本文選題：肝癌干細(xì)胞 + 耐藥　； 參考：《浙江大學(xué)》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(jiàn)的惡性腫瘤之一。中國(guó)是乙肝大國(guó),亦是全球肝癌最高發(fā)的地區(qū)之一,國(guó)內(nèi)每年新發(fā)肝癌病例近50萬(wàn)例,每年因肝癌致死亡病例約為11萬(wàn),肝癌具有高發(fā)病率和高死亡率的流行病學(xué)特征。近年來(lái),得益于肝移植技術(shù)的成熟和精準(zhǔn)醫(yī)學(xué)的快速發(fā)展,已形成了以外科手術(shù)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(shù)后腫瘤復(fù)發(fā)轉(zhuǎn)移仍然嚴(yán)重制約著肝癌患者的總體預(yù)后。即便是被公認(rèn)為肝癌根治手段的肝移植,其符合米蘭標(biāo)準(zhǔn)的5年無(wú)瘤生存率僅達(dá)73%。腫瘤干細(xì)胞理論認(rèn)為,在腫瘤組織中存在少量具有無(wú)限自我更新能力,并且能產(chǎn)生不同異質(zhì)性腫瘤細(xì)胞群的腫瘤干細(xì)胞。這種干細(xì)胞具有更強(qiáng)的成瘤能力和分化能力,在腫瘤的發(fā)生發(fā)展、復(fù)發(fā)轉(zhuǎn)移和耐藥過(guò)程中起著關(guān)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(shí)了腫瘤干細(xì)胞的存在。針對(duì)肝癌術(shù)后復(fù)發(fā)轉(zhuǎn)移這一關(guān)鍵問(wèn)題,干細(xì)胞理論給出的解釋是,腫瘤干細(xì)胞作為"火種",擁有更強(qiáng)的生命力和耐藥性,在腫瘤復(fù)發(fā)轉(zhuǎn)移過(guò)程中扮演關(guān)鍵角色。隨著相關(guān)分子生物學(xué)研究的深入,MicroRNA(MiRNA)在腫瘤干細(xì)胞自我更新、轉(zhuǎn)移復(fù)發(fā)等生理過(guò)程中的調(diào)節(jié)作用得以浮現(xiàn)。這類(lèi)保守的非編碼單鏈RNA分子通過(guò)互補(bǔ)配對(duì)與靶基因mRNA的3'UTR相結(jié)合,進(jìn)而調(diào)控靶基因mRNA表達(dá)。肝癌的發(fā)生發(fā)展、耐藥、轉(zhuǎn)移等生物學(xué)行為均與MiRNA的異常表達(dá)相關(guān),其中關(guān)系到肝癌細(xì)胞與周?chē)h(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(hào)通路的異�；罨�。圍繞重要信號(hào)通路上的調(diào)節(jié)性非編碼RNA進(jìn)行篩選分子位點(diǎn)并驗(yàn)證相關(guān)功能,有助于進(jìn)一步解釋肝癌發(fā)生發(fā)展的復(fù)雜機(jī)理,對(duì)發(fā)現(xiàn)肝癌治療的潛在靶點(diǎn)也有著重要意義。以肝癌術(shù)后高復(fù)發(fā)率和對(duì)輔助治療高耐受為背景,我們前期利用測(cè)序技術(shù)分析肝癌細(xì)胞的MicroRNA表達(dá)譜時(shí)發(fā)現(xiàn)肝癌細(xì)胞中miR-25表達(dá)水平異常升高。本課題旨在進(jìn)一步研究miR-25對(duì)肝癌細(xì)胞尤其是肝癌干細(xì)胞生物學(xué)行為的影響,并探索其作用機(jī)制,以便尋找針對(duì)肝癌干細(xì)胞治療的關(guān)鍵靶點(diǎn),并為進(jìn)一步拓展肝癌綜合治療手段提供潛在依據(jù)。目的:本研究旨在尋找和驗(yàn)證肝癌細(xì)胞中差異表達(dá)的MiRNA,用實(shí)時(shí)定量PCR的方法驗(yàn)證差異表達(dá)的miR-25在肝癌細(xì)胞系中的表達(dá)水平,并實(shí)驗(yàn)研究miR-25表達(dá)與肝癌細(xì)胞尤其是肝癌干細(xì)胞生物學(xué)行為的關(guān)系。在此基礎(chǔ)上,通過(guò)進(jìn)一步的獲得性和缺失性功能實(shí)驗(yàn)明確miR-25在肝癌細(xì)胞中的生物學(xué)功能,驗(yàn)證其上下游信號(hào)通路和基因靶點(diǎn),探索其在腫瘤致病過(guò)程中的分子生物學(xué)機(jī)制。方法:1.用實(shí)時(shí)定量PCR驗(yàn)證miR-25在肝癌細(xì)胞系和正常永生化肝細(xì)胞系中的表達(dá)情況:肝癌細(xì)胞系及永生化肝細(xì)胞系作為研究對(duì)象,分別提取RNA,反轉(zhuǎn)錄得到cDNA。以實(shí)時(shí)定量PCR技術(shù)分別檢測(cè)其miR-25表達(dá)水平。