本文選題：核糖核酸還原酶 + HPVE7　； 參考：《華中科技大學(xué)》2014年博士論文

【摘要】：宮頸癌是導(dǎo)致全世界女性死亡的第二位惡性腫瘤,流行病學(xué)研究表明持續(xù)感染高危型HPV(human papillomaviruses)16/18是導(dǎo)致宮頸癌發(fā)生的主要原因。高危型HPV主要依賴病毒基因組編碼的兩個(gè)病毒癌基因E6/E7來誘導(dǎo)感染細(xì)胞惡性轉(zhuǎn)化,而高危型HPVE7又是導(dǎo)致宮頸癌發(fā)生的主要癌蛋白。E7蛋白的致癌活性主要是依賴與視網(wǎng)膜母細(xì)胞瘤蛋白(pRb)的結(jié)合,使其泛素化降解,從而釋放具有活性的E2F,激活E2F的靶基因表達(dá),從而促進(jìn)宮頸癌的發(fā)生發(fā)展。然而,在宮頸癌中E7/E2F下游與宮頸癌發(fā)生發(fā)展相關(guān)的精確信號(hào)轉(zhuǎn)導(dǎo)通路尚不清楚。因此該論文旨在探索HPVE7/E2F的下游靶通路,為宮頸癌的診斷與治療提供分子基礎(chǔ)。 通過分析四個(gè)公開報(bào)道的宮頸癌相關(guān)基因表達(dá)芯片數(shù)據(jù),選取在四個(gè)數(shù)據(jù)庫中均表達(dá)上調(diào)2倍以上的基因。然后運(yùn)用TFSEARCH (ver.1.3)軟件預(yù)測上述哪些基因的啟動(dòng)子序列上含有E2F的結(jié)合位點(diǎn)。我們發(fā)現(xiàn)核糖核苷酸還原酶亞基M2基因RRM2在四個(gè)數(shù)據(jù)庫中均表達(dá)上調(diào)2倍以上,RRM2的啟動(dòng)子區(qū)域包含兩個(gè)E2F可能的結(jié)合位點(diǎn)。這些提示RRM2可能是HPVE7調(diào)控的靶基因。 RRM2蛋白是核苷酸還原酶的重要組成成分,是DNA合成過程中的限速酶。核苷酸還原酶的活性與癌細(xì)胞的分裂分化密切相關(guān)。許多研究表明RRM2與腫瘤的惡性生物學(xué)行為緊密相關(guān),如過表達(dá)RRM2與腫瘤細(xì)胞的侵襲、轉(zhuǎn)移和血管生成有關(guān)。RRM2能夠激活一系列原癌基因促進(jìn)腫瘤的生長,包括NF-κB、c-myc、v-fes和鳥氨酸脫羧酶等,但是RRM2調(diào)控這些原癌基因的機(jī)制尚不清楚。RRM2過表達(dá)可以促進(jìn)活性氧自由基的產(chǎn)生,活性氧能夠激活腫瘤生長過程中所需的MAPK、NF-κB和AP1的活性,在腫瘤的生長、轉(zhuǎn)移和血管生成中起著重要作用。然而,目前還不清楚ROS是否參與到RRM2所介導(dǎo)的惡性腫瘤發(fā)生過程。本研究通過生物信息學(xué)的預(yù)測,發(fā)現(xiàn)在RRM2啟動(dòng)子區(qū)有兩個(gè)保守的E2F結(jié)合位點(diǎn),熒光素酶活性試驗(yàn)證明了RRM2是HPVE7下游的靶基因。HPVE7是通過結(jié)合Rb蛋白,使釋放的E2F轉(zhuǎn)錄因子結(jié)合到RRM2的啟動(dòng)子區(qū)調(diào)節(jié)其轉(zhuǎn)錄活性。對(duì)宮頸癌組織的免疫組化分析表明RRM2的蛋白表達(dá)水平和E7的表達(dá)呈正相關(guān)。這些結(jié)果證明了RRM2可能是HPVE7的下游靶基因。 其次本研究首次闡明了HPVE7通過RRM2調(diào)控HIF-la和VEGF表達(dá)的分子機(jī)制。有研究報(bào)道,HPVE7能上調(diào)HIF-la和VEGF的表達(dá),而具體分子機(jī)制不明。另有研究報(bào)道,RRM2可以通過上調(diào)VEGF的表達(dá),促進(jìn)腫瘤細(xì)胞的血管生成,但其具體的調(diào)控機(jī)制尚不清楚。于是我們假設(shè)RRM2作為HPVE7下游的靶基因,可能是通過促進(jìn)VEGF的表達(dá)來影響宮頸癌的血管生成,從而在宮頸癌的發(fā)展中發(fā)揮作用。本文研究發(fā)現(xiàn)在宮頸癌細(xì)胞系中過表達(dá)RRM2,可以促進(jìn)活性氧(reactive oxygen species,ROS)的生成,ROS能夠激活ERKl/2信號(hào)通路使HIF-1α和VEGF的表達(dá)增加。當(dāng)加入活性氧清除劑NAC后,由過表達(dá)RRM2所引起的ERK1/2信號(hào)通路激活以及HIF-1α和VEGF的表達(dá)上調(diào)被抑制。另外體外血管形成實(shí)驗(yàn)表明,由E7調(diào)控的RRM2過表達(dá)能夠促進(jìn)HUVECs的血管形成,并且RRM2促進(jìn)血管的形成是VEGF依賴的。在異種移植小鼠模型中,過表達(dá)RRM2的C33A細(xì)胞注射小鼠后,促進(jìn)了腫瘤的生長,同時(shí)也增加了血管的形成密度。所以,本項(xiàng)目研究證實(shí)RRM2是受HPVE7調(diào)控的下游靶基因,HPVE7通過上調(diào)RRM2,產(chǎn)生ROS激活ERK1/2信號(hào)通路,促進(jìn)HIF-1α和VEGF的表達(dá),進(jìn)而促進(jìn)腫瘤的血管新生和最終導(dǎo)致癌癥發(fā)生。這些結(jié)果證明了一個(gè)新的HPV相關(guān)的宮頸癌發(fā)生的信號(hào)通路即HPVE7-RRM2-ROS-ERK-HIF-1α-VEGF信號(hào)通路。因此,抑制RRM2的活性可能成為治療人類宮頸癌的一個(gè)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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.33

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相關(guān)期刊論文 前1條

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