ACY-1在结直肠癌中的表达及作用机制

发布时间：2018-07-06 20:43

本文选题：结直肠癌 + ACY-1　；参考：《山东大学》2017年博士论文

【摘要】：背景结直肠癌为目前世界上最为常见的恶性肿瘤之一,其在消化道恶性肿瘤发病率中仅次于胃癌和食道癌。在我国,结直肠癌的发病率在近二十年来呈逐渐上升趋势,且发病人群的年龄逐渐趋于年轻化。随着我国居民的饮食结构以及生活方式的改变,结直肠癌的发病率在城市居民中增高尤其明显。结直肠癌具有高发病率、高死亡率以及高转移率的特点,因此对人类生命健康构成了巨大威胁。尽管随着现代医疗技术水平的不断发展和完善,在对结直肠癌的治疗上已经取得了突破性进展,但结直肠癌患者生存率依然未得到明显改善。结直肠癌患者的生存率与其诊断时所处的恶性化程度之间存在相关性,即Ⅰ期结直肠癌患者的五年生存率可高达93%,而Ⅲ期结直肠癌患者的5年生存率则仅为8%。引起结直肠癌患者死亡的大部分原因是由于肿瘤复发转移,目前临床上通过手术为主的综合治疗方法仅能够使约50%的结直肠癌患者在术后三年内无进展生存,但约有大于40%的结直肠癌患者在术后发生复发和转移,而且结直肠癌复发的高峰期往往集中在术后2年左右。因此,寻找与结直肠癌发生以及恶性化进程相关的分子标志物,同时对其相关功能及作用机制进行深入探索是目前医学界研究的重点和热点内容,对于结直肠癌患者的早期检测、治疗以及预后情况判断具有十分重要的意义。结直肠癌的恶性化过程通常是由体内外多种因素参与的多步骤的复杂过程,通过网络性调控影响结直肠癌的生物学行为。随着肿瘤分子生物学探索的不断深入,越来越多的基因被证实参与了结直肠癌的发生和发展过程。氨基酰化酶1(aminoacylase-1,ACY-1)为一种胞质酶,能够使细胞内蛋白的N末端肽链上的αα酰化氨基酸发生乙酰化,在此过程之后能够使该蛋白质的稳定性增强。当机体内的原癌基因活化或者抑癌基因失活时,细胞的生长以及分化过程将发生失控,从而向癌细胞转变。目前对于ACY-1的研究大部分集中在其具有的酰化氨基酸水解酶的活性作用方面,临床上通常将血清ACY-1作为判断肾移植预后的指标,但依然缺少其在恶性肿瘤发生和发展过程中作用的研究。目前已有研究表明,ACY-1在多种实体恶性肿瘤组织中的表达量发生下调,从而提示ACY-1可能参与了肿瘤的发生、发展。还有报道发现,ACY-1在神经母细胞瘤患者的血清中呈低表达状态时往往预示着患者的预后情况较差。但目前尚未有对于ACY-1在结直肠癌中表达以及作用机制的报道。转化生长因子β(TGF-β)为一个在人体多种生理病理过程具有生物学功能的生长因子超家族,主要功能是调控细胞增殖以及分化过程,同时还在胚胎发育过程中发挥重要作用。除此之外,TGF-β还具有促进细胞外基质的形成以及调控机体免疫反应等功能。TGF-β共包括6种异构体(TGF-β1～6),其中TGF-β1最为常见,几乎参与了生物机体内所有的病理以及生理过程,与临床上多种疾病的发生和发展密切相关。研究发现,TGF-β在恶性肿瘤的发生和发展、胚胎发育、细胞外基质的形成以及免疫调节过程中均扮演着十分重要的角色。目前已有研究证实,TGF-β1在乳腺癌、胃癌、结肠癌、前列腺癌、宫颈癌以及膀胱癌等恶性肿瘤组织中呈异常高表达状态,且其表达水平与上述肿瘤的进展以及转移相关。细胞外信号调节激酶(extracellular regulated kinase,ERK)为丝裂原活化蛋白激酶(mitogen-activatedprotein kinase,MAPK)家族的重要成员之一。MAPK通路能够被多种因素刺激而活化,其中包括细胞因子、神经递质、生长因子、激素、细胞应激以及细胞黏附等,其下游靶基因包括多种蛋白激酶、磷脂酶和转录因子等。当这些下游靶基因被活化之后能够直接使核转录因子以及其他蛋白激酶等底物发生磷酸化,从而对相关基因的转录过程进行调控,最终影响机体细胞的生长、发育、分裂,从而维持其正常生理功能的完整性。ERK1和ERK2是MAPK/ERK信号转导通路中的两个重要成员,主要对细胞生长、发育、分化、凋亡等生理过程进行调控,同时在细胞的恶性转化过程中亦扮演着重要角色。由ERK所介导的信号转导通路受到来自于细胞内外多种丝裂原的刺激和应激,从而使ERK的底物活化,最终实现调控细胞周期以及抑制细胞凋亡的作用,同时还能够促进细胞增殖、迁移以及侵袭等生理病理过程。ERK1/2在生物机体细胞内分布产生变化往往预示着MAPK信号转导通路的功能状态发生改变。研究表明,在未受到刺激的细胞中,ERK1/2大部分均存在于细胞质中;当细胞受到刺激时,部分ERK1/2向细胞核中聚集,从而形成p-ERK1/2。p-ERK1/2能够与细胞核内的多种转录因子发生相互作用,从而促进了癌基因的表达。ERK1/2信号通路与恶性肿瘤的发生和发展存在显著相关性。目的本研究通过检测ACY-1在结直肠癌组织中的表达,明确其临床意义,同时对其在结直肠癌细胞增殖、凋亡、侵袭以及转移过程中的相关作用机制进行研究,旨在探讨ACY-1与结直肠癌发生和发展的相关性,从而为寻找新的肿瘤分子标志物以及药物作用靶点奠定基础。方法(1)采集132例结直肠组织样本均来自于山东大学附属省立医院胃肠外科2015年6月～2016年6月结直肠癌手术切除组织样本,按照性别进行划分,其中男性为70例,女性为62例;按照年龄进行划分,其中≤50岁为57例,50岁为75例;按照是否淋巴结转移进行划分,其中发生转移的为52例,未发生转移的为80例;TNM分期进行划分,其中Ⅰ期为44例、Ⅱ期为62例、Ⅲ期+Ⅳ为26例。另外选取120例健康志愿者血清作为对照组。采集上述入选者的癌组织、相应癌旁正常组织或血清,分别运用荧光定量PCR、western blot法以及ELISA法检测ACY-1 mRNA及其蛋白的表达,并分析其与患者性别、年龄、TNM分期和淋巴结转移的相关性。(2)将携带有siRNA-ACY-1基因的慢病毒载体GV218-EGFP-ACY-1转染人293T细胞,待包装出病毒之后将之感染人结肠癌HT29细胞,以此构建ACY-1过抑制表达HT29细胞系。用倒置荧光显微镜对人293T细胞的转染效率进行观察。荧光定量PCR检测ACY-1表达抑制的HT29细胞中ACY-1 mRNA的表达;运用Transwell小室法检测该细胞系的侵袭能力;流式细胞术测定细胞的凋亡情况,CCK-8法检测细胞的增殖活性。(3)分别将 siRNA-NC 和 siRNA-ACY-1 转染 HT-29 细胞,运用 western blot法分别检测ACY-1抑制表达的人结肠癌细胞HT29细胞中p-ERK1/2和TGF-β1蛋白的表达。结果(1)荧光定量PCR检测表明,ACY-1 mRNA在癌旁相应正常组织(对照组)中的表达量为0.324±0.012,在结直肠癌组织中的表达量为0.614±0.051,显著高于对照组(P0.05)。western blot检测结果表明,ACY-1蛋白在癌旁相应正常组织中的表达量为0.231±0.065,在结直肠癌组织中的表达量为0.646±0.038,显著高于对照组(P0.05)。ELISA检测结果表明,癌旁正常组织中ACY-1的含量为36.88±5.65 pg/mL,结直肠癌患者血清中的含量为57.11±6.32pg/mL,明显高于健康志愿者(P0.05)。对ACY-1的表达与结直肠癌患者临床病例参数的相关性进行分析,结果表明ACY-1的表达与结直肠癌患者的性别和年龄之间无显著相关性(P0.05),而与TNM分期和是否发生淋巴结转移有关(P0.05)。