【摘要】：研究背景胃癌是嚴(yán)重威脅人類生命健康的惡性疾病之一,其全球發(fā)病率在惡性腫瘤中居于第四位。中國(guó)的胃癌發(fā)病率與死亡率居世界首位。早期胃癌發(fā)病多隱匿,多數(shù)患者出現(xiàn)明顯臨床癥狀已處于病程晚期。由于許多胃癌患者到醫(yī)院就診時(shí)已錯(cuò)過(guò)最佳手術(shù)時(shí)機(jī),傳統(tǒng)的放化療副作用較大,嚴(yán)重?fù)p害了患者的生存質(zhì)量。因此,揭示胃癌發(fā)生機(jī)制,發(fā)現(xiàn)關(guān)鍵的腫瘤標(biāo)志物,開(kāi)發(fā)新型治療藥物,早期進(jìn)行防治和干預(yù)是降低胃癌病死率的重要環(huán)節(jié)。胃癌發(fā)生是多因素作用累積引發(fā)的質(zhì)變過(guò)程,幽門螺桿菌感染是胃癌漸進(jìn)發(fā)展的重要環(huán)境誘因。長(zhǎng)期感染幽門螺桿菌可引起胃黏膜損傷,炎性細(xì)胞浸潤(rùn),從而引發(fā)慢性胃炎,持續(xù)性炎癥刺激可導(dǎo)致胃黏膜萎縮,腸上皮化生,最終胃壁細(xì)胞出現(xiàn)不典型增生以致癌變。與此同時(shí),胃癌是一種高度異質(zhì)性疾病,只有極少數(shù)幽門螺桿菌感染患者最終會(huì)發(fā)展到不可逆的惡性階段。由此可見(jiàn)宿主在接受幽門螺桿菌感染等外界環(huán)境因素刺激后,其自身特異性的基因表達(dá)調(diào)控變化在胃癌的發(fā)生發(fā)展中起著關(guān)鍵性的作用。因此探尋感染炎癥向惡性癌變進(jìn)程中存在差異性表達(dá)的節(jié)點(diǎn)分子可為胃癌的早期診斷和防治奠定理論基礎(chǔ)。轉(zhuǎn)錄調(diào)節(jié)因子在控制細(xì)胞基因表達(dá),維系細(xì)胞生命活動(dòng)中起重要作用。核受體超家族是人類細(xì)胞內(nèi)表達(dá)最為豐富的轉(zhuǎn)錄調(diào)節(jié)因子之一,在控制細(xì)胞增殖、機(jī)體發(fā)育分化及新陳代謝等方面發(fā)揮著重要作用。核受體可與相應(yīng)配體結(jié)合后激活調(diào)控相應(yīng)靶基因轉(zhuǎn)錄,也可與其他轉(zhuǎn)錄因子結(jié)合形成轉(zhuǎn)錄復(fù)合體結(jié)合到DNA序列上。在胃癌的相關(guān)研究中,目前對(duì)核受體超家族成員關(guān)注較少。通過(guò)顯微切割技術(shù)和全基因組測(cè)序,我們發(fā)現(xiàn)在胃炎向胃癌的演進(jìn)過(guò)程中,核受體超家族成員—肝核因子4 a (HNF4a)呈升高趨勢(shì)。HNF4 α可以同源二聚體的形式結(jié)合于DNA重復(fù)序列(AGGTCA)調(diào)節(jié)靶基因轉(zhuǎn)錄,進(jìn)而參與細(xì)胞代謝、分化等生物學(xué)進(jìn)程。本研究立足于揭示HNF4 α在胃癌細(xì)胞中的生物學(xué)功能及其在胃組織炎癌跨越過(guò)程中的具體作用機(jī)制。Fox家族是另一類重要的轉(zhuǎn)錄調(diào)控因子。其成員之一FoxMl在人類多種實(shí)體瘤中表達(dá)升高,抑制其表達(dá)可顯著延緩腫瘤進(jìn)程,其可通過(guò)結(jié)合于DNA序列(5’-A(C/T)AAA(C/T)AA-3')調(diào)控下游靶基因表達(dá)。實(shí)驗(yàn)室前期結(jié)果亦顯示轉(zhuǎn)錄因子FoxMl在胃癌中表達(dá)升高,幽門螺桿菌感染可誘導(dǎo)FoxMl表達(dá)。目前,多種腫瘤治療相關(guān)研究將促癌蛋白FoxMl作為治療靶點(diǎn),小分子化合物Siomycin A可抑制FoxMl的表達(dá)及其對(duì)下游基因的正性調(diào)控。本研究進(jìn)一步探究了FoxMl在胃癌中的具體機(jī)制及其藥物靶標(biāo)價(jià)值。研究目的(一) 探討HNF4 α在幽門螺桿菌感染引起的胃部炎癥惡性轉(zhuǎn)化過(guò)程中的作用與機(jī)制；(二)探討FoxMl促進(jìn)胃癌發(fā)生的機(jī)制與藥物靶標(biāo)價(jià)值。方法和結(jié)果(一)幽門螺桿菌感染可通過(guò)NF-κ B信號(hào)通路上調(diào)HNF4 α表達(dá),HNF4α可與其靶分子IL-1R1形成炎性反饋環(huán)路介導(dǎo)幽門螺桿菌感染引發(fā)的惡性轉(zhuǎn)化。1. 通過(guò)實(shí)時(shí)定量PCR (qRT-PCR)和免疫組化實(shí)驗(yàn)發(fā)現(xiàn)萎縮性胃炎標(biāo)本中HNF4α表達(dá)水平顯著低于胃癌標(biāo)本。胃癌細(xì)胞系中HNF4α表達(dá)明顯高于相對(duì)正常的永生化胃黏膜上皮細(xì)胞系GES-1。通過(guò)體外克隆形成實(shí)驗(yàn)和體內(nèi)裸鼠成瘤實(shí)驗(yàn)發(fā)現(xiàn)胃癌細(xì)胞系中HNF4 α被敲除后增殖能力減低。2. 實(shí)時(shí)定量PCR和western blot實(shí)驗(yàn)證實(shí)不同菌株來(lái)源的幽門螺桿菌感染均可誘導(dǎo)胃癌細(xì)胞系中HNF4 α表達(dá)。其毒力因子cagA亦可上調(diào)HNF4 α表達(dá)。幽門螺桿菌在小鼠體內(nèi)亦可使HNF4 α表達(dá)上調(diào)。3. Ensemble數(shù)據(jù)庫(kù)提示HNF4 α P1及P2啟動(dòng)子區(qū)存在兩個(gè)NF-κB信號(hào)通路關(guān)鍵轉(zhuǎn)錄因子p65的結(jié)合元件。使用NF-κ B信號(hào)通路抑制劑或p65 siRNA均可逆轉(zhuǎn)幽門螺桿菌感染導(dǎo)致的HNF4α表達(dá)上調(diào)。雙熒光素酶及CHIP實(shí)驗(yàn)證實(shí),p65可結(jié)合于HNF4α兩個(gè)啟動(dòng)子區(qū)。因此,我們證實(shí)幽門螺桿菌可通過(guò)NF-κB信號(hào)通路激活HNF4 α表達(dá)。4. 幽門螺桿菌感染可促使炎性細(xì)胞分泌炎性因子,細(xì)胞表面受體是上皮細(xì)胞接受外界炎性因子刺激的門戶。通過(guò)全基因組表達(dá)芯片技術(shù)對(duì)胃組織中表達(dá)的細(xì)胞表面受體分析發(fā)現(xiàn),IL-1R1在胃炎向胃癌的轉(zhuǎn)化過(guò)程中表達(dá)上調(diào)最為顯著。收集臨床標(biāo)本進(jìn)行實(shí)時(shí)定量PCR檢測(cè)發(fā)現(xiàn)IL-1R1與HNF4α表達(dá)存在正相關(guān)性。Western blot及細(xì)胞免疫熒光證實(shí)干擾HNF4 α表達(dá)IL-1R1表達(dá)隨之下調(diào),反之亦然。雙熒光素酶實(shí)驗(yàn)證實(shí)HNF4 α可結(jié)合于IL-1R1上游啟動(dòng)子區(qū)。HNF4 α對(duì)IL-1R1的靶向性調(diào)控,可增敏胃部細(xì)胞對(duì)外源性細(xì)胞炎性因子IL-1β的反應(yīng)。