长链非编码RNA TUG1介导P-gp和MDR1调控肝细胞癌多药耐药机制的研究

发布时间：2018-04-27 12:11

本文选题：肝细胞癌 + 肿瘤多药耐药　；参考：《浙江大学》2017年博士论文

【摘要】：肝细胞癌(hepatocellular carcinoma,HCC)是全球最常见的恶性肿瘤之一,死亡率仅次于肺癌和胃癌,居全球癌症相关死亡率的第三位。我国每年约有38.3万人死于肝癌,占全球肝癌死亡人数一半以上,给社会和患者家庭带来了沉重的负担。尽管放疗、手术、靶向治疗等治疗手段在不断的进步,化疗依然是目前肝癌综合治疗的主要方法之一,但肝细胞癌的化疗效果仍不容乐观。其中,肿瘤多药耐药(Multidrug resistance,MDR)是化疗治疗效果不佳的主要影响因素。降低或逆转癌细胞的多药耐药已经成为肝癌临床治疗中亟需解决的问题。目的:长链非编码 RNA(long non-coding RNA,lncRNA)TUG1 的表达在 HCC 等多种癌症中显著上调,其异常表达与癌细胞增殖及转移相关,我们推测其可能也与肝细胞癌的多药耐药性相关,因此本文拟通过分子生物学实验方法研究TUG1与肝细胞癌阿霉素耐药的相关性及其可能的调控机制。方法:在本研究中,我们首先采用实时定量PCR(Quantitative Real-time PCR,qRT-PCR)法检测了 TUG1在肝细胞癌组织以及癌旁组织中的表达模式,同时检测两种耐阿霉素肝癌细胞SMMC-7721/ADM和HepG2/ADM中TUG1的表达水平。进一步在耐药细胞系中沉默或过表达TUG1,分别采用CCK-8法和流式细胞仪检测阿霉素处理后细胞的存活力和凋亡率,研究TUG1对阿霉素耐药性的影响。此外,使用蛋白质印记法(Western blotting)检测了沉默或过表达TUG1后,耐药性肝癌细胞中凋亡相关基因PARP、caspase-3的蛋白表达水平。最后,采用qRT-PCR和western blotting方法分别研究MDR1和P-gp的表达水平变化。结果:结果表明,TUG1在肝细胞癌组织和肝癌细胞系中表达上调。此外,在两种耐阿霉素肝癌细胞系(SMMC-7721/ADM,HepG2/ADM)中沉默TUG1并用阿霉素处理过后,两种细胞的成活率均降低而凋亡率均上升。与TUG1沉默结果相反,TUG1过表达后进行阿霉素处理的两种肝癌细胞系存活率均提高而凋亡受抑制。Western blotting结果显示,TUG1沉默后肝癌细胞中细胞凋亡相关基因PARP和caspase-3的蛋白表达水平会显著下调,过表达则相反。最后,我们发现TUG1的下调可抑制P-gp和MDR1的表达,反之会促进P-gp和MDR1的表达。可见,在HCC中TUG1可直接或间接的调控P-gp和MDR1的表达。结论:抑制TUG1的表达可以有效逆转肝癌细胞的阿霉素耐药性,过表达则抑制阿霉素诱导的细胞凋亡,增强肝癌细胞耐药性。TUG1可通过调控P-gp及MDR1的表达,参与HCC多药耐药性的发展,表明TUG1可能是潜在的逆转肝癌耐药性的治疗靶点。
[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.
【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

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