卵巢癌顺铂耐药相关基因间长非编码RNA

发布时间：2018-01-22 21:51

本文关键词： 卵巢癌顺铂耐药 lincRNA H19　出处：《浙江大学》2016年博士论文　论文类型：学位论文

【摘要】：卵巢癌是女性常见的恶性肿瘤之一,其死亡率居妇科恶性肿瘤之首。化疗原发和继发耐药是导致卵巢癌患者存活率低的重要因素。顺铂和DNA可以形成链内和链间交联,从而引起DNA损伤。研究表明一些基因间长片段非编码RNA(lincRNAs)广泛参与了肿瘤细胞DNA损伤应答的各个环节。因此,lincRNAs可能参与了卵巢癌铂类耐药发生。然而,目前关于"lincRNAs如何参与卵巢癌顺铂耐药的产生"的认识依旧有限。1)本论文通过顺铂间隔刺激法建立了卵巢癌顺铂耐药细胞株。通过克隆形成率法分析,发现细胞株A2780-DR对顺铂、紫杉醇和双氧水的抗性都明显增强。通过转录组测序分析卵巢癌顺铂敏感和耐药细胞株,结合生物信息学分析获得了 436个差异表达的lincRNAs。经荧光定量 PCR验证,linc-RECK-3,H19,LUCAT1,LINC00961 和 linc-CARS2-2等在耐药细胞株中明显增加,而linc-TNFRSF19-1和LINC00515在耐药株中明显降低。H19和LUACT1在癌组织中高表达,且H19高表达的卵巢癌患者容易复发。2)H19在敏感细胞株中能被顺铂诱导表达,而在耐药细胞株中不能被诱导。耐药细胞株中H19被干扰后,细胞内H19的表达量明显下降,同时细胞对顺铂的敏感性也明显增加。裸鼠实验也证实了耐药细胞中H19被干扰后增加了对顺铂的敏感性。3)采用非标记定量蛋白质组学分析寻找被H19调控并参与顺铂耐药发生的蛋白。耐药细胞株与敏感细胞株中差异表达蛋白有113个,其中有27个是NRF2靶蛋白。KEGG分析发现有6个NRF2靶蛋白参与还原型谷胱甘肽(GSH)代谢。Western-blot实验发现有7个蛋白(ALDH1A1,NQO1,GSR,G6PD,GCLC,GCLM和NRF2)均在耐药细胞中高表达。与耐药细胞比较,敏感细胞(A2780和A2780-DR/H19si)对双氧水更敏感,且细胞内GSH水平更低。耐药细胞NRF2干扰或GSH合成酶抑制剂丁硫氨酸亚砜胺(BSO)处理后均增加了对顺铂的敏感性。本论文研究发现了一些与顺铂耐药有潜在关系的lincRNAs,为后续深入研究这些lincRNAs参与顺铂耐药分子机制提供基础。论文首次提出H19通过NRF2通路调节GSH代谢促进顺铂耐药,加深了我们对顺铂耐药机制的理解。为肿瘤化疗效果的提升提供了理论基础。
[Abstract]:Ovarian cancer is one of the most common malignant tumors in women. Chemotherapeutic primary and secondary drug resistance is an important factor leading to low survival rate of ovarian cancer patients. Cisplatin and DNA can form intrachain and inter-chain crosslinking. It is shown that some noncoding RNAs (lincRNASs) are involved in the DNA damage response of tumor cells. LincRNAs may be involved in the development of platinum resistance in ovarian cancer. There is still limited understanding of how lincRNAs is involved in the production of cisplatin resistance in ovarian cancer. In this paper, cisplatin resistant ovarian cancer cell lines were established by cisplatin interval stimulation. It was found that the resistance of cell line A2780-DR to cisplatin, paclitaxel and hydrogen peroxide was significantly increased. 436 differentially expressed lincRNAs were obtained by bioinformatics analysis. Linc-RECK-3 H19 LUCAT1 was confirmed by fluorescence quantitative PCR. LINC00961 and linc-CARS2-2 were significantly increased in drug-resistant cell lines. However, linc-TNFRSF19-1 and LINC00515 significantly decreased the expression of. H19 and LUACT1 in cancer tissues. Moreover, high expression of H19 in ovarian cancer patients can be easily relapsed. 2H19 can be induced by cisplatin in sensitive cell line, but not in drug-resistant cell line. H19 in drug-resistant cell line is interfered with. The expression of H19 decreased significantly. At the same time, the sensitivity of cells to cisplatin was also significantly increased. The results of nude mice also confirmed that H19 in drug-resistant cells increased the sensitivity to cisplatin after being interfered with. 3). Unlabeled quantitative proteomic analysis was used to search for proteins regulated by H19 and involved in cisplatin resistance. There were 113 differentially expressed proteins in drug resistant and sensitive cell lines. Among them, 27 were NRF2 target proteins. KEGG analysis showed that 6 NRF2 target proteins were involved in GSH of reduced glutathione. The results of metabolism. Western-blot analysis showed that there were 7 ALDH1A1 proteins. NQO1GSRG6PDGCLM and NRF2) were highly expressed in drug-resistant cells, compared with those in drug-resistant cells. Sensitive cells such as A2780 and A2780-DRR / H19si) were more sensitive to hydrogen peroxide. NRF2 interference or GSH synthase inhibitor butyronine sulfoxide amines (BSOs) was also found in drug-resistant cells. After treatment, the sensitivity to cisplatin was increased. In this study, we found some lincRNAs which have potential relationship with cisplatin resistance. In order to provide a basis for further study of the molecular mechanism of cisplatin resistance, H19 was proposed to regulate the metabolism of GSH through the NRF2 pathway to promote cisplatin resistance. Our understanding of the mechanism of cisplatin resistance is deepened, which provides a theoretical basis for the improvement of chemotherapy effect.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.31

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