IL-1β與IL-1R1結(jié)合可進(jìn)一步激活NF-κ B信號(hào)通路,進(jìn)而誘導(dǎo)HNF4 α表達(dá),形成正反饋環(huán)路促進(jìn)胃部細(xì)胞惡性轉(zhuǎn)化。5. 免疫組化證實(shí)HNF4α與IL-1R1隨萎縮性胃炎嚴(yán)重程度表達(dá)逐步上調(diào),胃癌組織中兩者表達(dá)進(jìn)一步升高,且兩者均高表達(dá)患者預(yù)后較差。(二)證實(shí)FoxMl可直接靶向調(diào)控端粒酶逆轉(zhuǎn)錄酶TERT促進(jìn)胃癌細(xì)胞增殖,小分子化合物siomycin A可通過(guò)抑制FoxMl-TERT表達(dá),阻滯胃癌細(xì)胞增殖。1. 通過(guò)收集臨床標(biāo)本進(jìn)行實(shí)時(shí)定量PCR和免疫組化檢測(cè),我們發(fā)現(xiàn)FoxMl與腫瘤發(fā)生發(fā)展密切相關(guān)的分子一端粒酶逆轉(zhuǎn)錄酶TERT的表達(dá)存在正相關(guān)性。2.實(shí)時(shí)定量PCR與western blot實(shí)驗(yàn)證實(shí)胃癌細(xì)胞系中干擾FoxMl表達(dá)TERT水平隨之降低。同時(shí),克隆形成實(shí)驗(yàn)證實(shí)FoxMl表達(dá)降低可顯著抑制胃癌細(xì)胞增殖能力。TERT高表達(dá)可逆轉(zhuǎn)FoxMl敲除對(duì)胃癌細(xì)胞增殖能力的抑制作用。3.通過(guò)Ensemble數(shù)據(jù)庫(kù)對(duì)TERT上游啟動(dòng)子區(qū)序列進(jìn)行分析,我們發(fā)現(xiàn)在距離開(kāi)放閱讀框架起始位點(diǎn)上游1285bp位置存在FoxMl的經(jīng)典結(jié)合原件。雙熒光素酶實(shí)驗(yàn)證實(shí)FoxMl干擾后,TERT啟動(dòng)子熒光素酶報(bào)告質(zhì)�；钚越档�,CHIP實(shí)驗(yàn)直接證明FoxMl可結(jié)合于TERT啟動(dòng)子區(qū)。4.克隆形成實(shí)驗(yàn)和裸鼠成瘤實(shí)驗(yàn)證實(shí)FoxMl抑制劑siomycin A在體內(nèi)外均可顯著抑制胃癌細(xì)胞的增殖能力,同時(shí)抑制FoxMl和TERT在細(xì)胞中的表達(dá)水平。研究結(jié)論(一)HNF4α可促進(jìn)胃癌細(xì)胞增殖。幽門螺桿菌感染可通過(guò)激活NF-K B信號(hào)通路上調(diào)HNF4α表達(dá),HNF4α可作為轉(zhuǎn)錄因子靶向調(diào)控IL-1R1表達(dá),增敏上皮細(xì)胞對(duì)外界炎性因子IL-1β的反應(yīng),進(jìn)一步激活NF-κB信號(hào)通路。從而形成正性炎性反饋環(huán)路介導(dǎo)胃組織惡性轉(zhuǎn)化。(二)FoxM1和TERT在胃癌臨床標(biāo)本中表達(dá)呈正相關(guān),FoxM1可作為轉(zhuǎn)錄因子直接調(diào)控TERT表達(dá)。Siomycin A在體內(nèi)外均可抑制細(xì)胞增殖,該作用部分依賴于其對(duì)FoxMl-TERT通路的抑制作用。
[Abstract]:The study of the background of gastric cancer is one of the malignant diseases which is a serious threat to the health of human life. The global incidence of gastric cancer is the fourth in the malignant tumor. The incidence and mortality of gastric cancer in China are the first in the world. In the early stage of gastric cancer, the incidence of gastric cancer is more and more insidious, and most of the patients have obvious clinical symptoms which are in the late stage of Due to the fact that many patients with gastric cancer have missed the best operation time in the hospital, the traditional chemotherapy and chemotherapy side effects are large, and the survival quality of the patients is seriously impaired. Therefore, it is an important link to reduce the mortality of gastric cancer by revealing the mechanism of gastric cancer, finding the key tumor markers and developing new therapeutic drugs. Helicobacter pylori infection is an important environmental cause of the progressive development of gastric cancer. The long-term infection of Helicobacter pylori can cause gastric mucosal injury and inflammatory cell infiltration, thus leading to chronic gastritis, and persistent inflammatory stimulation can lead to the atrophy of the gastric mucosa, intestinal metaplasia, and the appearance of the final gastric wall cells is not typical and canceration. At the same time, gastric cancer is a highly heterogeneous disease, and only a very small number of patients with H. pylori infection will eventually develop to an irreversible, malignant stage. It can be seen that, after the host is stimulated by external environmental factors such as H. pylori infection, the regulation of gene expression of its own specificity plays a key role in the development of gastric cancer. Therefore, it