同源盒基因HOXB7对胃癌发生发展的作用及其调控机制的初步研究

发布时间：2018-05-02 20:42

本文选题：同源盒基因 + HOXB7　；参考：《浙江大学》2016年博士论文

【摘要】：胃癌是全世界发病率最高的恶性肿瘤之一,是中国第二常见肿瘤。全球每年胃癌新发病例数约100万,中国占40%之多,且其患病率和死亡率均是世界平均水平的2倍。近年来胃癌发病人口亦趋于年轻化,其预防和治疗均需高度重视。随着胃癌规范化诊治的不断完善和推广,以手术为主的综合治疗对改善胃癌患者预后、提高生存率起到了一定的促进作用。然而现阶段提高肿瘤治愈率最有效的方法仍然是早期诊断、早期干预,因此如何更加早期、有效地诊治胃癌仍然是胃癌防治工作的重点。已有多项研究表明,胃癌的发生发展涉及癌基因活化和抑癌基因失活等多个方面,故筛选诊断和预后生物学靶标,阐明胃癌发生发展过程中的分子机制,是胃癌转化医学研究工作的重点方向。同源盒基因(Homeobox genes)是细胞增殖和分化的主控基因,其编码的蛋白作为转录因子,在脊椎动物胚胎发育和器官形成中起到重要作用；作用靶标通常为粘附分子、膜蛋白受体、生长因子等与肿瘤进展、转移密切相关的基因家族。同时对肿瘤干细胞的相关特性如自我更新、无限增殖等恶性生物学行为具有调控作用。目前在肺癌、乳腺癌、结肠癌等多种肿瘤中均可检测到HOX基因的异常表达。其中,位于第17号染色体的HOXB7已被证实属于癌基因的范畴。已有文献表明,HOXB7可通过上调碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF),激活Ras/RhoA蛋白通路,从而促进乳腺细胞发生上皮间质转化(epithelial-mesenchymal transition, EMT),促使肿瘤的发生。但有关HOXB7基因在胃癌中的表达情况以及其是否参与调控胃癌肿瘤细胞恶性进展与转移的能力,目前尚未见报道。本研究通过检测胃癌细胞株及胃癌患者肿瘤组织中HOXB7基因的表达情况,结合临床病理及随访资料,初步探讨HOXB7表达水平的临床意义；再进一步利用RNA干扰及慢病毒载体敲低HOXB7表达,检测胃癌细胞和裸鼠荷瘤的恶性生物学行为变化；采用表达谱芯片技术筛选HOXB7基因调控的下游关键基因并进行验证,进一步阐明其对胃癌促进作用及相关信号通路的分子调控机制。研究方法：1. HOXB7在胃癌中的表达及临床资料相关性分析①QT-PCR检测96对胃癌及相应常组织中HOXB7基因mRNA表达水平；免疫组化检测分析胃癌及相应正常组织中HOXB7的表达水平；②QT-PCR和Western blot检测8种胃癌细胞株中的HOXB7的表达；③根据QT-PCR结果分组分析HOXB7表达高低与患者临床病理参数之间的关系。2. HOXB7对胃癌的生物学行为的影响①体外转染小干扰RNA,敲减HOXB7表达量,通过MTS、Edu、流式细胞周期实验、流式细胞凋亡实验、Transwell实验以及Western blot检测HOXB7对胃癌细胞周期、增殖、侵袭、迁移能力的影响；②通过体外病毒载体构建HOXB7低表达细胞株,经裸鼠皮下荷瘤模型验证干扰HOXB7对胃癌生长的影响。3. HOXB7调控靶基因及相关信号通路的研究①利用基因表达谱芯片技术筛选HOXB7调控的下游靶基因,并对其中18个差异较明显候选靶基因在稳定转染HOXB7的胃癌细胞中进行QT-PCR验证;②Western blot检测HOXB7对细胞周期增殖调控通路关键蛋白p21、PIK3R3、AKT表达的影响。研究结果：1. HOXB7在胃癌中表达明显高于正常组织,且与病理分化程度及TNM分期相关① HOXB7在胃癌组织中表达水平较相应正常组织明显上调∽0.01)；② HOXB7在胃癌细胞株中MKN45、BGC-823、MGC-803、SGC-7901均呈现显著高表达,但在AGS及HGC-27中呈低表达；③HOXB7表达高低与肿瘤病理分化程度(P=0.02)、TNM分期(P=0.02)密切相关,与患者性别、年龄、肿瘤大小、位置无关；研究随访结果尚未显示其表达高低与胃癌患者生存期具有相关性。2.体内外实验证实HOXB7促进胃癌的生长和转移①干扰HOXB7能明显抑制胃癌细胞株BGC-823和SGC-7901的增殖、发生细胞阻滞,促进细胞凋亡,并降低侵袭、迁移能力(P0.01),改变EMT特征蛋白表达。②裸鼠胃癌移植瘤实验发现敲减HOXB7能显著抑制肿瘤的生长(P＜0.01)。3. HOXB7调控多个重要下游基因,并参与PI3K/AKT信号通路调控①筛选到HOXB7的下游调控基因：SGC-7901细胞系中发现60个上调基因,158个下调基因,BGC-823细胞系中发现149个上调基因,231个下调基因(倍数2,P0.05)。②经QT-PCR验证了包括NRXN3、NFAT5、KRAS、Smad2、WNT5A等;18个差异显著的基因,变化趋势与芯片结果一致。③Western blot检测p21、CyclinD1、PIK3R3、pAKT、AKT蛋白水平,发现干扰HOXB7抑制了PI3K/AKT信号通路,可能是胃癌发生发展过程中一个重要的分子机制。研究结论：1. HOXB7在胃癌细胞系及患者肿瘤组织中异常高表达,且与肿瘤分期等病理参数相关,虽然目前随访数据未显示其表达高低与胃癌进展及病人预后相关,但提示HOXB7仍是胃癌发生发展中一个有意义的分子靶标,其与疾病预后的关系仍需进一步研究。2.HOXB7参与调控胃癌肿瘤细胞体外增殖、细胞周期、凋亡、侵袭和体内成瘤能力,影响肿瘤恶性生物学行为。3.进一步深入研究HOXB7对胃癌发生发展的作用机制,筛选到其调控的一系列下游基因,这些基因参与肿瘤细胞恶性生物学过程,可为胃癌的临床防治提供新的靶点。
[Abstract]:Gastric cancer is one of the most common malignant tumors in the world, and it is the second common tumor in China. The number of new cases of gastric cancer is about 1 million in the world, and more than 40% in China, and its morbidity and mortality are 2 times of the world average. In recent years, the incidence of gastric cancer is also becoming younger, and the prevention and treatment of gastric cancer should be highly valued. With gastric cancer, the incidence of gastric cancer should be highly valued. The continuous improvement and popularization of standardized diagnosis and treatment, surgery based comprehensive treatment to improve the prognosis of gastric cancer patients and improve the survival rate has played a certain role in promoting. However, the most effective method to improve the cure rate of cancer is still early diagnosis and early intervention, so how to treat gastric cancer more early and effectively is still the prevention of gastric cancer. Many