本文選題：SIX1 + 細胞增殖�。� 參考：《華中科技大學》2014年博士論文

【摘要】：目的：惡性增殖和轉(zhuǎn)移是腫瘤細胞的最主要特征。本項目研究Sine oculis homeobox homolog1(SIX1)對于宮頸癌細胞增殖和腫瘤轉(zhuǎn)移的促進作用及其機制。 方法：real-time RT-PCR檢測基因的表達水平。蛋白印跡、酶聯(lián)免疫吸附測定(ELISA)和免疫組化檢測基因的蛋白水平。單熒光流式細胞術檢測表面分子的蛋白水平。免疫組化分析體內(nèi)淋巴管密度。采用Bioconductor edgeR、Onto-Tools以及Gene set enrichment analysis軟件包對The Cancer Genome Atlas (TCGA)數(shù)據(jù)庫來源的人宮頸癌測序數(shù)據(jù)進行生物信息學分析。流式細胞術檢測細胞在不同細胞周期中所占的比例。Cell Counting kit-8和軟瓊脂集落實驗用來分析腫瘤細胞的體外增殖。Transwell實驗分析腫瘤細胞的體外遷移和侵襲能力。粘附實驗分析細胞與細胞外基質(zhì)(ECM)的體外粘附能力。明膠酶譜檢測細胞分泌活性MMP2和MMP9的水平。流式細胞術檢測檢測腫瘤細胞的失巢凋亡。人淋巴管內(nèi)皮細胞(HLECs)的遷移和成管實驗分析SIX1對體外淋巴管生成的作用。宮頸癌原位移植模型和爪墊模型檢測腫瘤細胞的體內(nèi)淋巴結(jié)轉(zhuǎn)移能力。實驗性轉(zhuǎn)移模型檢測腫瘤細胞的對靶器官的粘附、滲出和形成轉(zhuǎn)移灶的能力�；铙w生物發(fā)光成像以及熒光成像檢測腫瘤的生長和轉(zhuǎn)移。免疫沉淀研究蛋白質(zhì)之間的相互作用。染色質(zhì)免疫沉淀檢測蛋白質(zhì)與基因啟動子的結(jié)合。結(jié)果：(一)在宮頸上皮內(nèi)瘤變和宮頸癌中,SIX1的表達由人乳頭瘤病毒的E7癌蛋白所誘導。SIX1表達的增高導致與DNA復制有關的多基因表達的上調(diào),包括微小染色體維持蛋白復合體(MCM2、MCM3、MCM6), DNA聚合酶α-引物酶復合體(POLA1、PRIM1、PRIM2), clamp loader (RFC3, RFC4, RFC5), DNA聚合酶δ復合體(POLD3)以及DNA聚合酶ε復合體(POLE2)。與此相一致,SIX1的高表達促進G1至S期細胞周期進程,并促進腫瘤細胞的增殖和腫瘤的生長。相應的,沉默SIX1可以減慢G1至S期細胞周期進程,并抑制腫瘤細胞的增殖和腫瘤的生長。重要的是,SIX1可以更有效地促進錨定非依賴的細胞生長,從而可能在腫瘤的侵襲浸潤生長和轉(zhuǎn)移灶中腫瘤細胞的生長起到顯著的促進作用。(二)組織標本免疫組化顯示,宮頸癌的淋巴管生成和淋巴結(jié)轉(zhuǎn)移與腫瘤細胞高表達SIX1密切相關。SIX1的高表達在體內(nèi)、體外都是通過增強VEGF-C的表達從而增強腫瘤細胞對淋巴管生成的促進作用。SIX1增強TGF-β誘導的SMAD2和SMAD3的激活,并且與SMAD通路協(xié)同促進VEGF-C的表達。SIX1與TGF-β協(xié)同促進腫瘤細胞表達VEGF-C的效應遠遠高于兩者單獨的作用。盡管TGF-β能促進VEGF-C表達,但是TGF-β1通過直接抑制淋巴管內(nèi)皮細胞的成管,從而抑制淋巴管生成。然而,TGF-β1與SIX1協(xié)同促進的VEGF-C表達不僅直接促進淋巴管內(nèi)皮細胞的遷移和成管,而且抵抗了TGF-β1對淋巴管內(nèi)皮細胞的抑制效應。(三)SIX1促進α5和β1亞基的表達,而不調(diào)控其他與多種腫瘤預后相關的整合素的表達。S1X1通過上調(diào)α5β1表達從而促進腫瘤細胞的體外粘附和體內(nèi)循環(huán)系統(tǒng)中細胞滯留的能力。通過上調(diào)α5β1的表達,SIX1也增強了ECM-α5β1介導的基因表達調(diào)控,包括提高活性MMP2和MMP9的分泌、上調(diào)抗凋亡基因(BCL-XL和BCL2)、下調(diào)促凋亡基因(BIM和BAX),從而增強宮頸癌細胞的轉(zhuǎn)移能力。阻斷α5β1,可消除SIX1對這些基因的表達調(diào)控,也消除了SIX1對腫瘤細胞侵襲能力的促進作用。并且,沉默a5也消除了SIX1在實驗性轉(zhuǎn)移和自發(fā)性轉(zhuǎn)移模型中對發(fā)展轉(zhuǎn)移灶的促進作用。 結(jié)論：SIX1在DNA復制中起到主調(diào)控因子的作用,促進腫瘤細胞的增殖。SIX1通過與TGF-β通路協(xié)同促進VEGF-C的表達,從而促進淋巴管生成和淋巴結(jié)轉(zhuǎn)移。同時,SIX1通過上調(diào)α5β1的表達,增強宮頸癌細胞的侵襲、轉(zhuǎn)移能力。這些發(fā)現(xiàn)說明SIX1在宮頸癌的發(fā)生、發(fā)展和侵襲轉(zhuǎn)移中起關鍵作用。這也提示S1X1可能作為評價宮頸癌增殖和轉(zhuǎn)移能力的標志分子,也可考慮作為一個潛在的抗腫瘤治療的靶點。
[Abstract]:Objective: malignant proliferation and metastasis is the most important feature of tumor cells. This project is to study the promoting effect and mechanism of Sine oculis homeobox homolog1 (SIX1) on cervical cancer cell proliferation and metastasis.
Methods: to detect the expression of real-time gene of RT-PCR. Western blot, enzyme-linked immunosorbent assay (ELISA) protein levels and immunohistochemical detection of gene. The protein level of single fluorescence detection of surface molecular flow cytometry. Immunohistochemical analysis of in vivo LymphVessels. Using Bioconductor edgeR, Onto-Tools Gene and set enrichment analysis software the The Cancer Genome Atlas package (TCGA) in human cervical cancer database sources of sequencing data were analyzed with bioinformatics. Flow cytometry cells in different cell cycle accounted for the proportion of.Cell Counting kit-8 and soft agar colony assay was used to analyze the migration and invasion of tumor cells in vitro proliferation of.Transwell tumor cells in vitro experiment the analysis of cells and extracellular matrix adhesion assay (ECM) in vitro adhesion. Gelatinase activity of MMP2 cells and MMP9 detection The level of flow cytometry to detect tumor cell anoikis. Human lymphatic endothelial cells (HLECs) migration and tube formation of SIX1 tube experiments in vitro. In vivo lymph lymph tumor cell detection in cervical cancer orthotopic transplantation model and footpad model node metastasis ability of experimental metastasis of tumor. Cell detection model of target organ adhesion, infiltration and metastasis formation ability. Bioluminescence imaging and fluorescence imaging of tumor growth and metastasis. The interaction between the immunoprecipitation