SIX1促进宫颈癌细胞增殖和转移

发布时间：2018-04-13 07:48

本文选题：SIX1 + 细胞增殖　；参考：《华中科技大学》2014年博士论文

【摘要】：目的：恶性增殖和转移是肿瘤细胞的最主要特征。本项目研究Sine oculis homeobox homolog1(SIX1)对于宫颈癌细胞增殖和肿瘤转移的促进作用及其机制。方法：real-time RT-PCR检测基因的表达水平。蛋白印迹、酶联免疫吸附测定(ELISA)和免疫组化检测基因的蛋白水平。单荧光流式细胞术检测表面分子的蛋白水平。免疫组化分析体内淋巴管密度。采用Bioconductor edgeR、Onto-Tools以及Gene set enrichment analysis软件包对The Cancer Genome Atlas (TCGA)数据库来源的人宫颈癌测序数据进行生物信息学分析。流式细胞术检测细胞在不同细胞周期中所占的比例。Cell Counting kit-8和软琼脂集落实验用来分析肿瘤细胞的体外增殖。Transwell实验分析肿瘤细胞的体外迁移和侵袭能力。粘附实验分析细胞与细胞外基质(ECM)的体外粘附能力。明胶酶谱检测细胞分泌活性MMP2和MMP9的水平。流式细胞术检测检测肿瘤细胞的失巢凋亡。人淋巴管内皮细胞(HLECs)的迁移和成管实验分析SIX1对体外淋巴管生成的作用。宫颈癌原位移植模型和爪垫模型检测肿瘤细胞的体内淋巴结转移能力。实验性转移模型检测肿瘤细胞的对靶器官的粘附、渗出和形成转移灶的能力。活体生物发光成像以及荧光成像检测肿瘤的生长和转移。免疫沉淀研究蛋白质之间的相互作用。染色质免疫沉淀检测蛋白质与基因启动子的结合。结果：(一)在宫颈上皮内瘤变和宫颈癌中,SIX1的表达由人乳头瘤病毒的E7癌蛋白所诱导。SIX1表达的增高导致与DNA复制有关的多基因表达的上调,包括微小染色体维持蛋白复合体(MCM2、MCM3、MCM6), DNA聚合酶α-引物酶复合体(POLA1、PRIM1、PRIM2), clamp loader (RFC3, RFC4, RFC5), DNA聚合酶δ复合体(POLD3)以及DNA聚合酶ε复合体(POLE2)。与此相一致,SIX1的高表达促进G1至S期细胞周期进程,并促进肿瘤细胞的增殖和肿瘤的生长。相应的,沉默SIX1可以减慢G1至S期细胞周期进程,并抑制肿瘤细胞的增殖和肿瘤的生长。重要的是,SIX1可以更有效地促进锚定非依赖的细胞生长,从而可能在肿瘤的侵袭浸润生长和转移灶中肿瘤细胞的生长起到显著的促进作用。(二)组织标本免疫组化显示,宫颈癌的淋巴管生成和淋巴结转移与肿瘤细胞高表达SIX1密切相关。SIX1的高表达在体内、体外都是通过增强VEGF-C的表达从而增强肿瘤细胞对淋巴管生成的促进作用。SIX1增强TGF-β诱导的SMAD2和SMAD3的激活,并且与SMAD通路协同促进VEGF-C的表达。SIX1与TGF-β协同促进肿瘤细胞表达VEGF-C的效应远远高于两者单独的作用。尽管TGF-β能促进VEGF-C表达,但是TGF-β1通过直接抑制淋巴管内皮细胞的成管,从而抑制淋巴管生成。然而,TGF-β1与SIX1协同促进的VEGF-C表达不仅直接促进淋巴管内皮细胞的迁移和成管,而且抵抗了TGF-β1对淋巴管内皮细胞的抑制效应。(三)SIX1促进α5和β1亚基的表达,而不调控其他与多种肿瘤预后相关的整合素的表达。S1X1通过上调α5β1表达从而促进肿瘤细胞的体外粘附和体内循环系统中细胞滞留的能力。通过上调α5β1的表达,SIX1也增强了ECM-α5β1介导的基因表达调控,包括提高活性MMP2和MMP9的分泌、上调抗凋亡基因(BCL-XL和BCL2)、下调促凋亡基因(BIM和BAX),从而增强宫颈癌细胞的转移能力。阻断α5β1,可消除SIX1对这些基因的表达调控,也消除了SIX1对肿瘤细胞侵袭能力的促进作用。并且,沉默a5也消除了SIX1在实验性转移和自发性转移模型中对发展转移灶的促进作用。结论：SIX1在DNA复制中起到主调控因子的作用,促进肿瘤细胞的增殖。SIX1通过与TGF-β通路协同促进VEGF-C的表达,从而促进淋巴管生成和淋巴结转移。同时,SIX1通过上调α5β1的表达,增强宫颈癌细胞的侵袭、转移能力。这些发现说明SIX1在宫颈癌的发生、发展和侵袭转移中起关键作用。这也提示S1X1可能作为评价宫颈癌增殖和转移能力的标志分子,也可考虑作为一个潜在的抗肿瘤治疗的靶点。
[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.

【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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