HPVE7调控的RRM2促进宫颈癌血管形成的机制研究

发布时间：2018-07-03 07:40

本文选题：核糖核酸还原酶 + HPVE7　；参考：《华中科技大学》2014年博士论文

【摘要】：宫颈癌是导致全世界女性死亡的第二位恶性肿瘤,流行病学研究表明持续感染高危型HPV(human papillomaviruses)16/18是导致宫颈癌发生的主要原因。高危型HPV主要依赖病毒基因组编码的两个病毒癌基因E6/E7来诱导感染细胞恶性转化,而高危型HPVE7又是导致宫颈癌发生的主要癌蛋白。E7蛋白的致癌活性主要是依赖与视网膜母细胞瘤蛋白(pRb)的结合,使其泛素化降解,从而释放具有活性的E2F,激活E2F的靶基因表达,从而促进宫颈癌的发生发展。然而,在宫颈癌中E7/E2F下游与宫颈癌发生发展相关的精确信号转导通路尚不清楚。因此该论文旨在探索HPVE7/E2F的下游靶通路,为宫颈癌的诊断与治疗提供分子基础。通过分析四个公开报道的宫颈癌相关基因表达芯片数据,选取在四个数据库中均表达上调2倍以上的基因。然后运用TFSEARCH (ver.1.3)软件预测上述哪些基因的启动子序列上含有E2F的结合位点。我们发现核糖核苷酸还原酶亚基M2基因RRM2在四个数据库中均表达上调2倍以上,RRM2的启动子区域包含两个E2F可能的结合位点。这些提示RRM2可能是HPVE7调控的靶基因。 RRM2蛋白是核苷酸还原酶的重要组成成分,是DNA合成过程中的限速酶。核苷酸还原酶的活性与癌细胞的分裂分化密切相关。许多研究表明RRM2与肿瘤的恶性生物学行为紧密相关,如过表达RRM2与肿瘤细胞的侵袭、转移和血管生成有关。RRM2能够激活一系列原癌基因促进肿瘤的生长,包括NF-κB、c-myc、v-fes和鸟氨酸脱羧酶等,但是RRM2调控这些原癌基因的机制尚不清楚。RRM2过表达可以促进活性氧自由基的产生,活性氧能够激活肿瘤生长过程中所需的MAPK、NF-κB和AP1的活性,在肿瘤的生长、转移和血管生成中起着重要作用。然而,目前还不清楚ROS是否参与到RRM2所介导的恶性肿瘤发生过程。本研究通过生物信息学的预测,发现在RRM2启动子区有两个保守的E2F结合位点,荧光素酶活性试验证明了RRM2是HPVE7下游的靶基因。HPVE7是通过结合Rb蛋白,使释放的E2F转录因子结合到RRM2的启动子区调节其转录活性。对宫颈癌组织的免疫组化分析表明RRM2的蛋白表达水平和E7的表达呈正相关。这些结果证明了RRM2可能是HPVE7的下游靶基因。其次本研究首次阐明了HPVE7通过RRM2调控HIF-la和VEGF表达的分子机制。有研究报道,HPVE7能上调HIF-la和VEGF的表达,而具体分子机制不明。另有研究报道,RRM2可以通过上调VEGF的表达,促进肿瘤细胞的血管生成,但其具体的调控机制尚不清楚。于是我们假设RRM2作为HPVE7下游的靶基因,可能是通过促进VEGF的表达来影响宫颈癌的血管生成,从而在宫颈癌的发展中发挥作用。本文研究发现在宫颈癌细胞系中过表达RRM2,可以促进活性氧(reactive oxygen species,ROS)的生成,ROS能够激活ERKl/2信号通路使HIF-1α和VEGF的表达增加。当加入活性氧清除剂NAC后,由过表达RRM2所引起的ERK1/2信号通路激活以及HIF-1α和VEGF的表达上调被抑制。另外体外血管形成实验表明,由E7调控的RRM2过表达能够促进HUVECs的血管形成,并且RRM2促进血管的形成是VEGF依赖的。在异种移植小鼠模型中,过表达RRM2的C33A细胞注射小鼠后,促进了肿瘤的生长,同时也增加了血管的形成密度。所以,本项目研究证实RRM2是受HPVE7调控的下游靶基因,HPVE7通过上调RRM2,产生ROS激活ERK1/2信号通路,促进HIF-1α和VEGF的表达,进而促进肿瘤的血管新生和最终导致癌症发生。这些结果证明了一个新的HPV相关的宫颈癌发生的信号通路即HPVE7-RRM2-ROS-ERK-HIF-1α-VEGF信号通路。因此,抑制RRM2的活性可能成为治疗人类宫颈癌的一个新策略。
[Abstract]:Cervical cancer is the second malignant tumor that causes the death of women in the world. Epidemiological studies have shown that persistent infection high risk HPV (human papillomaviruses) 16/18 is the main cause of cervical cancer. High risk HPV mainly relies on the two viral cancer bases encoded by the virus genome to induce malignant transformation of infected cells, and high risk. Type HPVE7 is the main carcinogenic activity of.E7 protein, the main cancer protein that causes cervical cancer, mainly depends on the binding of the retinoblastoma protein (pRb) to make it degrade, release the active E2F, activate the target gene expression of E2F, and promote the development of cervical cancer. However, in the cervical cancer, the lower E7/E2F and the cervix of the cervix are in the cervical cancer. The precise signal transduction pathway related to carcinogenesis and development is not clear. Therefore, the aim of this paper is to explore the downstream target of HPVE7/E2F and provide a molecular basis for the diagnosis and treatment of cervical cancer.
By analyzing the data of four publicly reported cervical cancer related gene expression chips, the genes of up to 2 times up-regulated in the four databases were selected. Then TFSEARCH (ver.1.3) software was used to predict the binding sites of E2F in the promoter sequences of the above genes. We found that the ribonucleotide reductase subunit M2 gene RRM2 is at the same time. The expression of the four databases is up to 2 times up, and the promoter region of RRM2 contains two E2F binding sites. These suggest that RRM2 may be the target gene for HPVE7 regulation.
