基于抑癌基因启动子的环境遗传毒性筛选系统的建立及其在抗肿瘤药物筛选中的应用

发布时间：2018-04-29 11:54

本文选题：环境因素 + 遗传因素　；参考：《昆明理工大学》2017年博士论文

【摘要】：癌症是人类复杂疾病中最难攻克的一种,目前我国的癌症发病率持续上升,恶性肿瘤已经成为致死率最高的疾病。癌症的发生和发展是遗传因素和环境因素共同作用的结果,环境因素包括多种化学致癌物、物理辐射、微生物类如病毒感染等,遗传因素则包括原癌基因的激活及抑癌基因的突变或表达被抑制导致其功能丢失或降低。在攻克癌症的道路上,各国科学家致力于抑癌基因的研究和探索,通过充分了解其作用机制进而尝试对其进行多方面的调控,从而完成对肿瘤的抑制。在众多抑癌基因中,p53作为最重要的抑癌基因之一,在肿瘤抑制中发挥重要作用。p53通过周期阻滞完成对DNA损伤的修复,在修复无法完成时诱导细胞凋亡和衰老,进而抑制肿瘤的发生和发展。在多种恶性肿瘤中p53水平很低,其表达水平受到多种因素的调控,其中转录水平的调控是基因表达的关键环节。基因转录水平的升高离不开上游转录因子对其启动子的转录激活,因此我们利用p53基因启动子特有的DNA损伤感应能力,克隆了p53基因核心启动子在内的起始密码子上游约750bp的序列,并构建了以绿色荧光蛋白(GFP)作为报告基因的遗传毒性筛选系统。该系统能对环境中的多种化合物进行高通量筛选,快速鉴别出具有遗传毒性的化合物。除了遗传毒性筛选之外,有研究证明在p53基因未突变的肿瘤细胞中再激活p53可以有效抑制肿瘤的发展甚至诱导肿瘤消退,因此我们预期可以通过该筛选系统对多种天然提取化合物进行筛选,以期筛选到以p53基因启动子作为靶点的低毒性抗肿瘤药物。除了 p53这一重要的抑癌基因外,研究发现在多种恶性肿瘤中存在抑癌基因Ink4a/ARF的变异,包括基因的纯合性缺失、杂合性缺失、突变以及启动子甲基化等。p16和p19蛋白功能分别属于Rb和p53信号途径,在监控细胞周期,抑制肿瘤发生等过程中起到了重要作用。有研究证明p16Ink4a基因的表达异常和恶性肿瘤的发生和发展有关,例如在黑色素瘤中经常发现p16Ink4a基因启动子甲基化导致的基因沉默。而ARF基因被证明能被多种原癌基因激活信号所激活,通过p53介导的肿瘤抑制对抗癌症的发生和发展。因此我们同样构建了以GFP作为报告基因,分别以p16Ink4a和p19ARF基因的启动子区域作为靶点的抗肿瘤药物筛选系统,以期能够筛选到能够有效激活Ink4a/ARF)基因启动子的低毒性抗肿瘤天然化合物。在本研究中,我们选择的第四代启动子报告载体pGL4.82是在前几代的基础上进行了改良,减少了过多的转录因子结合位点并提高了其在细胞内表达的稳定性。为了提高检测效率,我们将载体上的荧光素酶报告基因替换为GFP,这样我们可以通过荧光显微镜对报告基因进行快速观察,构建得到的载体命名为pGL4-GFP。随后我们将上面三种重要的抑癌基因启动子构建入载体,并稳定转染到遗传背景清晰的细胞系里,通过抗性筛选得到基于三种抑癌基因启动子的稳定筛选细胞系 p53-GFP,p16-GFP 和 p19-GFP。筛选系统建立后,我们首先对p53-GFP细胞系功能进行验证:对p53-GFP筛选系统我们选择了阿霉素、环磷酰胺、顺铂、苯并芘等DNA毒性剂进行测试。在阿霉素处理6h后就可以在荧光显微镜下观察到绿色荧光蛋白GFP的表达,且随着药物处理时间的增加,荧光强度逐步升高,在24h时达到高峰,而阴性对照则没有观察GFP表达。其他几种DNA毒性剂也观察到了相同的结果,蛋白免疫印迹确证了 GFP蛋白的表达。这一实验证明了该系统可以有效响应DNA损伤,因此我们可以将其应用于环境遗传毒性化合物的监测。同时,临床上使用的大量癌症化疗药物都属于DNA损伤剂,这些药物对于活跃增殖的正常细胞,如骨髓造血细胞、小肠上皮细胞等具有较大的杀伤,从而普遍存在药物毒副作用较强的问题。我们构建的这一高通量筛选系统是基于p53启动子的转录激活,因此我们预期能够筛选到能有效激活抑癌基因表达同时细胞毒性较弱的天然化合物。接下来,我们应用该系统筛选了多种天然化合物包括昆明植物研究所提供的升麻类提取物及黄杨类生物碱。结果表明,多种升麻类提取物均不能有效激活p53启动子,对野生型MEF细胞的毒性也较小。而黄杨生物碱化合物中有部分化合物如 KBA01、KBA02、KBA03、KBA07、KBR11 等能有效激活 GFP 表达,为了寻找到细胞毒性较低,又能在细胞内再激活p53的天然化合物,我们进一步将这些筛选出的化合物对野生型MEF细胞进行了处理,均有效升高了 p53水平。随后我们对这些化合物处理后的细胞形态进行拍摄并通过流式细胞仪对细胞凋亡水平和周期分布进行检测,发现其中多数化合物均对细胞增殖产生了抑制,且凋亡细胞明显增多,例如KBA03诱导了细胞G1期的阻滞,KBR18处理后凋亡细胞明显增多。大多数筛选出的黄杨生物碱均带有不同程度的基因毒性,但我们发现其中的KBA07能有效激活p53基因启动子,且不具有明显的遗传毒性,其处理后的野生型MEF细胞在周期和凋亡水平与对照均没有显著差异。我们认为该化合物可能是通过p53基因上游的某些转录因子的激活或相互作用激活了p5 基因启动子,但本身并不具有强遗传毒性,对KBA07需要进一步研究其作用机制。在实验室之前的普洱茶抗肿瘤机制的研究中,我们发现普洱茶能显著提高p16的mRNA水平,用普洱茶对p16-GFP细胞进行处理后,能观察到报告基因GFP的表达升高,证明普洱茶是通过作用于p16Ink4p基因启动子激活了 p16的表达,但并不存在剂量效应。在多种恶性肿瘤中均发现p16的失活,因此能激活p16启动子的化合物就具备一定的抗肿瘤潜能。我们用该系统筛出了如升麻提取物KY01、KY40等能有效激活p16的化合物,对它们的作用机制还需要进一步研究。在验证p19-GFP筛选系统时,我们对该筛选系统转染了 pBabe-RasG12V外源表达载体,过表达的RasG12V能有效激活p1p基因启动子,在转染后24h就观察到明显的GFP表达。之前有研究证明p19基因启动子对原癌基因激活信号敏感且存在特异性,这一特质也是该基因启动子的宝贵特点,区别于p53会被多种损伤信号激活从而有可能造成过度应激。通过对多种化合物进行筛选也发现鲜有能激活p19启动子的化合物,但我们欣喜的发现KBA07能有效激活p19启动子,从而我们觉得KBA07具有较高的研究价值。为了进一步研究KBA07的作用机制,我们用KBA07处理了野生型MEF细胞和多种遗传背景清晰的肿瘤细胞系,发现p53和p1p基因的转录水平明显升高,但升高的p53并没有激活其下游分子p21,证明其并不具备强遗传毒性,其对p53的激活主要是通过升高其转录水平。在p53为野生型的Hela细胞进行KBA07处理后,观察到p53升高,对细胞增殖产生了抑制。当KBA07与阿霉素和顺铂联合使用时对细胞增殖的抑制更为明显,提示我们KBA07可能可以作为一种化疗的辅助药物,通过提高细胞内的p53蛋白本底水平,并在DNA毒性剂作用下,快速激发细胞阻滞、凋亡和衰老进而完成肿瘤细胞的抑制和清除。为了进一步研究KBA07对p53基因启动子的作用机制,我们通过数据库预测和查询找到了 10个位于p53启动子的转录因子结合位点,通过人工合成这些转录因子结合序列并转染到p53-GFP细胞中,从而和启动子序列产生竞争抑制。研究发现在转入了NF-κB与HIF-1这两种转录因子结合序列的p53-GFP细胞中,KBA07对报告基因的转录激活受到了明显抑制,证明KBA07的作用机制可能与这两个转录因子有关。通过蛋白免疫印迹证明KBA07能有效激活HIF-1,进一步的研究还在进行中。综上所述,我们构建了分别基于三种重要的抑癌基因p53、Ink4a、ARF启动子的以绿色荧光蛋白GFP作为报告基因的稳定筛选细胞系p53-GFP,p16-GFP和p19-GFP,并对这三个筛选系统进行了验证实验,证明了它们能够快速、特异性的对作用于这三种抑癌基因的启动子的天然化合物小分子产生响应。该系统能对环境中的遗传毒性物质进行快速筛选并能对多种化合物或小分子进行筛选,快速找到靶向性明确的具有抗肿瘤潜力的化合物,对于抗肿瘤药物的开发具有重要意义。同时我们筛选出了一些有抗肿瘤潜力的化合物例如黄杨生物碱KBA07,并将其进一步处理多种遗传背景清晰的肿瘤细胞,对其抗肿瘤活性进行研究,证明了其抗肿瘤潜力。同时通过针对p53基因启动子的转录因子结合序列竞争实验,对KBA07的分子机制进行了初探。