is necessary to lay a theoretical foundation for the early diagnosis and prevention of gastric cancer. Transcriptional regulatory factors play an important role in the control of cell gene expression and the maintenance of cell life. The nuclear receptor superfamily is one of the most abundant transcriptional regulation factors in human cells, and plays an important role in controlling cell proliferation, body development differentiation and metabolism. The nuclear receptor can be activated to regulate the transcription of the corresponding target gene after binding with the corresponding ligand, or may be combined with other transcription factors to form a transcription complex to bind to the DNA sequence. In the related research of gastric cancer, the attention of the nuclear receptor superfamily members is less. By means of microdissection and full-genome sequencing, we have found that in the course of the evolution of gastritis to gastric cancer, the nuclear factor 4a (HNF4a) in the superfamily of the nuclear receptor has a tendency to increase. HNF4 can be used in the form of homodimer to regulate the transcription of the target gene in the form of a DNA repeat sequence (AGGTCA), so as to participate in the biological processes of cell metabolism, differentiation and the like. This study is based on the study of the biological function of HNF4 in gastric cancer cells and its specific mechanism in the treatment of gastric cancer. The Fox family is another type of important transcription factor. One of its members, FoxMl, expressed an increase in the expression of FoxMl in a variety of human solid tumors, which could significantly delay the progression of the tumor by regulating the expression of the downstream target gene by binding to a DNA sequence (5 '-A (C/ T) AAA (C/ T) AA-3'). Helicobacter pylori infection can induce the expression of FoxMl. The expression of FoxMl and its positive regulation on the downstream gene can be inhibited by Simin A, a small-molecule compound. The specific mechanism of FoxMl in gastric cancer and its target value are further investigated. (2) To study the mechanism of FoxMl to promote gastric cancer and the value of drug target. 1. The expression of HNF4 in the specimens of atrophic gastritis was significantly lower than that of the gastric cancer specimens by real-time quantitative PCR (qRT-PCR) and immunohistochemistry. The expression of HNF4 in the gastric cancer cell line was significantly higher. The proliferation ability of HNF4 in gastric cancer cell lines was reduced by in vitro cloning and in vivo nude mice.2. The results of real-time quantitative PCR and western blot proved that the infection of H. pylori in different strains of gastric cancer can be induced. The expression of HNF4 in the gastric cancer cell line can also be up-regulated by the virulence factor cagA. The expression of HNF4 is also up-regulated by the virulence factor cagA. -both the B-signal pathway inhibitor or the p65 siRNA can reverse the up-regulation of the HNF4 gene expression resulting from the Helicobacter pylori infection. The double luciferase and the CHIP experiment confirm that p65 can be bound to the two promoter regions of the HNF4. Therefore, We