HIF-2在HPPCn促进肾癌细胞生长迁移中所起作用的研究

发布时间：2018-05-08 15:45

本文选题：肾脏透明细胞癌 + HPPCn　；参考：《济南大学》2014年硕士论文

【摘要】：目的意义： HPPCn(Hepatopoietin Cn)是在我们实验室一种新发现的再生肝脏的细胞生长因子，哺乳动物为其主要来源，在多种组织中广泛表达。作为一类多功能的核蛋白，它可以表达于肿瘤组织中，同时也表达于自我具有更新能力的细胞中和正常组织中，同时在很多方面发挥了重要的作用，其中包括在蛋白质的降解、细胞骨架动力学、细胞之间的信号转导、和细胞的形态变化方面和其他许多生物过程。在CRCC细胞中HIF-1α和HIF-2α的转录靶基因并不完全相同，并清楚定义了HIF-1α和HIF-2α的特异靶基因。多种癌基因如CyclinD1、TGF-α、VEGF等是HIF-2α的特异靶基因，而促凋亡基因BNIP3是HIF-1α的特异靶基因。而且HIF-1α和HIF-2α与不同的信号转导通路交联。激活的HIF-1α能够降低c-Myc依赖的细胞增殖，而HIF-2α则与之相反。除此以外，HIF-1α比HIF-2α对VHL缺失CRCC细胞中蛋白酶体的降解更敏感。CRCC的多血管特征常与HIF-2α上调密切相关，而CRCC中高表达HIF-1α患者的生存期要长于其他类型患者。因此，HIF，特别是HIF-2α可能在CRCC发展中发挥了更为重要的作用近年来的研究发现HPPCn的表达和活性与肿瘤的生长与预后密切相关。HPPCn能够抑制多种化疗药物（Sorafinib，ADM，CDD，TSA）诱导的肝癌细胞凋亡，并经过调控mRNA的表达促进肿瘤细胞上皮-间质转化（EMT）和血管内皮细胞迁移，促进肿瘤的转移和浸润。转基因小鼠的肝癌诱发实验显示HPPCn能够在诱导早期增加原癌基因c-myc的表达，并显著增加肿瘤的肺转移率。肝癌细胞尾静脉注射实验显示HPPCn高表达能够增加肝癌细胞的肺转移和肿瘤中血管的数量。同时，初步的研究还发现HPPCn在肾脏透明细胞癌中的表达明显高于癌旁组织和正常肾脏组织。并且发现HPPCn可促进HIF-2的表达。那么，肾脏透明细胞癌的发生、发展过程中与HPPCn之间会有着什么样的关系呢？HIF-2又在这之中是否有作用？围绕上述问题，本实验目的是研究HPPCn调控HIF-2a促进肾癌细胞生长转移的作用和机制。为此做了如下的研究工作。方法: 构建干涉HIF-2的慢病毒载体，应用Western blot和Q-PCR检测HIF-2的干涉效率。再通过干涉肿瘤细胞的HIF-2表达，应用MTS，划痕实验、Transwell实验验证HIF-2在HPPCn调控肾癌细胞发生发展中的作用。结果: 我们通过慢病毒载体构建技术构建了PLKO.1-shHIF-2，并将其包装成慢病毒，它可以有效的干涉细胞内的HIF-2的表达，干涉效率在蛋白质水平和mRNA水平上均大于80%，病毒感染细胞后经过克隆扩大培养，获得了稳定干涉HIF-2的786-0细胞，慢病毒的获取和稳定细胞的筛选为下一步阐述HPPCn在肾脏透明细胞癌中的作用奠定了基础。随后，我们验证了HPPCn蛋白通过对肾脏透明细胞癌中HIF-2的调控进而影响肾透明细胞癌细胞生物特性。我们用稳定干涉HIF-2的786-0细胞来进行实验，证明干涉786-0细胞内HIF-2表达后，HPPCn蛋白对细胞的增殖能力，迁移能力，细胞的凋亡率都消失。基于以上的研究，，我们得出结论HPPCn在肾脏透明细胞癌的生物特性方面有重要的作用，HPPCn是通过对HIF-2的调节来促进肿瘤细胞的增殖，迁移、活化和转移等生物过程，该研究将为肾脏透明细胞癌的分子靶向治疗提供新的理论支持。
[Abstract]:Objective significance:
HPPCn (Hepatopoietin Cn) is a newly discovered cell growth factor of the regenerated liver in our laboratory, which is widely expressed in many tissues as its main source. As a class of multifunctional nucleoprotein, it can be expressed in tumor tissues, and also in cells and normal tissues that have the ability to renew themselves. At the same time, it plays an important role in many aspects, including protein degradation, cytoskeleton dynamics, signal transduction between cells, and cell morphological changes and many other biological processes.
The transcriptional target genes of HIF-1 alpha and HIF-2 a are not exactly the same in CRCC cells, and the specific target genes of HIF-1 A and HIF-2 alpha are clearly defined. A variety of oncogenes, such as CyclinD1, TGF- a, VEGF and so on, are the specific target genes of HIF-2 alpha, while BNIP3 is the specific target gene of HIF-1 alpha, and HIF-1 A and alpha are different signal transduction pathways. Cross linking. Activated HIF-1 alpha can reduce the proliferation of c-Myc dependent cells, while HIF-2 alpha is the opposite. In addition to this, HIF-1 alpha is more sensitive than HIF-2 a to the degradation of proteasome in VHL missing CRCC cells. The multiple vascular features of.CRCC are closely related to the up regulation of HIF-2 alpha, while the survival period of the high expression HIF-1 alpha patient in CRCC is longer than that of other types of patients. Therefore, HIF, especially HIF-2 alpha, may play a more important role in the development of CRCC.