[Abstract]:Cancer is the most difficult to attack in human complex diseases. At present, the incidence of cancer in our country is rising, and the malignant tumor has become the most fatal disease. The occurrence and development of cancer are the result of the combination of genetic and environmental factors, and the environmental factors include a variety of carcinogens, physical radiation, and the sense of microorganism like virus. The genetic factors include the activation of the proto oncogene, the mutation of the tumor suppressor gene or the inhibition of the expression of the gene, which leads to the loss or reduction of its function. In the road to attack cancer, the scientists of all countries are committed to the research and exploration of the tumor suppressor genes. In many tumor suppressor genes, p53 is one of the most important tumor suppressor genes and plays an important role in tumor inhibition..p53 can repair DNA damage through periodic block, induce cell apoptosis and senescence when the repair is unable to complete, and then inhibit the occurrence and development of tumor. In many malignant tumors, the level of p53 is very low. The level of the transcriptional level is the key link in gene expression. The elevation of the transcriptional level can not be separated from the transcriptional activation of the upstream transcription factor to its promoter. Therefore, we use the specific DNA damage induction ability of the p53 gene promoter to clone the initial density of the core promoter of the p53 gene. A sequence of about 750bp upstream, and a genotoxicity screening system with green fluorescent protein (GFP) as a reporter gene is constructed. This system can screen a variety of compounds in the environment by high throughput screening, and quickly identify the compounds with genetic toxicity. Besides the genetic toxicity screening, there is a study to prove that the p53 gene is not mutated. The reactivation of p53 in the tumor cells can effectively inhibit the development of tumor and even induce tumor regression. Therefore, we expect to screen various natural extracts through this screening system, in order to screen the low toxic antitumor drugs that target the p53 gene promoter as a target. In addition to the important tumor suppressor gene of p53, we have found that Mutations in the tumor suppressor gene Ink4a/ARF exist in a variety of malignant tumors, including homozygous deletion of genes, deletion of heterozygosity, mutation and promoter methylation of.P16 and P19 proteins, which belong to Rb and p53 signaling pathways, which play an important role in monitoring cell cycle and inhibiting the occurrence of tumor. The abnormal expression is associated with the occurrence and development of malignant tumors, such as the gene silencing caused by p16Ink4a gene promoter methylation in melanoma, and the ARF gene has been proved to be activated by a variety of proto oncogene activation signals, and p53 mediated tumor inhibition against the occurrence and development of cancer. The anti tumor drug screening system with GFP as the reporter gene, using the promoter region of the p16Ink4a and p19ARF genes as the target, is expected to be able to screen the low toxic antitumor natural compounds that can effectively activate the Ink4a/ARF gene promoter. In this study, our selected fourth generation promoter report carrier pGL4.82 is in the front of this study. In order to improve the detection efficiency, we replace the luciferase reporter gene on the carrier to GFP so that we can quickly observe the reporter gene through a fluorescence microscope and construct the obtained vector. The three important tumor suppressor promoter were built into the carrier and transfected into the cell lines with clear genetic background, and the stable screening cell line p53-GFP based on the promoter of the tumor suppressor gene was obtained by resistance screening. After the establishment of the p16-GFP