转录调节分子HNF4α和FoxM1参与胃癌发生的功能与机制
发布时间：2019-07-04 10:29
【摘要】：研究背景胃癌是严重威胁人类生命健康的恶性疾病之一,其全球发病率在恶性肿瘤中居于第四位。中国的胃癌发病率与死亡率居世界首位。早期胃癌发病多隐匿,多数患者出现明显临床症状已处于病程晚期。由于许多胃癌患者到医院就诊时已错过最佳手术时机,传统的放化疗副作用较大,严重损害了患者的生存质量。因此,揭示胃癌发生机制,发现关键的肿瘤标志物,开发新型治疗药物,早期进行防治和干预是降低胃癌病死率的重要环节。胃癌发生是多因素作用累积引发的质变过程,幽门螺杆菌感染是胃癌渐进发展的重要环境诱因。长期感染幽门螺杆菌可引起胃黏膜损伤,炎性细胞浸润,从而引发慢性胃炎,持续性炎症刺激可导致胃黏膜萎缩,肠上皮化生,最终胃壁细胞出现不典型增生以致癌变。与此同时,胃癌是一种高度异质性疾病,只有极少数幽门螺杆菌感染患者最终会发展到不可逆的恶性阶段。由此可见宿主在接受幽门螺杆菌感染等外界环境因素刺激后,其自身特异性的基因表达调控变化在胃癌的发生发展中起着关键性的作用。因此探寻感染炎症向恶性癌变进程中存在差异性表达的节点分子可为胃癌的早期诊断和防治奠定理论基础。转录调节因子在控制细胞基因表达,维系细胞生命活动中起重要作用。核受体超家族是人类细胞内表达最为丰富的转录调节因子之一,在控制细胞增殖、机体发育分化及新陈代谢等方面发挥着重要作用。核受体可与相应配体结合后激活调控相应靶基因转录,也可与其他转录因子结合形成转录复合体结合到DNA序列上。在胃癌的相关研究中,目前对核受体超家族成员关注较少。通过显微切割技术和全基因组测序,我们发现在胃炎向胃癌的演进过程中,核受体超家族成员—肝核因子4 a (HNF4a)呈升高趋势。HNF4 α可以同源二聚体的形式结合于DNA重复序列(AGGTCA)调节靶基因转录,进而参与细胞代谢、分化等生物学进程。本研究立足于揭示HNF4 α在胃癌细胞中的生物学功能及其在胃组织炎癌跨越过程中的具体作用机制。Fox家族是另一类重要的转录调控因子。其成员之一FoxMl在人类多种实体瘤中表达升高,抑制其表达可显著延缓肿瘤进程,其可通过结合于DNA序列(5’-A(C/T)AAA(C/T)AA-3')调控下游靶基因表达。实验室前期结果亦显示转录因子FoxMl在胃癌中表达升高,幽门螺杆菌感染可诱导FoxMl表达。目前,多种肿瘤治疗相关研究将促癌蛋白FoxMl作为治疗靶点,小分子化合物Siomycin A可抑制FoxMl的表达及其对下游基因的正性调控。本研究进一步探究了FoxMl在胃癌中的具体机制及其药物靶标价值。研究目的(一) 探讨HNF4 α在幽门螺杆菌感染引起的胃部炎症恶性转化过程中的作用与机制；(二)探讨FoxMl促进胃癌发生的机制与药物靶标价值。方法和结果(一)幽门螺杆菌感染可通过NF-κ B信号通路上调HNF4 α表达,HNF4α可与其靶分子IL-1R1形成炎性反馈环路介导幽门螺杆菌感染引发的恶性转化。1. 通过实时定量PCR (qRT-PCR)和免疫组化实验发现萎缩性胃炎标本中HNF4α表达水平显著低于胃癌标本。胃癌细胞系中HNF4α表达明显高于相对正常的永生化胃黏膜上皮细胞系GES-1。通过体外克隆形成实验和体内裸鼠成瘤实验发现胃癌细胞系中HNF4 α被敲除后增殖能力减低。2. 实时定量PCR和western blot实验证实不同菌株来源的幽门螺杆菌感染均可诱导胃癌细胞系中HNF4 α表达。其毒力因子cagA亦可上调HNF4 α表达。幽门螺杆菌在小鼠体内亦可使HNF4 α表达上调。3. Ensemble数据库提示HNF4 α P1及P2启动子区存在两个NF-κB信号通路关键转录因子p65的结合元件。使用NF-κ B信号通路抑制剂或p65 siRNA均可逆转幽门螺杆菌感染导致的HNF4α表达上调。