羽扇豆醇诱导microRNAs调控HeLa细胞耐药响应通路分析

发布时间：2017-12-28 03:08

本文关键词：羽扇豆醇诱导microRNAs调控HeLa细胞耐药响应通路分析　出处：《哈尔滨工业大学》2015年硕士论文　论文类型：学位论文

【摘要】：化疗是目前癌症治疗的主要手段,顺铂是癌症化疗中最常使用的一种铂类化合药物。但是,肿瘤细胞耐药性的形成却成为长期治疗恶性肿瘤的障碍。近年来,越来越多的证据表明micro RNAs与肿瘤形成过程中的多种生物学活动有关,同时,其在肿瘤细胞耐药性获得过程中也起着重要作用。研究显示,通过膳食控制能预防超过三分之二的肿瘤发生,蔬菜、水果、膳食纤维和某些微营养成分均具有一定的防癌作用,果蔬中含有一种三萜类生活性物质——羽扇豆醇,其抗氧化、诱导凋亡、抗恶性肿瘤增殖、抗基因突变以及抗炎症和抑制肿瘤细胞生长等多种功效使得其在肿瘤的预防方面引起广泛关注。目前,部分micro RNAs对肿瘤耐药性的作用已逐渐被揭示,与此同时,羽扇豆醇对肿瘤细胞的抑制机制也需要不断完善,因此,研究耐药细胞中羽扇豆醇对micro RNAs的表达调控作用对肿瘤的治疗具有一定的指导意义,同时也能进一步完善羽扇豆醇的抗肿瘤机制。本研究通过浓度梯度法诱导出四种不同程度的顺铂抗性的He La细胞(He La/CR1、He La/CR2、He La/CR3和He La/CR4),检测其耐药倍数,对He La细胞耐药性活动过程进行模拟。根据前期研究和已有理论知识,挑选了五种候选耐药性相关micro RNAs(mi R-183、mi R-182、mi R-30a、mi R-15b和mi R-16)以及其潜在靶标作为研究对象。采用RT-q PCR对micro RNAs和相应的m RNAs表达量进行检测。随后用高剂量的羽扇豆醇处理挑选出耐药性较好的He La/CR4,并设置四个不同的时间节点(6h、12h、18h以及24h),并对药物处理细胞中的候选micro RNAs及其靶标进行荧光定量PCR,对其表达变化进行检测,进而对羽扇豆醇诱导耐药细胞中micro RNAs相关的抑癌通路进行分析。结果表明,在四种耐药细胞中,mi R-182和mi R-15b表达上调;但是mi R-183和mi R-30a表达却出现显著下调。与此同时,根据靶标RT-PCR结果可以得出KIAA1199是mi R-183的主要靶标,PDCD4为mi R-182的靶标,但是mi R-30a的调节靶标还需要进一步验证。与非耐药细胞相比,耐药细胞中micro RNAs表达量的显著变化,表明He La细胞耐药性的发展与一部分micro RNAs相关。羽扇豆醇处理耐药细胞后的结果表明,相关的micro RNAs及其靶标表达再次出现显著变化,而且变化趋势耐药性发展过程中的变化趋势相反,这表明羽扇豆醇可能通过调节相关micro RNAs的表达而达到抑制肿瘤的目的,同时,micro RNAs可能通过调节细胞对化疗的敏感性而成为肿瘤治疗的研究方向。结合实验结果和已有理论知识得出,在耐药He La细胞中,羽扇豆醇调节micro RNAs参与的相关抑癌信号通路及下游的信号分子有Wnt/β-catenin、PI3K/PTEN/AKT、BAX、YB-1、NF-κB、Caspase家族、PGRMC1、ATG以及TGF-TGF-β等,从分析可以看出,羽扇豆醇能通过多靶标,多信号通路来抑制肿瘤细胞迁移和细胞增殖、促进细胞凋亡以及增强药物敏感性等。
[Abstract]:Chemotherapy is the main means of cancer treatment at present, and cisplatin is one of the most commonly used platinum - like drugs in cancer chemotherapy. However, the formation of drug resistance in tumor cells is an obstacle to the long-term treatment of malignant tumors. In recent years, more and more evidences show that micro RNAs is related to many biological activities in tumor formation, and it plays an important role in the process of drug resistance acquisition. Research shows that, through dietary control could prevent more than 2/3 of the tumor, vegetables, fruits, dietary fiber and some micro nutrients have anti-cancer effects, fruits and vegetables contain a three terpenoid material life -- lupeol, its antioxidant, inducing apoptosis, anti proliferation, anti malignant tumor and gene mutation the anti-inflammatory and inhibiting tumor cell growth and other effects which caused widespread concern in the prevention of tumor. At present, part of micro RNAs on tumor drug resistance has been gradually revealed. At the same time, the inhibition mechanism of lupeol on tumor cells also need to constantly improve, therefore, has a certain guiding significance for research on expression regulation effect of lupeol in resistant cells on micro RNAs of tumor, but also to further improve the mechanism of tumor anti lupeol. In this study, four different levels of cisplatin resistant He La cells (He La/CR1, He La/CR2, He La/CR3 and He La/CR4) were induced by concentration gradient method. The drug resistance times were detected, and the process of drug resistance activity of He cells was simulated. According to previous studies and existing theoretical knowledge, five candidate resistance related micro RNAs (MI R-183, MI R-182, MI R-30a, MI R-15b and MI R-16) and their potential targets were selected as research objects. RT-q PCR was used to detect the expression of micro RNAs and corresponding M RNAs. Followed by high doses of lupeol treatment selected better He resistance La/CR4, and set the four different time points (6h, 12h, 18h and 24h), and fluorescence quantitative PCR on drug candidate micro in RNAs cells and on the expression of target, change detection, analysis of tumor suppressor pathway and then the lupeol induced micro resistant cells related to RNAs. The results showed that the expression of MI R-182 and MI R-15b was up-regulated in four kinds of drug-resistant cells, but the expression of MI R-183 and MI R-30a decreased significantly. Meanwhile, according to the result of target RT-PCR, it can be concluded that KIAA1199 is the main target of MI R-183, PDCD4 is the target of MI R-182, but the target of MI R-30a needs further verification. Compared with non drug resistant cells, the significant changes in the expression of micro RNAs in drug-resistant cells showed that the development of He La cell resistance was associated with a part of micro RNAs. The results of lupeol treatment resistant cells after the indicated that the expression of micro RNAs and its related target again significant changes, and the changes of development trend in the trend of drug resistance instead, suggesting that lupeol may lead to inhibition of tumor by regulating expression of micro, RNAs and micro RNAs may become a research direction treatment of cancer by regulating cell sensitivity to chemotherapy. According to the experimental results and theoretical knowledge obtained in He resistant La cells, lupeol regulation related inhibitory pathway and the downstream signal molecule micro RNAs cancer in Wnt/, PI3K/PTEN/AKT, BAX, -catenin beta YB-1, NF-, Caspase, PGRMC1 kappa B family, ATG and TGF-TGF- beta, as can be seen from the analysis lupeol, through multi targets, multi signal pathway to inhibit tumor cell migration and cell proliferation, promote cell apoptosis and enhance the drug sensitivity.
【学位授予单位】：哈尔滨工业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R73-36

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