90 positive cells while Caspase-8 mRNA were up-regulated. There were no changes in p65,β-catenin, p53, Tg737, bcl-XL mRNA.β-catenin, Tg737, bcl-2, bcl-XL, caspase-3 were down-regulated in miR-548c-5p transfected groups in CD90 positive cells. bcl-2, bcl-XL, caspase-3 protein were down-regulated in miR-548c-5p transfected groups. Cellular invasive capacity was depressed.ConclusionsmiR-1301 may inhibitor tumor process through p53, bcl-2, and bcl-XL. miR-548c-5p may affect the level of caspase-3, bcl-2, and bcl-XL mRNAs and their proteins.Expression level of miRNAs were alterd in hepatocellular carcinoma and in liver cancer stem-like cells. p65,β-catenin, p53, Tg737, bcl-2, bcl-XL, caspase-3, caspase-8 mRNA were up-regulated or down-regulated in experiment groups. We may suppose that miRNAs may have great effects on hepatocellular carcinoma and liver cancer stem-like cells. Disorder of apotosis mechanism may plays important role in liver cancer stem cells.更多还原