MiR-25在肝癌干细胞中的功能及其相关机制的研究

发布时间：2018-05-07 17:13

本文选题：肝癌干细胞 + 耐药　；参考：《浙江大学》2017年博士论文

【摘要】：背景原发性肝癌的发病率位居全球第5位,病死率在全球范围排名第3位,是最为常见的恶性肿瘤之一。中国是乙肝大国,亦是全球肝癌最高发的地区之一,国内每年新发肝癌病例近50万例,每年因肝癌致死亡病例约为11万,肝癌具有高发病率和高死亡率的流行病学特征。近年来,得益于肝移植技术的成熟和精准医学的快速发展,已形成了以外科手术或原位肝移植为主,放化疗及免疫靶向治疗为辅助的综合治疗体系。然而,术后肿瘤复发转移仍然严重制约着肝癌患者的总体预后。即便是被公认为肝癌根治手段的肝移植,其符合米兰标准的5年无瘤生存率仅达73%。肿瘤干细胞理论认为,在肿瘤组织中存在少量具有无限自我更新能力,并且能产生不同异质性肿瘤细胞群的肿瘤干细胞。这种干细胞具有更强的成瘤能力和分化能力,在肿瘤的发生发展、复发转移和耐药过程中起着关键作用。在白血病、肺癌、前列腺癌、乳腺癌、胰腺癌、肝癌等恶性肿瘤中均已证实了肿瘤干细胞的存在。针对肝癌术后复发转移这一关键问题,干细胞理论给出的解释是,肿瘤干细胞作为"火种",拥有更强的生命力和耐药性,在肿瘤复发转移过程中扮演关键角色。随着相关分子生物学研究的深入,MicroRNA(MiRNA)在肿瘤干细胞自我更新、转移复发等生理过程中的调节作用得以浮现。这类保守的非编码单链RNA分子通过互补配对与靶基因mRNA的3'UTR相结合,进而调控靶基因mRNA表达。肝癌的发生发展、耐药、转移等生物学行为均与MiRNA的异常表达相关,其中关系到肝癌细胞与周围微环境和宿主免疫的交互作用,并涉及PTEN/PI3K通路、HGF/Met通路、FAK通路等等各种信号通路的异常活化。围绕重要信号通路上的调节性非编码RNA进行筛选分子位点并验证相关功能,有助于进一步解释肝癌发生发展的复杂机理,对发现肝癌治疗的潜在靶点也有着重要意义。以肝癌术后高复发率和对辅助治疗高耐受为背景,我们前期利用测序技术分析肝癌细胞的MicroRNA表达谱时发现肝癌细胞中miR-25表达水平异常升高。本课题旨在进一步研究miR-25对肝癌细胞尤其是肝癌干细胞生物学行为的影响,并探索其作用机制,以便寻找针对肝癌干细胞治疗的关键靶点,并为进一步拓展肝癌综合治疗手段提供潜在依据。目的:本研究旨在寻找和验证肝癌细胞中差异表达的MiRNA,用实时定量PCR的方法验证差异表达的miR-25在肝癌细胞系中的表达水平,并实验研究miR-25表达与肝癌细胞尤其是肝癌干细胞生物学行为的关系。在此基础上,通过进一步的获得性和缺失性功能实验明确miR-25在肝癌细胞中的生物学功能,验证其上下游信号通路和基因靶点,探索其在肿瘤致病过程中的分子生物学机制。方法:1.用实时定量PCR验证miR-25在肝癌细胞系和正常永生化肝细胞系中的表达情况:肝癌细胞系及永生化肝细胞系作为研究对象,分别提取RNA,反转录得到cDNA。以实时定量PCR技术分别检测其miR-25表达水平。2.以TRAIL诱导肝癌细胞凋亡,双荧光素酶报告检测TRAIL处理后各组肝癌细胞的活性,同时与miR-25表达水平作相关性分析,初探miR-25对肝癌生物学行为的影响。3.对LCSCs转染miR-25模拟物与miR-25抑制物,以模拟获得性和缺失性功能。以细胞活力检测、裸鼠体内实验等方法研究miR-25对肝癌干细胞增殖、耐药等生物学行为的影响。4.在线数据库预测miR-25可能的靶基因,以线粒体分离、细胞色素C检测、凋亡检测、ROS检测等子实验,通过双荧光素酶报告、免疫沉淀、Western blot等技术鉴定miR-25的信号通路的作用位点和机理。从而进一步探讨miR-25调控肝癌干细胞行为的分子机制。结果:1.分别检测了 miR-25在3株肝癌细胞系及一株永生化肝细胞系L-02中的表达水平,发现在肝癌细胞系中miR-25表达异常上调,而以肝癌干细胞中上调尤为明显。2.肝癌干细胞的miR-25表达水平与其对TRAIL治疗敏感性相关。对肝癌予单用TRAIL治疗后,miR-25高表达的肝癌干细胞因对TRAIL耐受而在治疗后比例升高,这可能是肝癌耐药和早期复发的细胞学层面原因。3.转染miR-25抑制物调低miR-25表达可以提高裸鼠体内肝癌细胞对TRAIL治疗的敏感性。4.靶基因预测分析与细胞表型相结合,确定抑癌基因PTEN为候选靶基因。在LCSCs中以PTEN siRNA抑制PTEN基因表达后,可以观察到miR-25抑制物上调的TRAIL敏感性被逆转,故提示PTEN是miR-25的作用靶点。5.构建pGL3-PTEN-UTR野生型及突变型荧光素酶报告载体,将miR-25模拟物与野生型荧光素酶报告载体共转染HepG2细胞后,荧光素酶报告基因活性明显下降;共转染miR-25抑制物可增加荧光素酶报告基因活性;而共转染突变型荧光素酶报告载体或空白质粒则不影响荧光素酶报告基因活性。表明miR-25过表达可直接作用于PTEN3'UTR的靶序列,下调靶基因的表达。6.miR-25调低可以抑制下游通路PI3K、Akt、Bad等分子的磷酸化,并在胞浆中检测出线粒体释放出的凋亡诱导复合物。表明anti-miR-25通过PTEN/PI3K/Akt/Bad信号通路促进肝癌干细胞对TRAIL的敏感性。结论:1.miR-25在肝癌细胞中表达水平异常上调,其中以肝癌干细胞中表达升高尤为明显。2.miR-25表达与肝癌对TRAIL耐受性相关,对肝癌予单用TRAIL治疗后,miR-25高表达的肝癌干细胞因对TRAIL耐受而在治疗后比例升高,这可能是肝癌耐药和早期复发的细胞学层面原因。3.下调miR-25表达可以在体外和体内增加肝癌干细胞对TRAIL的敏感性。4.PTEN是miR-25的功能靶点。5.下调miR-25表达可以通过PTEN/PI3K/Akt/Bad信号通路传导凋亡信号,与TRAIL治疗协同诱导肝癌干细胞凋亡。6.miR-25抑制物逆转肝癌干细胞耐药性的特点使其有可能成为潜在抗肿瘤治疗靶点。
[Abstract]:Background the incidence of primary hepatocellular carcinoma is the fifth largest in the world, and the mortality rate is ranked third in the world. It is one of the most common malignant tumors. China is one of the largest hepatitis B countries, and it is one of the highest incidence of liver cancer in the world. There are nearly 500 thousand cases of new hepatocellular carcinoma in China every year. The number of deaths caused by liver cancer is about 110 thousand every year. In recent years, thanks to the maturation of liver transplantation and the rapid development of precision medicine, a comprehensive treatment system, assisted by surgical or orthotopic liver transplantation, chemotherapy and immunotherapy, has been formed. However, postoperative tumor recurrence and metastasis still severely restrict the total number of patients with liver cancer. Body prognosis. Even as a liver transplant accepted as a radical cure for liver cancer, the 5 year tumor free survival rate of 5 years conforms to the standard of Milan. The tumor stem cell theory suggests that there are a small number of tumor stem cells with unlimited self renewal capacity in