of protein. Chromatin immunoprecipitation combined with detection of protein and gene promoter. Results: (a) in cervical intraepithelial neoplasia variable and cervical cancer, the expression of SIX1 by human papilloma virus E7 protein induced by the increased expression of.SIX1 and DNA resulted in the expression of multiple genes related to replication increases, including minichromosome maintenance Protein complexes (MCM2, MCM3, MCM6), DNA polymerase alpha primase complex (POLA1, PRIM1, PRIM2), clamp loader (RFC3, RFC4, RFC5), DNA polymerase complex (POLD3) and DNA polymerase epsilon complex (POLE2). Consistent with this, the high expression of SIX1 can promote G1 to S during the process of cell cycle, and promote tumor cell proliferation and tumor growth. The silencing of SIX1 can slow down the G1 to S phase of cell cycle progression, and inhibition of tumor cell proliferation and tumor growth. It is important that SIX1 can effectively promote the growth of anchorage dependent cells, which may be infiltrative growth and to a significant role in promoting tumor cells from metastatic lesion growth in tumor invasion. (two) tissue immunohistochemistry showed that high expression of SIX1 is closely related to.SIX1 formation and lymph node metastasis and tumor cells of cervical cancer lymph tube high expression in vivo and in vitro are By enhancing the expression of VEGF-C can enhance the formation of tumor cells promotes the activation of.SIX1 enhances TGF- induced SMAD2 and SMAD3 of lymph nodes, and the SMAD signaling pathway and effect of the expression of VEGF-C.SIX1 and TGF- beta synergistically promote tumor cell expression of VEGF-C is much higher than that of the two individual effects. Although TGF- can promote the expression of VEGF-C beta however, TGF- beta 1 through direct inhibition of tube formation of lymphatic endothelial cells, thus inhibiting lymphangiogenesis. However, TGF- beta 1 and SIX1 promoted the expression of VEGF-C not only directly promote lymphatic endothelial cell migration and tube formation, and resistance to TGF- beta 1 on lymphatic endothelial cells (three inhibitory effect. SIX1) alpha 5 and beta 1 promotes the expression of subunits, the expression of.S1X1 and the regulation of other related with tumor prognosis through upregulation of integrin alpha 5 beta 1 expression thereby promoting tumor cell adhesion in vitro and in vivo The ability of retaining cells in the circulatory system. Through upregulation of the expression of alpha 5 beta 1, SIX1 also enhanced the ECM- alpha gene 5 beta 1 mediated expression, including improving the activity of MMP2 and MMP9, anti apoptotic genes up-regulated (BCL-XL and BCL2), down regulated Pro apoptotic genes (BIM and BAX), metastasis in order to increase cervical cancer cells. Blocking alpha 5 beta 1, can eliminate the SIX1 expression of these gene regulation, but also eliminate the effect of SIX1 on invasion of tumor cells. Moreover, silencing of A5 also eliminates the SIX1 on the development of metastases in experimental metastasis and spontaneous metastasis model in the promotion.
Conclusion: SIX1 plays a main role in regulating factor DNA replication, promoting the proliferation of tumor cells through the expression of.SIX1 and TGF- beta VEGF-C pathway to promote synergy, thereby promoting lymphangiogenesis and lymph node metastasis. At the same time, the expression of SIX1 by up regulating the expression of alpha 5 beta 1, enhanced cervical cancer cell invasion and metastasis. These findings suggest that SIX1 in the occurrence of cervical cancer, which plays a key role in the development and invasion. It also suggested that S1X1 may serve as a marker to evaluate the proliferation of cervical cancer and metastasis, can also be considered as a potential target for anticancer therapy.

【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R737.33

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