studies have shown that the development of gastric cancer involves the activation of oncogene and the inactivation of tumor suppressor genes. Therefore, the screening of the biological targets for diagnosis and prognosis and the clarifying the molecular mechanism in the development of gastric cancer is the key direction of the research of gastric cancer transformation medicine. The homologous box gene (Homeobox genes) is fine. The main controlled gene of cell proliferation and differentiation, its encoded protein as a transcription factor, plays an important role in the development of vertebrate embryos and organ formation; the target target is usually the gene family closely related to the progression of tumor, such as adhesion molecules, membrane protein receptors, growth factors, etc.. The abnormal expression of HOX gene can be detected in many kinds of tumor, such as lung cancer, breast cancer, colon cancer and so on. Among them, HOXB7 on chromosome seventeenth has been proved to belong to the category of oncogene. It has been shown that HOXB7 can increase the growth of basic fibroblast cells. Basic fibroblast growth factor (bFGF) activates the Ras/RhoA protein pathway to promote the epithelial mesenchymal transition (epithelial-mesenchymal transition, EMT) to promote the occurrence of tumor, but the expression of the HOXB7 gene in gastric cancer and whether it participates in the regulation of malignant progression and metastasis of cancer cells of gastric cancer. In this study, the expression of HOXB7 gene in the tumor tissue of gastric cancer cell lines and gastric cancer patients was detected, and the clinical significance of HOXB7 expression was preliminarily studied by clinical pathology and follow-up data. Further, RNA interference and lentivirus vector were used to detect the expression of HOXB7 in the gastric cancer cells and nude mice. The change of malignant biological behavior; the screening of the key downstream key genes regulated by HOXB7 gene by expression spectrum chip technology and verification to further elucidate its role in promoting gastric cancer and the molecular regulation mechanism of related signal pathways. Research methods: expression of 1. HOXB7 in gastric cancer and correlation analysis of bed data (1) QT-PCR detection of 96 pairs of stomach The expression level of HOXB7 gene mRNA in cancer and the corresponding normal tissues; immunohistochemical detection and analysis of the expression level of HOXB7 in gastric cancer and corresponding normal tissues; (2) QT-PCR and Western blot to detect the expression of HOXB7 in 8 kinds of gastric cancer cell lines; (3) the relationship between the high and low expression of HOXB7 and the clinicopathological parameters of the patients was analyzed by QT-PCR results,.2.. The effect of HOXB7 on the biological behavior of gastric cancer (1) transfection of small interference RNA, knock down HOXB7 expression, MTS, Edu, flow cytometry, flow cytometry, Transwell experiment and Western blot to detect the effect of HOXB7 on the cell cycle, proliferation, invasion and migration of gastric cancer; secondly, to construct HOXB7 low by the vector of virus in vitro Expression of cell lines, the effect of interfering HOXB7 on the growth of gastric cancer by subcutaneous tumor model in nude mice, the study of.3. HOXB7 regulation target gene and related signaling pathway (1) screening the downstream