氯福克酚和索拉非尼协同作用前列腺癌PC3细胞的机制研究以及miR-4293 rs12220909多态性与肺癌易感性的关联

发布时间：2017-12-28 06:33

本文关键词：氯福克酚和索拉非尼协同作用前列腺癌PC3细胞的机制研究以及miR-4293 rs12220909多态性与肺癌易感性的关联分析　出处：《苏州大学》2016年硕士论文　论文类型：学位论文

【摘要】：前列腺癌是男性生殖系肿瘤中常见疾病之一,其发病率和死亡率呈逐年增长趋势,这一趋势在中国尤为明显。在前列腺癌的治疗中,早期患者通过前列腺切除术合并辅助放射来治疗;对于晚期患者来说,主要为激素治疗,但经过一段治疗期后,前列腺癌从对激素依赖逐渐变为激素抵抗型,这会使治疗更加困难。最后当去势治疗或传统的激素治疗失败后,就只能采用化疗。然而多次化疗会使患者对化疗药物产生耐药性,导致化疗总体疗效不佳。因此,开发更加有效的化疗药物是当前治疗前列腺癌的新趋势。由于药物发现和开发是一个昂贵和耗时的过程,以及旧药新用给我们提供的捷径,所以前期本实验利用约翰·霍普金斯大学建立的临床药库进行大规模筛选,初步鉴定出一种目前已用于临床治疗呼吸道感染的抗生素药物—氯福克酚(Clofoctol),它能够抑制前列腺癌激素不敏感细胞系PC3增殖。鉴于单药治疗时剂量增加造成的副反应以及药物联用治疗肿瘤的优势,前期本实验室选用四十种在临床上已经应用且对前列腺癌细胞有抑制作用的小分子药物,与氯福克酚(Clofoctol)分别联用处理PC3细胞,根据Chou-Talalay计算公式及剂量-反应实验进行筛选,表明氯福克酚(Clofoctol)和索拉非尼(Sorafenib)能够协同抑制PC3细胞的增殖。索拉非尼(Sorafenib)是一种多靶点多激酶抑制剂,经美国FDA批准被用于治疗晚期肾细胞癌和肝癌,目前大量研究证实索拉非尼(Sorafenib)对前列腺癌的增殖具有抑制作用。本研究我们以前列腺癌PC3为细胞模型,研究氯福克酚(Clofoctol)和索拉非尼(Sorafenib)协同作用的机制。通过细胞凋亡、细胞周期、Western blot以及qRT-PCR等技术,我们确定10μM氯福克酚(Clofoctol)和6μM索拉非尼(Sorafenib)联合用药能够使78.84%的细胞被阻滞在G1期,且细胞协同凋亡率达到16.65%,高于Clofoctol或Sorafenib单独用药时的凋亡率;qRT-PCR结果显示药物联用后ER-stress通路中DDIT3/CHOP、GADD34、ATF6和ATF4 mRNA表达水平明显增高,对应的Western blot结果表明8小时处DDIT3/CHOP和ATF4的蛋白水平显著性协同上调,并且在联合处理24h小时处,XBP1前体明显被剪接成有转录活性的XBP1s。也就是说,Clofoctol和Sorafenib联用激活了ER-stress的IRE-1a和ATF6通路。接下来我们又观察到氯福克酚(Clofoctol)单独用药后PC3细胞会出现类似RNAi技术沉默VCP/p97后的内质网液泡化和泛素化蛋白累积现象。鉴于VCP/p97在内质网相关的蛋白酶体系统中起重要作用,因此我们推测氯福克酚(Clofoctol)可能靶向VCP/p97,抑制其功能的发挥。基于实验室的另外一个研究方向,我们还进行了microRNA的单核苷酸多态性与肺癌易感性的关联分析。肺癌是发病率和死亡率增长最快,对人群健康和生命威胁最大的恶性肿瘤之一,而遗传因素是影响肺癌发病的重要原因之一。越来越多的研究证明,microRNA发挥癌基因或抑癌基因的功能,且microRNA的单核苷酸多态性与肿瘤易感性存在一定关联。我们通过前期的统计分析及文献查阅后以miR-4293rs12220909多态性位点为研究对象,拟通过病例-对照的研究方法来分析该mirSNP与中国人群肺癌易感性的关联。结果表明,在我们所研究的998例肺癌病例和1471例正常对照组中,miR-4293rs12220909多态性位点与肺癌的发病风险具有显著相关性:突变基因型GC/CC能够显著降低肺癌的发病风险(OR=0.687;95%CI=0.564-0.837),等位基因C是肺癌易感性的保护因素(OR=0.734;95%CI=0.616-0.874);对遗传模型进行分层分析后发现,GC基因型在≥62岁、62岁、男性、吸烟、非吸烟、肺腺癌及肺鳞癌人群中均能降低肺癌患病风险。接下来,我们对于miR-4293 rs12220909多态性位点降低肺癌的发病风险进行了机制研究,发现野生型miR-4293可能通过调控抑癌基因PRKAA1和ADAMTSL3来增强肺癌易感性,从而在肺癌的发生、发展中起到促进作用。
[Abstract]:Prostate cancer is one of the common diseases in male reproductive system tumors. The incidence and mortality of prostate cancer are increasing year by year. This trend is particularly obvious in China. In the treatment of prostate cancer in patients with early by prostate resection combined with radiotherapy for advanced treatment; patients, such as hormone therapy, but after a period after the treatment period, from prostate cancer for hormone dependent became steroid resistant, which makes the treatment more difficult. Finally, chemotherapy is used only after the treatment of castration or the failure of traditional hormone therapy. However, multiple chemotherapy can cause drug resistance to chemotherapeutic drugs, resulting in the overall effectiveness of chemotherapy. Therefore, the development of more effective chemotherapeutic drugs is a new trend in the treatment of prostate cancer. Due to drug discovery and development is a costly and time-consuming process, and to provide us with new old drugs, so this experiment using the pre clinical pharmacy store established by Johns Hopkins University for large-scale screening, preliminary identification of an antibiotic has already been used in the clinical treatment of respiratory tract infection clofoctol (Clofoctol), it can inhibiting hormone insensitive prostate cancer cell line PC3 proliferation. In view of the single drug treatment dose increased adverse reactions caused by drug use and treatment of tumor early advantage, the laboratory with forty kinds of small molecule drugs in clinical application and has inhibitory effects on prostate cancer cells, and clofoctol (Clofoctol) respectively connected to use PC3 cells, according to the Chou-Talalay calculation