本文選題：同源盒基因 + HOXB7　； 參考：《浙江大學(xué)》2016年博士論文

【摘要】：胃癌是全世界發(fā)病率最高的惡性腫瘤之一,是中國第二常見腫瘤。全球每年胃癌新發(fā)病例數(shù)約100萬,中國占40%之多,且其患病率和死亡率均是世界平均水平的2倍。近年來胃癌發(fā)病人口亦趨于年輕化,其預(yù)防和治療均需高度重視。隨著胃癌規(guī)范化診治的不斷完善和推廣,以手術(shù)為主的綜合治療對(duì)改善胃癌患者預(yù)后、提高生存率起到了一定的促進(jìn)作用。然而現(xiàn)階段提高腫瘤治愈率最有效的方法仍然是早期診斷、早期干預(yù),因此如何更加早期、有效地診治胃癌仍然是胃癌防治工作的重點(diǎn)。已有多項(xiàng)研究表明,胃癌的發(fā)生發(fā)展涉及癌基因活化和抑癌基因失活等多個(gè)方面,故篩選診斷和預(yù)后生物學(xué)靶標(biāo),闡明胃癌發(fā)生發(fā)展過程中的分子機(jī)制,是胃癌轉(zhuǎn)化醫(yī)學(xué)研究工作的重點(diǎn)方向。同源盒基因(Homeobox genes)是細(xì)胞增殖和分化的主控基因,其編碼的蛋白作為轉(zhuǎn)錄因子,在脊椎動(dòng)物胚胎發(fā)育和器官形成中起到重要作用；作用靶標(biāo)通常為粘附分子、膜蛋白受體、生長因子等與腫瘤進(jìn)展、轉(zhuǎn)移密切相關(guān)的基因家族。同時(shí)對(duì)腫瘤干細(xì)胞的相關(guān)特性如自我更新、無限增殖等惡性生物學(xué)行為具有調(diào)控作用。目前在肺癌、乳腺癌、結(jié)腸癌等多種腫瘤中均可檢測(cè)到HOX基因的異常表達(dá)。其中,位于第17號(hào)染色體的HOXB7已被證實(shí)屬于癌基因的范疇。已有文獻(xiàn)表明,HOXB7可通過上調(diào)堿性成纖維細(xì)胞生長因子(basic fibroblast growth factor,bFGF),激活Ras/RhoA蛋白通路,從而促進(jìn)乳腺細(xì)胞發(fā)生上皮間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition, EMT),促使腫瘤的發(fā)生。但有關(guān)HOXB7基因在胃癌中的表達(dá)情況以及其是否參與調(diào)控胃癌腫瘤細(xì)胞惡性進(jìn)展與轉(zhuǎn)移的能力,目前尚未見報(bào)道。本研究通過檢測(cè)胃癌細(xì)胞株及胃癌患者腫瘤組織中HOXB7基因的表達(dá)情況,結(jié)合臨床病理及隨訪資料,初步探討HOXB7表達(dá)水平的臨床意義；再進(jìn)一步利用RNA干擾及慢病毒載體敲低HOXB7表達(dá),檢測(cè)胃癌細(xì)胞和裸鼠荷瘤的惡性生物學(xué)行為變化；采用表達(dá)譜芯片技術(shù)篩選HOXB7基因調(diào)控的下游關(guān)鍵基因并進(jìn)行驗(yàn)證,進(jìn)一步闡明其對(duì)胃癌促進(jìn)作用及相關(guān)信號(hào)通路的分子調(diào)控機(jī)制。研究方法：1. HOXB7在胃癌中的表達(dá)及臨床資料相關(guān)性分析①Q(mào)T-PCR檢測(cè)96對(duì)胃癌及相應(yīng)常組織中HOXB7基因mRNA表達(dá)水平；免疫組化檢測(cè)分析胃癌及相應(yīng)正常組織中HOXB7的表達(dá)水平；②QT-PCR和Western blot檢測(cè)8種胃癌細(xì)胞株中的HOXB7的表達(dá)；③根據(jù)QT-PCR結(jié)果分組分析HOXB7表達(dá)高低與患者臨床病理參數(shù)之間的關(guān)系。2. HOXB7對(duì)胃癌的生物學(xué)行為的影響①體外轉(zhuǎn)染小干擾RNA,敲減HOXB7表達(dá)量,通過MTS、Edu、流式細(xì)胞周期實(shí)驗(yàn)、流式細(xì)胞凋亡實(shí)驗(yàn)、Transwell實(shí)驗(yàn)以及Western blot檢測(cè)HOXB7對(duì)胃癌細(xì)胞周期、增殖、侵襲、遷移能力的影響；②通過體外病毒載體構(gòu)建HOXB7低表達(dá)細(xì)胞株,經(jīng)裸鼠皮下荷瘤模型驗(yàn)證干擾HOXB7對(duì)胃癌生長的影響。