HIF-2α在低氧微环境下对结肠癌c-Myc癌基因调控研究

发布时间：2018-03-17 09:06

本文选题：HIF-2α　切入点：低氧　出处：《浙江大学》2015年硕士论文　论文类型：学位论文

【摘要】：全球结肠癌的发病率占所有恶性肿瘤第三位,病死率居第四位。由于结直肠癌的早期缺乏特异性症状,一旦确诊大部分已属中、晚期。结直肠癌的治疗以手术为主,配合化疗、放疗和靶向分子治疗等综合治疗手段。寻找生物学靶标用于癌变监测和干预是结直肠癌防治研究的一个重要方向。结肠癌的发生发展与癌基因的突变、抑癌基因的失活密切相关。c-Myc作为重要原癌基因在多种恶性肿瘤中都呈高表达。c-Myc基因对细胞具有双重作用,可刺激细胞增殖,也可促进细胞凋亡。因此,研究c-Myc在肿瘤中的作用及相关调控机制对结肠癌的诊治有重要价值。结肠肿瘤组织生长中心区会不同程度出现缺氧和坏死。低氧环境对肿瘤组织的生长具有重要影响。低氧微环境参与肿瘤细胞增殖、凋亡、侵袭转移及血管生成等调控。低氧诱导因子HIF广泛表达于哺乳动物的各种组织细胞中,以促进机体对低氧的适应过程,是细胞在基因转录水平协调缺氧变化的最主要调节因子。本研究将通过系统研究低氧微环境下结肠癌细胞株中HIF-1α/HIF-2α与c-Myc的表达情况；同时利用siRNA干扰技术,了解HIF-1α/HIF-2α对c-Myc的调控作用；利用蛋白酶体抑制剂MG132预处理后结肠癌细胞低氧培养c-Myc蛋白的表达,以了解泛素化是否参与了c-Myc蛋白的降解过程。研究方法： 1、不同低氧处理时间点,提取结肠癌细胞HCT116和SW480的总蛋白,Western blot检测HIF-1α、HIF-2α、c-Myc及p-c-Myc的表达变化。 2、不同低氧处理时间点,提取结肠癌细胞HCT116和SW480的总RNA,qPCR检测c-Myc mRNA表达的变化。低氧处理24h后,双荧光素酶报告基因检测c-Myc启动子转录活性的变化。 3、采用siRNA靶向干扰HIF-1α或HIF-2α的表达,低氧培养24h后,Western blot检测c-Myc蛋白表达的变化。 4、采用MG132处理结肠癌细胞HCT116和SW480,分别常氧和低氧下培养,Western blot检测c-Myc蛋白表达的变化。研究结果： 1、低氧微环境下结肠癌细胞株中HIF-1α/HIF-2α与c-Myc蛋白表达的变化。随低氧时间延长,HIF-1α在4h左右达到高峰,继续延长低氧时间表达水平逐渐下降；而HIF-2α蛋白在24h表达保持持续升高。c-Myc和p-c-Myc蛋白水平随低氧时间延长逐步减弱。 2、低氧微环境下结肠癌细胞株中c-Myc mRNA及转录活性的变化。随着低氧处理时间的延长,结肠癌细胞株HCT116和SW480中的c-Myc mRNA含量逐渐降低。低氧24h后c-Myc的转录活性明显下降。 3、低氧诱导因子对c-Myc表达的调节。结肠癌细胞株HCT116、SW480中,干扰HIF-1α或HIF-2a并低氧处理24h后,c-Myc的表达增加,且干扰HIF-2a后,c-Myc表达的增加更为明显。 4、 MG132对c-Myc蛋白表达的影响。结肠癌细胞株HCT116、SW480中加入MG132预处理后c-Myc蛋白的表达明显增加,且呈浓度依赖性。研究结论： 1、慢性低氧微环境下,结肠癌细胞中HIF-2α为优势表达的低氧诱导因子,伴随着c-Myc启动子转录活性、mRNA及蛋白质表达的下降。 2、慢性低氧微环境下,结肠癌细胞中c-Myc的表达受低氧诱导因子和泛素化的影响,其中HIF-2α对c-Myc的调控作用比HIF-1α更为明显。
[Abstract]:The global incidence of colorectal cancer accounted for third of all malignancies, the mortality rate ranks fourth. Due to the lack of early colorectal cancer specific symptoms, once the diagnosis is most advanced. In the treatment of colorectal cancer with surgery, chemotherapy, radiotherapy and targeted molecular therapy and other comprehensive treatment. Looking for biological targets for cancer surveillance and intervention is an important direction of research colorectal cancer prevention.
The occurrence and development of cancer gene mutation and colon cancer, activity is closely related to.C-Myc as an oncogene in a variety of malignant tumors with high expression of.C-Myc gene has a dual effect on cell loss of tumor suppressor that can stimulate cell proliferation, also promote cell apoptosis. Therefore, diagnosis and treatment of colon cancer and related regulation. The c-Myc in tumor mechanism are of great value.
Colon tumor growth center will be different degrees of hypoxia and necrosis. Has an important effect on the growth of tumor tissue hypoxia. Hypoxia is involved in tumor cell proliferation, apoptosis, invasion and angiogenesis. The regulation is widely expressed in mammalian animal factor HIF induced by hypoxia in various tissues and cells, to promote the body to hypoxia the adaptation process is the most cells in the major regulator of gene transcription. The expression of coordination of hypoxia in this study through the systematic study of hypoxia in human colon cell lines HIF-1 alpha /HIF-2 alpha and c-Myc; at the same time using siRNA interference technology, understand the regulatory role of HIF-1 alpha /HIF-2 alpha on c-Myc expression by pretreatment; proteasome inhibitor MG132 after hypoxia in cultured c-Myc colon cancer cell junction proteins, in order to understand the ubiquitination is involved in the degradation process of c-Myc.