the tumor tissue and can produce different heterogeneous tumor cell groups. Tumorigenesis and differentiation ability play a key role in the development of tumor, recurrence and metastasis and drug resistance. Cancer stem cells have been confirmed in leukemia, lung cancer, prostate cancer, breast cancer, pancreatic cancer, liver cancer and other malignant tumors. The explanation of stem cell theory for the key problem of relapse after hepatoma surgery Cancer stem cells, as "fire species", have stronger vitality and resistance, and play a key role in the process of tumor recurrence and metastasis. With the development of related molecular biology, MicroRNA (MiRNA) is emerging in the physiological processes such as self renewal, metastasis and recurrence of tumor stem cells. This conservative non coding single strand RN A molecules are combined with the target gene mRNA 3'UTR, and then regulate the expression of target gene mRNA. The biological behavior of liver cancer is related to the abnormal expression of MiRNA, which is related to the interaction of the hepatoma cells with the surrounding microenvironment and host immunity, and involves the PTEN/PI3K pathway, HGF/Met pathway, and FAK pass. Abnormal activation of various signaling pathways, such as road and so on. The screening of molecular sites around the regulatory non coded RNA on important signaling pathways and verification of related functions will help further explain the complex mechanism of the development of liver cancer, and also be of great significance for the discovery of potential targets for the treatment of liver cancer. In the background of high tolerance, we used sequencing technology to analyze the MicroRNA expression profiles of hepatoma cells and found that the expression level of miR-25 in hepatoma cells increased abnormally. The purpose of this study was to further study the effect of miR-25 on the biological behavior of hepatoma cells, especially the liver cancer stem cells, and to explore the mechanism of its action in order to find the liver cancer stem. The key target of cell therapy is to provide a potential basis for further development of comprehensive treatment of liver cancer. Objective: This study aims to find and verify the differential expression of MiRNA in hepatoma cells, and to verify the expression level of differentially expressed miR-25 in hepatoma cell lines by real-time quantitative PCR, and to study the expression of miR-25 and hepatoma cells. In particular, the relationship between the biological behavior of liver cancer stem cells and on this basis, the biological function of miR-25 in the hepatoma cells was confirmed by further acquired and missing function experiments, and its upstream and downstream signaling pathways and gene targets were verified and its molecular biological mechanism in the process of cancer pathogenicity was explored. Method: 1. real-time quantitative PCR assay was used. The expression of miR-25 in hepatoma cell lines and normal immortalized hepatocyte lines: liver cancer cell lines and immortalized hepatocyte lines were used as research objects to extract RNA respectively. Reverse transcriptional cDNA. was obtained by real-time quantitative PCR technique to detect miR-25 expression level.2. to induce apoptosis of liver cancer cells by TRAIL, and double Luciferase Report was used to detect TRAIL. The activity of liver cancer cells in each group and the correlation analysis with the expression level of miR-25 were analyzed, and the effects of miR-25 on the biological behavior of liver cancer were studied..3. was used to simulate the acquired and missing functions of LCSCs transfected miR-25 mimics and miR-25 inhibitors. The proliferation of