2.以TRAIL誘導(dǎo)肝癌細(xì)胞凋亡,雙熒光素酶報(bào)告檢測(cè)TRAIL處理后各組肝癌細(xì)胞的活性,同時(shí)與miR-25表達(dá)水平作相關(guān)性分析,初探miR-25對(duì)肝癌生物學(xué)行為的影響。3.對(duì)LCSCs轉(zhuǎn)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(xì)胞活力檢測(cè)、裸鼠體內(nèi)實(shí)驗(yàn)等方法研究miR-25對(duì)肝癌干細(xì)胞增殖、耐藥等生物學(xué)行為的影響。4.在線數(shù)據(jù)庫(kù)預(yù)測(cè)miR-25可能的靶基因,以線粒體分離、細(xì)胞色素C檢測(cè)、凋亡檢測(cè)、ROS檢測(cè)等子實(shí)驗(yàn),通過(guò)雙熒光素酶報(bào)告、免疫沉淀、Western blot等技術(shù)鑒定miR-25的信號(hào)通路的作用位點(diǎn)和機(jī)理。從而進(jìn)一步探討miR-25調(diào)控肝癌干細(xì)胞行為的分子機(jī)制。結(jié)果:1.分別檢測(cè)了 miR-25在3株肝癌細(xì)胞系及一株永生化肝細(xì)胞系L-02中的表達(dá)水平,發(fā)現(xiàn)在肝癌細(xì)胞系中miR-25表達(dá)異常上調(diào),而以肝癌干細(xì)胞中上調(diào)尤為明顯。2.肝癌干細(xì)胞的miR-25表達(dá)水平與其對(duì)TRAIL治療敏感性相關(guān)。對(duì)肝癌予單用TRAIL治療后,miR-25高表達(dá)的肝癌干細(xì)胞因?qū)RAIL耐受而在治療后比例升高,這可能是肝癌耐藥和早期復(fù)發(fā)的細(xì)胞學(xué)層面原因。3.轉(zhuǎn)染miR-25抑制物調(diào)低miR-25表達(dá)可以提高裸鼠體內(nèi)肝癌細(xì)胞對(duì)TRAIL治療的敏感性。4.靶基因預(yù)測(cè)分析與細(xì)胞表型相結(jié)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(dá)后,可以觀察到miR-25抑制物上調(diào)的TRAIL敏感性被逆轉(zhuǎn),故提示PTEN是miR-25的作用靶點(diǎn)。5.構(gòu)建pGL3-PTEN-UTR野生型及突變型熒光素酶報(bào)告載體,將miR-25模擬物與野生型熒光素酶報(bào)告載體共轉(zhuǎn)染HepG2細(xì)胞后,熒光素酶報(bào)告基因活性明顯下降;共轉(zhuǎn)染miR-25抑制物可增加熒光素酶報(bào)告基因活性;而共轉(zhuǎn)染突變型熒光素酶報(bào)告載體或空白質(zhì)粒則不影響熒光素酶報(bào)告基因活性。表明miR-25過(guò)表達(dá)可直接作用于PTEN3'UTR的靶序列,下調(diào)靶基因的表達(dá)。6.miR-25調(diào)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(cè)出線粒體釋放出的凋亡誘導(dǎo)復(fù)合物。表明anti-miR-25通過(guò)PTEN/PI3K/Akt/Bad信號(hào)通路促進(jìn)肝癌干細(xì)胞對(duì)TRAIL的敏感性。結(jié)論:1.miR-25在肝癌細(xì)胞中表達(dá)水平異常上調(diào),其中以肝癌干細(xì)胞中表達(dá)升高尤為明顯。2.miR-25表達(dá)與肝癌對(duì)TRAIL耐受性相關(guān),對(duì)肝癌予單用TRAIL治療后,miR-25高表達(dá)的肝癌干細(xì)胞因?qū)RAIL耐受而在治療后比例升高,這可能是肝癌耐藥和早期復(fù)發(fā)的細(xì)胞學(xué)層面原因。3.下調(diào)miR-25表達(dá)可以在體外和體內(nèi)增加肝癌干細(xì)胞對(duì)TRAIL的敏感性。4.PTEN是miR-25的功能靶點(diǎn)。5.下調(diào)miR-25表達(dá)可以通過(guò)PTEN/PI3K/Akt/Bad信號(hào)通路傳導(dǎo)凋亡信號(hào),與TRAIL治療協(xié)同誘導(dǎo)肝癌干細(xì)胞凋亡。6.miR-25抑制物逆轉(zhuǎn)肝癌干細(xì)胞耐藥性的特點(diǎn)使其有可能成為潛在抗腫瘤治療靶點(diǎn)。
[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(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R735.7

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本文編號(hào)：1857767