target genes regulated by HOXB7 by gene expression chip technology, and 18 of the more distinct candidate target genes in the stable transfected gastric cancer cells transfected with HOXB7 The effect of Western blot on the expression of key protein p21, PIK3R3 and AKT in cell cycle proliferation pathway was detected by Western blot. Results: the expression of 1. HOXB7 in gastric cancer was significantly higher than that of normal tissue, and the expression level of HOXB7 in gastric cancer group was up to be up to the corresponding normal tissue. MKN45, BGC-823, MGC-803 and SGC-7901 were highly expressed in gastric cancer cell lines, but low expression in AGS and HGC-27; (3) the level of HOXB7 expression was closely related to the degree of pathological differentiation (P=0.02) and TNM stages (P=0.02) of the tumor, and was not related to the sex, age, tumor size and position of the patients, and the follow-up results have not been shown yet. The results of the follow-up have not been shown yet. The relationship between the expression level and the survival time of gastric cancer patients.2. in vitro and in vivo experiments confirmed that HOXB7 promotes the growth and metastasis of gastric cancer, and the interference of HOXB7 can obviously inhibit the proliferation of BGC-823 and SGC-7901 in gastric cancer cell lines, cell block, promote cell apoptosis, and reduce the invasion, migration ability (P0.01), and change the expression of EMT characteristic protein. The tumor transplantation experiment found that subtraction HOXB7 could significantly inhibit the growth of the tumor (P < 0.01).3. HOXB7 regulation of multiple important downstream genes, and participate in the regulation of PI3K/AKT signaling pathway to screen the downstream regulatory genes of HOXB7: the SGC-7901 cell lines found 60 up genes, 158 down regulated genes, and 149 up regulated genes in BGC-823 cell lines, 2 31 down-regulated genes (multiple 2, P0.05). (2) QT-PCR, including NRXN3, NFAT5, KRAS, Smad2, WNT5A, etc., 18 genes with significant differences were found to be in agreement with the results of the chip. (3) Western blot detected p21, CyclinD1, PIK3R3, and protein levels, which may be in the development of gastric cancer. An important molecular mechanism. Conclusion: 1. HOXB7 is highly expressed in gastric cancer cell lines and tumor tissues, and is related to pathological parameters such as tumor staging. Although the current follow-up data does not show that the expression is related to the progression of gastric cancer and the prognosis of the patients, it suggests that HOXB7 is still a meaningful molecule in the development of gastric cancer. The relationship between the target and the prognosis of the disease still needs further study on the involvement of.2.HOXB7 in regulating the proliferation, cell cycle, apoptosis, invasion and tumorigenicity of tumor cells in vitro, affecting the malignant biological behavior of the tumor, and affecting the tumor's malignant biological behavior,.3. further studies the mechanism of HOXB7 for the development of gastric cancer, and screening a series of downstream genes regulated by it. Some genes are involved in malignant biological process of tumor cells, which can provide new targets for clinical prevention and treatment of gastric cancer.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

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