(2)荧光定量PCR检测结果表明,构建的ACY-1抑制表达HT29细胞中的ACY-1 mRNA的表达量明显低于对照组,表明成功构建ACY-1抑制表达HT29细胞系。CCK-8检测结果表明,转染siRNA-ACY-1后细胞的增殖活性明显低于对照组(P0.01)。流式细胞术检测结果表明,ACY-1抑制表达的HT29细胞的凋亡细胞数量显著高于对照组(P0.01)。Transwell检测结果表明,感染siRNA-ACY-1后HT29细胞的侵袭能力显著低于对照组(P0.05)。(3)western blot法检测结果表明,对照组ACY-1蛋白的相对表达量为0.78±0.11,siRNA-ACY-1 组为 0.16±0.04。与对照组相比,siRNA-ACY-1组ACY-1蛋白的表达量明显低于对照组(P0.01)。对照组p-ERK1/2蛋白的相对表达量为 0.89±0.13,TGF-β1 蛋白为 0.78±0.14;siRNA-ACY-1组p-ERK1/2蛋白的相对表达量为0.33±0.09,TGF-β1蛋白为0.21±0.06。与对照组相比,p-ERK1/2、TGF-β1蛋白的表达量均显著降低,差异具有统计学意义(P0.05)。结论ACY-1在结直肠癌组织中呈高表达,且其表达量与结直肠癌患者的TNM分期以及淋巴结转移密切相关。ACY-1能够通过直接调控TGF-β-ERK1/2信号通路来实现对结直肠癌细胞增殖、侵袭以及凋亡的调控。
[Abstract]:Background colorectal cancer is one of the most common malignant tumors in the world. It is second only to gastric cancer and esophagus cancer in the incidence of digestive tract malignancies. In China, the incidence of colorectal cancer has been increasing in the past twenty years, and the age of the population is gradually becoming younger. The incidence of colorectal cancer is particularly evident in urban residents. Colorectal cancer has a high incidence, high mortality, and high metastasis rate, which poses a great threat to human life and health. Although with the continuous development and improvement of modern medical technology, the treatment of colorectal cancer has been done. The survival rate of colorectal cancer patients remained unchanged. There was a correlation between the survival rate of colorectal cancer patients and the degree of malignancy at the time of diagnosis, that is, the five year survival rate of patients with stage I colorectal cancer was up to 93%, while the 5 year survival rate of patients with stage III colorectal cancer was only 8%.. Most of the causes of death in patients with colorectal cancer are due to tumor recurrence. Currently, a comprehensive treatment based on surgery can only make about 50% of the patients with colorectal cancer without progress within three years after the operation, but more than 40% of the colorectal cancer patients have recurrence and metastasis after operation, and the recurrence of colorectal cancer is at the peak period. It is often concentrated at about 2 years after the operation. Therefore, it is the key and hot topic in the medical field to find the molecular markers related to the process of colorectal cancer and the process of malignancy, and to explore the related functions and mechanisms of action. It is ten for the early detection, treatment and prognosis of colorectal cancer patients. It is of great significance. The malignant process of colorectal cancer is usually a multistep and complex process involved in a variety of factors in the body and the body. It affects the biological behavior of colorectal cancer through network regulation. With the continuous exploration of the molecular biology of the tumor, more and more genes have been proved to be involved in the process of the occurrence and development of rectal cancer. Aminoacylase 1 (aminoacylase-1, ACY-1) is a cytoplasmic enzyme that can make acetylation of alpha acylated amino acids on the N terminal peptide chain of the intracellular