本文選題：肝細胞癌 + 腫瘤多藥耐藥　； 參考：《浙江大學》2017年博士論文

【摘要】：肝細胞癌(hepatocellular carcinoma,HCC)是全球最常見的惡性腫瘤之一,死亡率僅次于肺癌和胃癌,居全球癌癥相關死亡率的第三位。我國每年約有38.3萬人死于肝癌,占全球肝癌死亡人數(shù)一半以上,給社會和患者家庭帶來了沉重的負擔。盡管放療、手術、靶向治療等治療手段在不斷的進步,化療依然是目前肝癌綜合治療的主要方法之一,但肝細胞癌的化療效果仍不容樂觀。其中,腫瘤多藥耐藥(Multidrug resistance,MDR)是化療治療效果不佳的主要影響因素。降低或逆轉癌細胞的多藥耐藥已經成為肝癌臨床治療中亟需解決的問題。目的:長鏈非編碼 RNA(long non-coding RNA,lncRNA)TUG1 的表達在 HCC 等多種癌癥中顯著上調,其異常表達與癌細胞增殖及轉移相關,我們推測其可能也與肝細胞癌的多藥耐藥性相關,因此本文擬通過分子生物學實驗方法研究TUG1與肝細胞癌阿霉素耐藥的相關性及其可能的調控機制。方法:在本研究中,我們首先采用實時定量PCR(Quantitative Real-time PCR,qRT-PCR)法檢測了 TUG1在肝細胞癌組織以及癌旁組織中的表達模式,同時檢測兩種耐阿霉素肝癌細胞SMMC-7721/ADM和HepG2/ADM中TUG1的表達水平。進一步在耐藥細胞系中沉默或過表達TUG1,分別采用CCK-8法和流式細胞儀檢測阿霉素處理后細胞的存活力和凋亡率,研究TUG1對阿霉素耐藥性的影響。此外,使用蛋白質印記法(Western blotting)檢測了沉默或過表達TUG1后,耐藥性肝癌細胞中凋亡相關基因PARP、caspase-3的蛋白表達水平。最后,采用qRT-PCR和western blotting方法分別研究MDR1和P-gp的表達水平變化。結果:結果表明,TUG1在肝細胞癌組織和肝癌細胞系中表達上調。此外,在兩種耐阿霉素肝癌細胞系(SMMC-7721/ADM,HepG2/ADM)中沉默TUG1并用阿霉素處理過后,兩種細胞的成活率均降低而凋亡率均上升。與TUG1沉默結果相反,TUG1過表達后進行阿霉素處理的兩種肝癌細胞系存活率均提高而凋亡受抑制。Western blotting結果顯示,TUG1沉默后肝癌細胞中細胞凋亡相關基因PARP和caspase-3的蛋白表達水平會顯著下調,過表達則相反。最后,我們發(fā)現(xiàn)TUG1的下調可抑制P-gp和MDR1的表達,反之會促進P-gp和MDR1的表達。可見,在HCC中TUG1可直接或間接的調控P-gp和MDR1的表達。結論:抑制TUG1的表達可以有效逆轉肝癌細胞的阿霉素耐藥性,過表達則抑制阿霉素誘導的細胞凋亡,增強肝癌細胞耐藥性。TUG1可通過調控P-gp及MDR1的表達,參與HCC多藥耐藥性的發(fā)展,表明TUG1可能是潛在的逆轉肝癌耐藥性的治療靶點。
[Abstract]:Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. About 383000 people die of liver cancer every year in China, accounting for more than half of the deaths of liver cancer in the world, which brings a heavy burden to the society and the families of patients. Although radiotherapy, surgery, targeted therapy and other treatment methods are progressing, chemotherapy is still one of the main methods in the comprehensive treatment of liver cancer, but the effect of chemotherapy on HCC is still not optimistic. Among them, multidrug resistance (MDR) is the main influencing factor for the poor effect of chemotherapy. Reducing or reversing multidrug resistance of cancer cells has become an urgent problem in the clinical treatment of liver cancer. Objective: the expression of TUG1 in long chain noncoding RNA(long non-coding RNAs was significantly up-regulated in many cancers such as HCC, and its abnormal expression was related to the proliferation and metastasis of cancer cells. We speculated that it might also be related to the multidrug resistance of hepatocellular carcinoma. Therefore, we intend to study the relationship between TUG1 and adriamycin resistance in hepatocellular carcinoma and its possible regulatory mechanism by molecular biological methods. Methods: in this study, we first detected the expression patterns of TUG1 in hepatocellular carcinoma and adjacent tissues by real-time quantitative PCR(Quantitative Real-time PCR qRT-PCR. at the same time, we detected the expression levels of SMMC-7721/ADM and HepG2/ADM in two kinds of adriamycin resistant hepatoma cells. Furthermore, TUG1 was silenced or overexpressed in drug-resistant cell lines. The viability and apoptosis rate of the cells treated with adriamycin were detected by CCK-8 assay and flow cytometry, respectively, and the effect of TUG1 on adriamycin resistance was studied. In addition, protein imprinting was used to detect the protein expression of the apoptosis-related gene PARPncaspase-3 in drug-resistant hepatocellular carcinoma cells after silencing or overexpression of TUG1. Finally, qRT-PCR and western blotting were used to study the expression level of MDR1 and P-gp. Results: the expression of TUG1 was up-regulated in hepatocellular carcinoma and hepatoma cell lines. In addition, after silencing TUG1 in two adriamycin-resistant hepatoma cell lines (SMMC-7721 / ADMG _ 2 / ADM) and treated with adriamycin, the survival rate of both cells decreased and the apoptosis rate increased. Contrary to the results of TUG1 silencing, the survival rate of the two hepatoma cell lines treated with doxorubicin was increased and apoptosis was inhibited. Western blotting results showed that PARP and caspase-3, the apoptosis-related genes, were expressed on the protein surface of the apoptosis-related gene PARP and caspase-3 in the hepatoma cells after the silencing of TUG1. The level will be significantly lowered. Overexpression is the opposite. Finally, we found that down-regulation of TUG1 can inhibit the expression of P-gp and MDR1, whereas it can promote the expression of P-gp and MDR1. Therefore, TUG1 can directly or indirectly regulate the expression of P-gp and MDR1 in HCC. Conclusion: inhibiting the expression of TUG1 can effectively reverse the adriamycin resistance of hepatoma cells, and over-expression can inhibit the apoptosis induced by doxorubicin, and enhance the expression of P-gp and MDR1 by enhancing the expression of drug resistance. The development of multidrug resistance in HCC suggests that TUG1 may be a potential therapeutic target for reversing drug resistance in hepatocellular carcinoma.
【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：1810684