confirm that H. pylori can activate the expression of HNF4 through the NF-B signal pathway. The cell surface receptor is a portal in which the epithelial cells are stimulated by an external inflammatory factor. The cell surface receptor analysis expressed in the stomach tissue is found by the full-genome expression chip technique, The expression of IL-1R1 was most significant in the course of the transformation of gastritis to gastric cancer. The positive correlation between the expression of IL-1R1 and HNF4 was detected by real-time quantitative PCR. The expression of IL-1R1 was reduced by Western blot and immunofluorescence. and vice versa. The double-luciferase experiment confirmed that the HNF4 antigen can be bound to the upstream promoter region of the IL-1R1. The targeting of the HNF4 antigen to the IL-1R1 can increase the response of the stomach cell to the inflammatory factor IL-1 of the exogenous cell. The IL-1R1 binding to the IL-1R1 can further activate the NF-1BB signal path, 5. The expression of HNF4 was further induced to form a positive feedback loop to promote the malignant transformation of the stomach cells. and (2) confirming that the FoxMl can directly target and control the telomerase reverse transcriptase to promote the proliferation of the gastric cancer cells, 1. the proliferation of the gastric cancer cells is retarded.1. real-time quantitative PCR and immunohistochemical detection are carried out through the collection of clinical samples, We found that the expression of FoxMl was positively correlated with the expression of telomerase reverse transcriptase, which was closely related to the development of tumorigenesis.2. The expression level of the interfering FoxMl in the gastric cancer cell line was reduced with the real-time quantitative PCR and western blot. The results showed that the expression of FoxMl could significantly inhibit the proliferation of gastric cancer cells. We found that a classical binding original of FoxMl was present at a location of 1285 bp upstream of the starting site of the open reading frame. The luciferase reporter plasmid activity was reduced after the double luciferase assay confirmed the FoxMl interference. The results showed that FoxMl could inhibit the proliferation of gastric cancer cells in vivo and in vivo. Conclusion (1) HNF4 can promote the proliferation of gastric cancer cells. H. pylori infection can increase the expression of HNF4 by activating NF-K B signal pathway, and the HNF4 gene can be used as a transcription factor to target the expression of IL-1R1. the reaction of the sensitizing epithelial cells to the external inflammatory factor IL-1 is further activated to form a positive inflammatory feedback loop to mediate the malignant transformation of the stomach tissue. (2) the expression of FoxM1 and the antigen in the clinical sample of the gastric cancer is positively correlated, FoxM1 can be directly regulated and expressed as a transcription factor.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.2

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1 馬琳;轉(zhuǎn)錄調(diào)節(jié)分子HNF4α和FoxM1參與胃癌發(fā)生的功能與機(jī)制[D];山東大學(xué);2016年

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本文編號(hào)：2509852