protein. After this process, the stability of the protein can be enhanced. When the oncogene is activated in the body or when Yi Aiji is inactivated, the cell growth and differentiation process will occur. The study of ACY-1 is mostly focused on the activity of its acylated amino acid hydrolase. The serum ACY-1 is usually used as a prognostic indicator of renal transplantation, but it still lacks the study of its role in the development and development of malignant tumors. Currently, research has shown that A The expression of CY-1 in a variety of solid malignant tumor tissues is down, suggesting that ACY-1 may be involved in the occurrence and development of the tumor. It is also reported that the low expression of ACY-1 in the serum of the patients with neuroblastoma often indicates that the prognosis of the patients is poor, but there is no table for ACY-1 in colorectal cancer. Transforming growth factor beta (TGF- beta) is a growth factor superfamily with biological functions in various physiological and pathological processes in human body. The main function is to regulate cell proliferation and differentiation, and also plays an important role in the process of embryonic development. In addition, TGF- beta also promotes the extracellular matrix. 6 isomers (TGF- beta 1~6) are included in the formation of the substance and the regulation of the body's immune response, including 6 isomers (TGF- beta 1~6). The TGF- beta 1 is the most common, almost involved in all the pathological and physiological processes in the biological machine. It is closely related to the occurrence and development of various clinical diseases. The study found that TGF- beta is in the occurrence and development of malignant tumors and embryos. Development, the formation of extracellular matrix and immunomodulation play a very important role in the process of immunoregulation. TGF- beta 1 has been proved to be highly expressed in malignant tumor tissues such as breast cancer, gastric cancer, colon cancer, prostate cancer, cervical cancer and bladder cancer, and its expression level is associated with the progression and metastasis of the above tumor. Extracellular regulated kinase (ERK), one of the important members of the mitogen activated protein kinase (MAPK) family, is one of the important members of the mitogen-activatedprotein kinase (MAPK) family. The.MAPK pathway can be activated by a variety of factors, including cytokines, neurotransmitters, growth factors, hormones, cell stress, and cell adhesion. The downstream target genes include a variety of protein kinases, phospholipases and transcription factors. When these downstream target genes are activated, they can directly phosphorylate the nuclear transcription factors and other protein kinase and other substrates, thereby regulating the transcriptional processes of the related genes, which ultimately affect the growth, development and division of the body cells. The integrity of.ERK1 and ERK2, with its normal physiological function, is the two important member of the MAPK/ERK signal transduction pathway, which regulates the physiological processes, such as cell