3. HOXB7調(diào)控靶基因及相關(guān)信號(hào)通路的研究①利用基因表達(dá)譜芯片技術(shù)篩選HOXB7調(diào)控的下游靶基因,并對(duì)其中18個(gè)差異較明顯候選靶基因在穩(wěn)定轉(zhuǎn)染HOXB7的胃癌細(xì)胞中進(jìn)行QT-PCR驗(yàn)證;②Western blot檢測(cè)HOXB7對(duì)細(xì)胞周期增殖調(diào)控通路關(guān)鍵蛋白p21、PIK3R3、AKT表達(dá)的影響。研究結(jié)果：1. HOXB7在胃癌中表達(dá)明顯高于正常組織,且與病理分化程度及TNM分期相關(guān)① HOXB7在胃癌組織中表達(dá)水平較相應(yīng)正常組織明顯上調(diào)∽0.01)；② HOXB7在胃癌細(xì)胞株中MKN45、BGC-823、MGC-803、SGC-7901均呈現(xiàn)顯著高表達(dá),但在AGS及HGC-27中呈低表達(dá)；③HOXB7表達(dá)高低與腫瘤病理分化程度(P=0.02)、TNM分期(P=0.02)密切相關(guān),與患者性別、年齡、腫瘤大小、位置無關(guān)；研究隨訪結(jié)果尚未顯示其表達(dá)高低與胃癌患者生存期具有相關(guān)性。2.體內(nèi)外實(shí)驗(yàn)證實(shí)HOXB7促進(jìn)胃癌的生長和轉(zhuǎn)移①干擾HOXB7能明顯抑制胃癌細(xì)胞株BGC-823和SGC-7901的增殖、發(fā)生細(xì)胞阻滯,促進(jìn)細(xì)胞凋亡,并降低侵襲、遷移能力(P0.01),改變EMT特征蛋白表達(dá)。②裸鼠胃癌移植瘤實(shí)驗(yàn)發(fā)現(xiàn)敲減HOXB7能顯著抑制腫瘤的生長(P＜0.01)。3. HOXB7調(diào)控多個(gè)重要下游基因,并參與PI3K/AKT信號(hào)通路調(diào)控①篩選到HOXB7的下游調(diào)控基因：SGC-7901細(xì)胞系中發(fā)現(xiàn)60個(gè)上調(diào)基因,158個(gè)下調(diào)基因,BGC-823細(xì)胞系中發(fā)現(xiàn)149個(gè)上調(diào)基因,231個(gè)下調(diào)基因(倍數(shù)2,P0.05)。②經(jīng)QT-PCR驗(yàn)證了包括NRXN3、NFAT5、KRAS、Smad2、WNT5A等;18個(gè)差異顯著的基因,變化趨勢(shì)與芯片結(jié)果一致。③Western blot檢測(cè)p21、CyclinD1、PIK3R3、pAKT、AKT蛋白水平,發(fā)現(xiàn)干擾HOXB7抑制了PI3K/AKT信號(hào)通路,可能是胃癌發(fā)生發(fā)展過程中一個(gè)重要的分子機(jī)制。研究結(jié)論：1. HOXB7在胃癌細(xì)胞系及患者腫瘤組織中異常高表達(dá),且與腫瘤分期等病理參數(shù)相關(guān),雖然目前隨訪數(shù)據(jù)未顯示其表達(dá)高低與胃癌進(jìn)展及病人預(yù)后相關(guān),但提示HOXB7仍是胃癌發(fā)生發(fā)展中一個(gè)有意義的分子靶標(biāo),其與疾病預(yù)后的關(guān)系仍需進(jìn)一步研究。2.HOXB7參與調(diào)控胃癌腫瘤細(xì)胞體外增殖、細(xì)胞周期、凋亡、侵襲和體內(nèi)成瘤能力,影響腫瘤惡性生物學(xué)行為。3.進(jìn)一步深入研究HOXB7對(duì)胃癌發(fā)生發(fā)展的作用機(jī)制,篩選到其調(diào)控的一系列下游基因,這些基因參與腫瘤細(xì)胞惡性生物學(xué)過程,可為胃癌的臨床防治提供新的靶點(diǎn)。
[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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.2

【相似文獻(xiàn)】

相關(guān)期刊論文 前2條

1 朱發(fā)霞;李明梅;湯夢(mèng)婕;向清明;林從堯;;同源異型盒基因HOXB7在乳腺癌組織中的表達(dá)及其臨床意義[J];微循環(huán)學(xué)雜志;2012年04期

2 ;[J];;年期

相關(guān)博士學(xué)位論文 前1條

1 蔡佳沁;同源盒基因HOXB7對(duì)胃癌發(fā)生發(fā)展的作用及其調(diào)控機(jī)制的初步研究[D];浙江大學(xué);2016年

，

本文編號(hào)：1835425