and p19-GFP. screening system, we first introduced p53-GFP to p53-GFP The function of cell line was verified: the p53-GFP screening system was tested with adriamycin, cyclophosphamide, cisplatin, benzopyrene and other DNA toxic agents. After adriamycin treatment 6h, the expression of green fluorescent protein GFP could be observed under the fluorescence microscope, and the fluorescence intensity increased gradually with the increase of the time of drug treatment, and reached the time of 24h. At the peak, the negative control did not observe the expression of GFP. Several other DNA toxicities also observed the same results. The protein immunoblotting confirmed the expression of GFP protein. This experiment proved that the system could respond effectively to DNA damage, so we can apply it to the monitoring of environmental genotoxic compounds. The large number of cancer chemotherapy drugs used are DNA damage agents. These drugs have a great killing effect on the active and proliferating normal cells, such as bone marrow hematopoietic cells, small intestinal epithelial cells, and so on. Therefore, there is a widespread problem of drug toxicity. The high flux screening system constructed by us is based on the transcription activation of the p53 promoter. We expect to be able to screen the natural compounds that can effectively activate the expression of the tumor suppressor gene with weak cytotoxicity. Next, we used this system to screen a variety of natural compounds, including the cohosh extracts and boxwood alkaloids provided by the Kunming Plant Research Institute. The active p53 promoter is less toxic to the wild type MEF cells. Some compounds in the alkaloid of Euonymus japonicus, such as KBA01, KBA02, KBA03, KBA07, KBR11, can effectively activate the GFP expression. In order to find the low cytotoxicity and can reactivate the natural compounds of p53 in the cells, we further apply these screened compounds to the compounds. Wild type MEF cells were treated and p53 levels were increased effectively. Subsequently, we detected the cell morphology after these compounds and detected the cell apoptosis and the cycle distribution by flow cytometry. Most of them inhibited the proliferation of cells, and the apoptotic cells were significantly increased, such as KB. A03 induced the cell G1 phase block, and the apoptotic cells increased significantly after KBR18 treatment. Most of the selected poplar alkaloids had different degree of gene toxicity, but we found that the KBA07 could effectively activate the p53 gene promoter and did not have obvious genetic toxicity, and the wild type MEF cells after treatment were in the cycle and apoptosis level. There is no significant difference from the control. We think that the compound may activate the P5 gene promoter through the activation or interaction of some transcription factors upstream of the p53 gene, but it does not have strong genotoxicity itself. It is necessary to further study the mechanism of action for KBA07. In the study of the antitumor mechanism of Pu'er tea before the laboratory, We found that Pu'er tea can significantly increase the mRNA level of p16. After treating p16-GFP cells with Pu'er tea, the expression of the reporter gene GFP can be observed. It proves that Pu'er tea is activated by p16Ink4p gene promoter to activate the expression of p16, but does not exist in the dose effect. The deactivation of p16 in many malignant tumors is found, therefore, the inactivation of p16 is found in many malignant tumors. The compounds that can activate the p16 promoter have a certain antitumor potential. We use this system to screen compounds such as cohosh extract KY01, KY40 and so on, which can effectively activate p16. The mechanism of their action needs