双荧光素酶及CHIP实验证实,p65可结合于HNF4α两个启动子区。因此,我们证实幽门螺杆菌可通过NF-κB信号通路激活HNF4 α表达。4. 幽门螺杆菌感染可促使炎性细胞分泌炎性因子,细胞表面受体是上皮细胞接受外界炎性因子刺激的门户。通过全基因组表达芯片技术对胃组织中表达的细胞表面受体分析发现,IL-1R1在胃炎向胃癌的转化过程中表达上调最为显著。收集临床标本进行实时定量PCR检测发现IL-1R1与HNF4α表达存在正相关性。Western blot及细胞免疫荧光证实干扰HNF4 α表达IL-1R1表达随之下调,反之亦然。双荧光素酶实验证实HNF4 α可结合于IL-1R1上游启动子区。HNF4 α对IL-1R1的靶向性调控,可增敏胃部细胞对外源性细胞炎性因子IL-1β的反应。IL-1β与IL-1R1结合可进一步激活NF-κ B信号通路,进而诱导HNF4 α表达,形成正反馈环路促进胃部细胞恶性转化。5. 免疫组化证实HNF4α与IL-1R1随萎缩性胃炎严重程度表达逐步上调,胃癌组织中两者表达进一步升高,且两者均高表达患者预后较差。(二)证实FoxMl可直接靶向调控端粒酶逆转录酶TERT促进胃癌细胞增殖,小分子化合物siomycin A可通过抑制FoxMl-TERT表达,阻滞胃癌细胞增殖。1. 通过收集临床标本进行实时定量PCR和免疫组化检测,我们发现FoxMl与肿瘤发生发展密切相关的分子一端粒酶逆转录酶TERT的表达存在正相关性。2.实时定量PCR与western blot实验证实胃癌细胞系中干扰FoxMl表达TERT水平随之降低。同时,克隆形成实验证实FoxMl表达降低可显著抑制胃癌细胞增殖能力。TERT高表达可逆转FoxMl敲除对胃癌细胞增殖能力的抑制作用。3.通过Ensemble数据库对TERT上游启动子区序列进行分析,我们发现在距离开放阅读框架起始位点上游1285bp位置存在FoxMl的经典结合原件。双荧光素酶实验证实FoxMl干扰后,TERT启动子荧光素酶报告质粒活性降低,CHIP实验直接证明FoxMl可结合于TERT启动子区。4.克隆形成实验和裸鼠成瘤实验证实FoxMl抑制剂siomycin A在体内外均可显著抑制胃癌细胞的增殖能力,同时抑制FoxMl和TERT在细胞中的表达水平。研究结论(一)HNF4α可促进胃癌细胞增殖。幽门螺杆菌感染可通过激活NF-K B信号通路上调HNF4α表达,HNF4α可作为转录因子靶向调控IL-1R1表达,增敏上皮细胞对外界炎性因子IL-1β的反应,进一步激活NF-κB信号通路。从而形成正性炎性反馈环路介导胃组织恶性转化。(二)FoxM1和TERT在胃癌临床标本中表达呈正相关,FoxM1可作为转录因子直接调控TERT表达。Siomycin A在体内外均可抑制细胞增殖,该作用部分依赖于其对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.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2
本文编号：2509852
[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.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2
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1 马琳;转录调节分子HNF4α和FoxM1参与胃癌发生的功能与机制[D];山东大学;2016年
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