本文選題：肝癌干細胞 + 耐藥　； 參考：《浙江大學》2017年博士論文

【摘要】：背景原發(fā)性肝癌的發(fā)病率位居全球第5位,病死率在全球范圍排名第3位,是最為常見的惡性腫瘤之一。中國是乙肝大國,亦是全球肝癌最高發(fā)的地區(qū)之一,國內每年新發(fā)肝癌病例近50萬例,每年因肝癌致死亡病例約為11萬,肝癌具有高發(fā)病率和高死亡率的流行病學特征。近年來,得益于肝移植技術的成熟和精準醫(yī)學的快速發(fā)展,已形成了以外科手術或原位肝移植為主,放化療及免疫靶向治療為輔助的綜合治療體系。然而,術后腫瘤復發(fā)轉移仍然嚴重制約著肝癌患者的總體預后。即便是被公認為肝癌根治手段的肝移植,其符合米蘭標準的5年無瘤生存率僅達73%。腫瘤干細胞理論認為,在腫瘤組織中存在少量具有無限自我更新能力,并且能產生不同異質性腫瘤細胞群的腫瘤干細胞。這種干細胞具有更強的成瘤能力和分化能力,在腫瘤的發(fā)生發(fā)展、復發(fā)轉移和耐藥過程中起著關鍵作用。在白血病、肺癌、前列腺癌、乳腺癌、胰腺癌、肝癌等惡性腫瘤中均已證實了腫瘤干細胞的存在。針對肝癌術后復發(fā)轉移這一關鍵問題,干細胞理論給出的解釋是,腫瘤干細胞作為"火種",擁有更強的生命力和耐藥性,在腫瘤復發(fā)轉移過程中扮演關鍵角色。隨著相關分子生物學研究的深入,MicroRNA(MiRNA)在腫瘤干細胞自我更新、轉移復發(fā)等生理過程中的調節(jié)作用得以浮現。這類保守的非編碼單鏈RNA分子通過互補配對與靶基因mRNA的3'UTR相結合,進而調控靶基因mRNA表達。肝癌的發(fā)生發(fā)展、耐藥、轉移等生物學行為均與MiRNA的異常表達相關,其中關系到肝癌細胞與周圍微環(huán)境和宿主免疫的交互作用,并涉及PTEN/PI3K通路、HGF/Met通路、FAK通路等等各種信號通路的異�；罨@重要信號通路上的調節(jié)性非編碼RNA進行篩選分子位點并驗證相關功能,有助于進一步解釋肝癌發(fā)生發(fā)展的復雜機理,對發(fā)現肝癌治療的潛在靶點也有著重要意義。以肝癌術后高復發(fā)率和對輔助治療高耐受為背景,我們前期利用測序技術分析肝癌細胞的MicroRNA表達譜時發(fā)現肝癌細胞中miR-25表達水平異常升高。本課題旨在進一步研究miR-25對肝癌細胞尤其是肝癌干細胞生物學行為的影響,并探索其作用機制,以便尋找針對肝癌干細胞治療的關鍵靶點,并為進一步拓展肝癌綜合治療手段提供潛在依據。目的:本研究旨在尋找和驗證肝癌細胞中差異表達的MiRNA,用實時定量PCR的方法驗證差異表達的miR-25在肝癌細胞系中的表達水平,并實驗研究miR-25表達與肝癌細胞尤其是肝癌干細胞生物學行為的關系。在此基礎上,通過進一步的獲得性和缺失性功能實驗明確miR-25在肝癌細胞中的生物學功能,驗證其上下游信號通路和基因靶點,探索其在腫瘤致病過程中的分子生物學機制。方法:1.用實時定量PCR驗證miR-25在肝癌細胞系和正常永生化肝細胞系中的表達情況:肝癌細胞系及永生化肝細胞系作為研究對象,分別提取RNA,反轉錄得到cDNA。以實時定量PCR技術分別檢測其miR-25表達水平。2.以TRAIL誘導肝癌細胞凋亡,雙熒光素酶報告檢測TRAIL處理后各組肝癌細胞的活性,同時與miR-25表達水平作相關性分析,初探miR-25對肝癌生物學行為的影響。3.對LCSCs轉染miR-25模擬物與miR-25抑制物,以模擬獲得性和缺失性功能。以細胞活力檢測、裸鼠體內實驗等方法研究miR-25對肝癌干細胞增殖、耐藥等生物學行為的影響。4.在線數據庫預測miR-25可能的靶基因,以線粒體分離、細胞色素C檢測、凋亡檢測、ROS檢測等子實驗,通過雙熒光素酶報告、免疫沉淀、Western blot等技術鑒定miR-25的信號通路的作用位點和機理。從而進一步探討miR-25調控肝癌干細胞行為的分子機制。結果:1.分別檢測了 miR-25在3株肝癌細胞系及一株永生化肝細胞系L-02中的表達水平,發(fā)現在肝癌細胞系中miR-25表達異常上調,而以肝癌干細胞中上調尤為明顯。2.肝癌干細胞的miR-25表達水平與其對TRAIL治療敏感性相關。對肝癌予單用TRAIL治療后,miR-25高表達的肝癌干細胞因對TRAIL耐受而在治療后比例升高,這可能是肝癌耐藥和早期復發(fā)的細胞學層面原因。3.轉染miR-25抑制物調低miR-25表達可以提高裸鼠體內肝癌細胞對TRAIL治療的敏感性。4.靶基因預測分析與細胞表型相結合,確定抑癌基因PTEN為候選靶基因。在LCSCs中以PTEN siRNA抑制PTEN基因表達后,可以觀察到miR-25抑制物上調的TRAIL敏感性被逆轉,故提示PTEN是miR-25的作用靶點。5.構建pGL3-PTEN-UTR野生型及突變型熒光素酶報告載體,將miR-25模擬物與野生型熒光素酶報告載體共轉染HepG2細胞后,熒光素酶報告基因活性明顯下降;共轉染miR-25抑制物可增加熒光素酶報告基因活性;而共轉染突變型熒光素酶報告載體或空白質粒則不影響熒光素酶報告基因活性。表明miR-25過表達可直接作用于PTEN3'UTR的靶序列,下調靶基因的表達。6.miR-25調低可以抑制下游通路PI3K、Akt、Bad等分子的磷酸化,并在胞漿中檢測出線粒體釋放出的凋亡誘導復合物。表明anti-miR-25通過PTEN/PI3K/Akt/Bad信號通路促進肝癌干細胞對TRAIL的敏感性。結論:1.miR-25在肝癌細胞中表達水平異常上調,其中以肝癌干細胞中表達升高尤為明顯。2.miR-25表達與肝癌對TRAIL耐受性相關,對肝癌予單用TRAIL治療后,miR-25高表達的肝癌干細胞因對TRAIL耐受而在治療后比例升高,這可能是肝癌耐藥和早期復發(fā)的細胞學層面原因。3.下調miR-25表達可以在體外和體內增加肝癌干細胞對TRAIL的敏感性。4.PTEN是miR-25的功能靶點。5.下調miR-25表達可以通過PTEN/PI3K/Akt/Bad信號通路傳導凋亡信號,與TRAIL治療協(xié)同誘導肝癌干細胞凋亡。6.miR-25抑制物逆轉肝癌干細胞耐藥性的特點使其有可能成為潛在抗腫瘤治療靶點。
[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.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R735.7

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本文編號：1857767