growth, development, differentiation and apoptosis, and plays an important role in the process of cell malignant transformation. The signal transduction pathway mediated by ERK is derived from and from the cell. The stimulation and stress of a variety of mitogens make the substrates of ERK activate, ultimately regulate cell cycle and inhibit cell apoptosis, and can also promote cell proliferation, migration and invasion and other physiological and pathological processes, which tend to indicate the function of the MAPK signal transduction pathway of.ERK1/2. The study showed that the majority of ERK1/2 existed in the cytoplasm in the cells that were not stimulated, and when the cells were stimulated, some of the ERK1/2 gathered in the nucleus, thus forming p-ERK1/2.p-ERK1/2 that could interact with a variety of transcription factors in the nucleus, thereby promoting the expression of the oncogene expression of.ERK1/2. There is a significant correlation between the occurrence and development of malignant tumors. Objective to investigate the clinical significance of the expression of ACY-1 in colorectal cancer tissue, and to study the mechanism of its role in the proliferation, apoptosis, invasion and metastasis of colorectal cancer cells, and to explore the occurrence and development of ACY-1 and colorectal cancer. The correlation of the exhibition lay the foundation for finding new biomarkers and drug targets. Methods (1) the samples of 132 colorectal tissues were collected from the gastrointestinal surgery of the Provincial Hospital Affiliated to Shandong University from June 2015 to June 2016, and were divided by sex. Among them, 70 cases were male. Women were 62 cases; in accordance with age, 57 cases were 50 years old and 75 cases were 50 years old. According to whether lymph node metastasis was divided, 52 cases were metastasize and 80 cases were not metastasize; TNM stage was divided into 44 cases, 62 cases in stage II, and 26 cases of stage III + IV, and another 120 healthy volunteers serum as the same. The expression of ACY-1 mRNA and its protein were detected by fluorescence quantitative PCR, Western blot and ELISA, and the correlation with sex, age, TNM stage and lymph node metastasis of the patients was analyzed by fluorescence quantitative PCR, Western and ELISA. (2) the lentivirus carrier with siRNA-ACY-1 gene was carried. 8-EGFP-ACY-1 transfected to human 293T cells and infected human colon cancer HT29 cells after the virus was packaged. In order to construct a ACY-1 overexpressed HT29 cell line, the transfection efficiency of human 293T cells was observed by inverted fluorescence microscopy. The expression of ACY-1 mRNA in the HT29 cells inhibited by ACY-1 expression was detected by fluorescence quantitative PCR. The cell method was used to detect the invasion ability of the cell line. Flow cytometry was used to determine the cell apoptosis and CCK-8 assay was used to detect the cell proliferation activity. (3) HT-29 cells were transfected by siRNA-NC and siRNA-ACY-1, and the expression of p-ERK1/2 and TGF- beta 1 protein in human colon cancer cell HT29 cells expressed by ACY-1 was detected