RRM2 protein is an important component of nucleotide reductase, a speed limiting enzyme in the process of DNA synthesis. The activity of nucleotides reductase is closely related to the division and differentiation of cancer cells. Many studies have shown that RRM2 is closely related to the malignant biological behavior of the tumor, such as the expression of RRM2 and the invasion of tumor cells, metastasis and angiogenesis related to.RRM2 A series of proto oncogenes are activated to promote tumor growth, including NF- kappa B, c-myc, v-fes and ornithine decarboxylase, but the mechanism of RRM2 regulation of these proto oncogenes is not clear that.RRM2 overexpression can promote the production of reactive oxygen free radicals, and active oxygen can activate the activity of MAPK, NF- kappa B and AP1 in tumor growth process, in tumor The growth, metastasis and angiogenesis play an important role. However, it is not clear whether ROS is involved in the process of malignant tumor mediated by RRM2. This study found that there were two conservative E2F binding sites in the RRM2 promoter region by bioinformatics, and the fluorescein activity test proved that RRM2 is the target gene for the downstream of HPVE7. .HPVE7 is combined with the Rb protein to modulate the transcriptional activity of the released E2F transcription factor binding to the promoter of RRM2. Immunohistochemical analysis of cervical cancer shows that the protein expression level of RRM2 is positively related to the expression of E7. These results show that RRM2 may be the downstream target gene of HPVE7.
Secondly, we first elucidate the molecular mechanism that HPVE7 regulates the expression of HIF-la and VEGF through RRM2. It is reported that HPVE7 can up regulate the expression of HIF-la and VEGF, but the specific molecular mechanism is unknown. Furthermore, RRM2 can promote the blood Guan Shengcheng of tumor cells by up regulating the expression of VEGF, but the specific regulatory mechanism is still unclear. We hypothesized that RRM2 is the target gene in the downstream of HPVE7, which may affect the angiogenesis of cervical cancer by promoting VEGF expression, and thus play a role in the development of cervical cancer. This study found that overexpression of RRM2 in cervical cancer cell lines can promote the formation of reactive oxygen species (reactive oxygen species, ROS) and ROS can activate ERKl/2. The signal pathway increased the expression of HIF-1 alpha and VEGF. When the active oxygen scavenger NAC was added, the ERK1/2 signaling pathway caused by the overexpressed RRM2 and the expression of HIF-1 alpha and VEGF were inhibited. In addition, the in vitro angiogenesis experiment showed that the RRM2 overexpression regulated by E7 could promote the angiogenesis of HUVECs and RRM2 promote the blood vessels. The formation is VEGF dependent. In the xenograft mouse model, the C33A cells that overexpressed RRM2 promoted the growth of the tumor and also increased the density of the blood vessels. Therefore, this project has confirmed that RRM2 is a downstream target gene regulated by HPVE7, and HPVE7 can activate the ERK1/2 signaling pathway through the upper modulation RRM2, and promotes HIF-1 a to promote HIF-1 a. And the expression of VEGF, which further promotes neovascularization and ultimately leads to cancer. These results demonstrate a new HPV related signaling pathway, the HPVE7-RRM2-ROS-ERK-HIF-1 alpha -VEGF signaling pathway. Therefore, inhibition of RRM2 activity may be a new strategy for the treatment of human cervical cancer.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.33

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