Research methods:
1, the total protein of HCT116 and SW480 in colon cancer cells was extracted by different hypoxia treatment time points, and Western blot was used to detect the changes in the expression of HIF-1 alpha, HIF-2 alpha, c-Myc and p-c-Myc.
2, at different hypoxic treatment time points, the total RNA of HCT116 and SW480 from colon cancer cells was extracted, and qPCR was used to detect the change of c-Myc mRNA expression. After hypoxia treatment 24h, dual luciferase reporter gene was used to detect c-Myc promoter transcriptional activity.
3, the expression of HIF-1 alpha or HIF-2 alpha was interfered with siRNA, and 24h was cultured in hypoxia, and the expression of c-Myc protein was detected by Western blot.
4, HCT116 and SW480 of colon cancer cells were treated with MG132, and the expression of c-Myc protein was detected by Western blot.
The results of the study:
1, changes in the colorectal cancer cell line HIF-1 alpha /HIF-2 alpha and c-Myc protein expression in node hypoxia. With prolonged hypoxia, alpha HIF-1 reached a peak at about 4h, continue to extend the time of hypoxia and the expression level decreased; HIF-2 alpha protein in 24h expression increasing.C-Myc and protein levels of p-c-Myc with prolonged hypoxia gradually weakened.
2, the changes of c-Myc mRNA and transcriptional activity in colon cancer cell lines under hypoxic microenvironment. With the prolongation of hypoxia treatment time, the content of c-Myc mRNA in colon cancer cell lines HCT116 and SW480 decreased gradually. C-Myc activity decreased significantly after hypoxia 24h.
3, hypoxia inducible factor regulates the expression of c-Myc. In colon cancer cell line HCT116 and SW480, the expression of c-Myc increases after interfering with HIF-1 alpha or HIF-2a and 24h after hypoxia treatment, and the increase of c-Myc expression is more obvious after interfering HIF-2a.
4, the effect of MG132 on the expression of c-Myc protein. The expression of c-Myc protein in the colon cancer cell line HCT116, MG132 pretreated with MG132 was significantly increased, and was in a concentration dependent manner.
The conclusions are as follows:
1, in chronic hypoxic microenvironment, HIF-2 alpha is a dominant hypoxia inducible factor, which is associated with the transcriptional activity of c-Myc promoter, and the decrease of mRNA and protein expression.
2, in chronic hypoxia microenvironment, the expression of c-Myc in colon cancer cells is influenced by hypoxia inducible factor and ubiquitination. HIF-2 alpha plays a more significant role in regulating c-Myc than HIF-1 alpha.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.35

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