by Western blot respectively. Fruit (1) fluorescence quantitative PCR detection showed that the expression of ACY-1 mRNA in the corresponding normal tissue (control group) was 0.324 + 0.012, and the expression in colorectal cancer tissues was 0.614 + 0.051, which was significantly higher than that of the control group (P0.05).Western blot detection results showed that the expression of ACY-1 egg white in the normal tissues adjacent to the cancer was 0.231 + 0.065. The expression in colorectal cancer tissue was 0.646 + 0.038, which was significantly higher than that of the control group (P0.05).ELISA detection results showed that the content of ACY-1 in the normal tissues adjacent to the cancer was 36.88 + 5.65 pg/mL, the content of the serum in the patients with colorectal cancer was 57.11 + 6.32pg/mL, obviously higher than that of the healthy volunteers (P0.05). The expression of ACY-1 and the clinical cases of colorectal cancer patients The correlation of the parameters was analyzed. The results showed that there was no significant correlation between the expression of ACY-1 and the sex and age of colorectal cancer patients (P0.05), but it was related to TNM staging and lymph node metastasis (P0.05). (2) the results of fluorescence quantitative PCR showed that the expression of ACY-1 mRNA in the constructed ACY-1 inhibited expression of HT29 cells was significantly lower. In the control group, the results of the successful construction of the ACY-1 inhibition expression HT29 cell line.CCK-8 showed that the proliferation activity of the cells after siRNA-ACY-1 transfection was significantly lower than that of the control group (P0.01). The results of flow cytometry showed that the number of apoptotic cells of the HT29 cells expressed by ACY-1 was significantly higher than that of the control group (P0.01).Transwell detection results. The invasion ability of HT29 cells after siRNA-ACY-1 infection was significantly lower than that of the control group (P0.05). (3) the results of Western blot assay showed that the relative expression of ACY-1 protein in the control group was 0.78 + 0.11, and the siRNA-ACY-1 group was 0.16 + 0.04. compared with the control group, the amount of the ACY-1 protein in the siRNA-ACY-1 group was significantly lower than that of the control group (P0.01). The control group p-ERK1/2 eggs The relative expression of white was 0.89 + 0.13, TGF- beta 1 protein was 0.78 + 0.14, the relative expression of p-ERK1/2 protein in siRNA-ACY-1 group was 0.33 + 0.09, TGF- beta 1 protein was 0.21 + 0.06. compared with the control group, and the expression of TGF- beta 1 protein was significantly decreased, and the difference was statistically significant (P0.05). Conclusion ACY-1 is high in colorectal cancer tissue. Expression, and its expression is closely related to TNM staging and lymph node metastasis in colorectal cancer patients,.ACY-1 can regulate the proliferation, invasion and apoptosis of colorectal cancer cells by direct regulation of TGF- beta -ERK1/2 signaling pathway.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.34

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