liver cancer stem cells by miR-25 was studied by cell viability detection and nude mice in vivo. The influence of drug resistance and other biological behaviors on the.4. online database to predict the possible target genes of miR-25, with mitochondrial separation, cytochrome C detection, apoptosis detection, ROS detection, and the identification of the miR-25 signaling pathway and mechanism by double Luciferase Report, immunoprecipitation, Western blot and so on, thus further exploring miR-25 The molecular mechanism of regulating the behavior of liver cancer stem cells. Results: 1. the expression level of miR-25 in 3 hepatocellular carcinoma cell lines and an immortalized hepatocyte line L-02 was detected, and the expression of miR-25 in the hepatoma cell line was unusually up-regulated, and the up-regulation of the miR-25 expression level of.2. liver cancer stem cells in the liver cancer stem cells was significantly higher than that of TRAIL. After single use of TRAIL for liver cancer, miR-25 high expression of HCC stem cells increased after treatment with TRAIL tolerance, which may be the cytological factor of drug resistance and early recurrence of liver cancer, and.3. transfection of miR-25 inhibitor to miR-25 expression can improve the sensitivity.4 of liver cancer cells in nude mice to TRAIL treatment. The target gene prediction analysis combined with cell phenotype to determine the tumor suppressor gene PTEN as the candidate target gene. After the inhibition of the PTEN gene expression with PTEN siRNA in LCSCs, the TRAIL sensitivity of the up regulation of miR-25 inhibitor can be observed to be reversed. Therefore, PTEN is the target of miR-25 and.5. for the construction of pGL3-PTEN-UTR wild type and mutant luciferase report. After CO transfection of miR-25 mimics with wild type luciferase reporter vector, the activity of luciferase reporter gene decreased significantly after CO transfection of HepG2 cells with wild type luciferase reporter vector, and co transfection of miR-25 inhibitor could increase the activity of luciferase reporter gene, while CO transfected mutant luciferase reporter carrier or blank plasmid did not affect the activity of luciferase reporter gene. The overexpression of miR-25 can directly affect the target sequence of PTEN3'UTR, and the downregulation of target gene expression.6.miR-25 lowers the phosphorylation of PI3K, Akt, Bad and other molecules in the downstream pathway, and detects the apoptosis induced complex released from mitochondria in the cytoplasm. It indicates that anti-miR-25 can promote the liver cancer stem cells to T through the PTEN/PI3K/Akt/Bad signaling pathway. RAIL sensitivity. Conclusion: the expression level of 1.miR-25 in HCC cells is abnormal up-regulated, especially in the liver cancer stem cells, especially the expression of.2.miR-25 is associated with the tolerance of liver cancer to TRAIL tolerance. After TRAIL treatment of liver cancer, the high expression of miR-25 in the liver cancer stem cells increases after the treatment of TRAIL tolerance, which may be The cytological cause of drug resistance and early recurrence of liver cancer.3. down regulation of miR-25 expression can increase the sensitivity of liver cancer stem cells to TRAIL in vitro and in vivo.4.PTEN is a functional target of miR-25,.5. downregulation miR-25 expression can transmit apoptosis signal through PTEN/PI3K/Akt/Bad signaling pathway, and co induce apoptosis of liver cancer stem cell.6. with TRAIL therapy.6.. MiR-25 inhibitors reverse the drug resistance characteristics of liver cancer stem cells, making it possible to become potential targets for anti-tumor therapy.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7

【参考文献】