Recent studies have found that the expression and activity of HPPCn are closely related to the growth and prognosis of tumors..HPPCn can inhibit the apoptosis of hepatoma cells induced by various chemotherapeutic drugs (Sorafinib, ADM, CDD, TSA), and promote the metastasis and immersion of tumor cells by regulating the expression of mRNA and promoting the epithelial mesenchymal transformation (EMT) and vascular endothelial cell migration. The induction of liver cancer in transgenic mice showed that HPPCn could increase the expression of proto oncogene c-myc in the early induction and significantly increase the lung metastasis rate of the tumor. The tail vein injection experiment of hepatoma cells showed that the high expression of HPPCn could increase the lung metastasis of hepatoma cells and the number of blood vessels in the tumor. At the same time, the preliminary study also found that HPPCn was in the tumor. The expression of the renal clear cell carcinoma is significantly higher than that of the paracancerous and normal renal tissues. And it is found that HPPCn can promote the expression of HIF-2. Then, what is the relationship between the occurrence of clear cell carcinoma of the kidney and the development of the kidney with HPPCn? Is there any effect of HIF-2 in this process?
The aim of this study is to study the role and mechanism of HPPCn regulating HIF-2a in promoting the growth and metastasis of renal cell carcinoma.
Method:
The interference efficiency of HIF-2 was detected by Western blot and Q-PCR. By interfering with the expression of HIF-2 in tumor cells, MTS, scratch test and Transwell experiment were used to verify the role of HIF-2 in the regulation of the development of renal cell carcinoma cells by HPPCn blot and Q-PCR.
Result:
We constructed PLKO.1-shHIF-2 through the construction of lentivirus vector and packaged it into a lentivirus, which could effectively interfere with the expression of HIF-2 in the cell. The interference efficiency was more than 80% at the protein level and the level of mRNA. After the virus infected cells, the cells were cloned and expanded, and the 786-0 cells, which were stable interfering with HIF-2, were obtained. Obtaining and stabilizing cell screening laid the foundation for the next step to explain the role of HPPCn in renal clear cell carcinoma.
Then we verify that HPPCn protein affects the biological characteristics of renal clear cell carcinoma cells through the regulation of HIF-2 in the renal clear cell carcinoma. We use 786-0 cells that interfere with HIF-2 to carry out the experiment. It is proved that after the expression of HIF-2 in the 786-0 cells, the proliferation ability, migration ability and apoptosis rate of HPPCn protein are eliminated. Lost.
Based on the above study, we conclude that HPPCn plays an important role in the biological characteristics of renal clear cell carcinoma. HPPCn is a biological process that promotes the proliferation, migration, activation and metastasis of tumor cells through the regulation of HIF-2, which will provide new theoretical support for the molecular targeting therapy of renal cell carcinoma.

【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.11

【参考文献】