formula and experiment dose response were selected that clofoctol (Clofoctol) and Sola Fini (Sorafenib) could inhibit the proliferation of PC3 cells. Sola Fini (Sorafenib) is a multi target multi kinase inhibitor, approved by the US FDA, which is used for the treatment of advanced renal cell carcinoma and liver cancer. Currently, a large number of studies have proved that Sola Fini (Sorafenib) has an inhibitory effect on the proliferation of prostate cancer. In this study, we studied the synergistic mechanism of chloramphenol (Clofoctol) and Sola Fini (Sorafenib) in the PC3 cell model of prostate cancer. Through cell apoptosis, cell cycle, Western blot and qRT-PCR technology, we identified 10 M clofoctol (Clofoctol) and 6 M (Sorafenib) combined with sorafenib treatment can make 78.84% of the cells were arrested in G1 phase, and the cell apoptosis rate of cooperation reached 16.65%, apoptosis was higher than that of Clofoctol or Sorafenib alone. Rate; qRT-PCR results showed that the drug combination of ER-stress after DDIT3/CHOP, GADD34, ATF6 pathway and ATF4 mRNA expression levels were significantly increased, the corresponding Western blot results showed that the protein level of 8 hours at DDIT3/CHOP and ATF4 were co upregulated, and in the combined treatment of 24h hours, XBP1 precursor was cut into a transcriptional activity XBP1s. That is to say, Clofoctol and Sorafenib are used to activate the IRE-1a and ATF6 paths of ER-stress. Next, we observed that after PC3 alone, RNAi cells appeared vacuolization and ubiquitin accumulation in endoplasmic reticulum after RNAi VCP/p97 silencing. In view of the fact that VCP/p97 plays an important role in endoplasmic reticulum related proteasome system, we speculate that chlorfokol (Clofoctol) may target VCP/p97 and inhibit its function. Based on another laboratory research direction, we also conducted an analysis of the association of single nucleotide polymorphisms in microRNA with susceptibility to lung cancer. Lung cancer is one of the most malignant tumors that cause the fastest growth of morbidity and mortality and the most serious threat to people's health and life. Genetic factors are one of the important factors that affect the incidence of lung cancer. More and more studies have demonstrated that microRNA plays the function of oncogene or tumor suppressor gene, and microRNA single nucleotide polymorphism is associated with tumor susceptibility. We analyzed the association between mirSNP and susceptibility to lung cancer in China by case control study. We used the miR-4293rs12220909 polymorphic loci as the object of study. The results showed that in 998 cases of lung cancer that we study the cases and 1471 cases of normal control group, the risk of miR-4293rs12220909 polymorphism and lung cancer was significantly correlated with the mutation genotype GC/CC can significantly reduce the risk of lung cancer (OR=0.687; 95%CI= 0.564-0.837), C allele is a protective factor for lung cancer susceptibility (OR=0.734; 95%CI=0.616-0.874); hierarchical analysis of genetic model and found that the GC genotype can reduce the risk of lung cancer in more than 62 years old, 62 years old, male, smoking and non smoking, lung adenocarcinoma and squamous cell carcinoma of the lung in the crowd. Next, we carried out research on the mechanism of the miR-4293 rs12220909 polymorphism to reduce the risk of lung cancer, found that wild type miR-4293 can regulate the tumor suppressor gene PRKAA1 and ADAMTSL3 to increase the susceptibility to lung cancer, resulting in lung cancer occurrence and development play a role in promoting.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.25

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