further study. When the p19-GFP screening system is verified, we transfect the pBabe-RasG12V exogenous expression to the screening system. The overexpressed RasG12V can effectively activate the p1p gene promoter and observe the obvious GFP expression in 24h after transfection. Previous studies have shown that the P19 gene promoter is sensitive and specific to the activation signal of the proto oncogene. This trait is also a valuable feature of the gene promoter, which is different from that p53 will be activated by a variety of damage signals. It may cause excessive stress. By screening a variety of compounds, there are few compounds that can activate the P19 promoter, but we are pleased to find that KBA07 can effectively activate the P19 promoter, so we think that KBA07 has high research value. In order to further study the mechanism of KBA07, we used KBA07 to treat the wild type MEF fine. Cell and a variety of genetic background clear tumor cell lines found that the transcriptional level of p53 and p1p genes increased significantly, but the elevated p53 did not activate its downstream molecule p21, which proved that it did not possess strong genotoxicity. The activation of p53 was mainly by increasing its transcriptional level. After p53 for the wild type Hela cells, the observation was observed. P53 increases and inhibits cell proliferation. When KBA07 is combined with adriamycin and cisplatin, the inhibition of cell proliferation is more obvious, suggesting that KBA07 may act as a adjuvant for chemotherapy by increasing the level of p53 protein background in cells and rapidly stimulating cell block, apoptosis and failure under the action of DNA toxicities. In order to further study the inhibition and removal of tumor cells, in order to further study the mechanism of KBA07's action on the p53 gene promoter, we found 10 transcription factor binding sites located in the p53 promoter by database prediction and query, and transfected into p53-GFP cells by synthesizing these transcription factors and transfected into p53-GFP cells. The subsequence produced competitive inhibition. The study found that in the p53-GFP cells transferred to the two transcription factor binding sequences of NF- kappa B and HIF-1, KBA07 inhibited the transcriptional activation of the reporter gene. It was proved that the mechanism of KBA07 may be related to the two transcription factors. By protein immunoblotting, it is proved that KBA07 can activate HIF-1 effectively. One step of the study is still in progress. To sum up, we have constructed a stable screening cell line p53-GFP, p16-GFP and p19-GFP based on three important tumor suppressor genes, p53, Ink4a and ARF promoters, using green fluorescent protein GFP as the reporter gene, and tested these three screening systems, proving that they can be fast and specific. Sexual response to small molecules of natural compounds that act on the promoter of these three tumor suppressor genes. The system can quickly screen the genotoxic substances in the environment and screen a variety of compounds or small molecules to quickly find targeted compounds with specific antitumor potential and develop antitumor drugs. It is of great significance. At the same time, we have screened some antitumor potential compounds, such as boxwood alkaloid KBA07, and further treated a variety of tumor cells with clear genetic background, and studied its anti-tumor activity and proved its anti-tumor potential. At the same time, the transcription factor binding sequence of the p53 gene promoter was competing with the needle. In this experiment, the molecular mechanism of KBA07 is discussed.

【学位授予单位】：昆明理工大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q523

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