HGF在SMAD4缺失的结直肠癌细胞中对Mcl-1的表达调控机制

发布时间：2018-07-09 19:39

本文选题：结直肠癌 + HGF　；参考：《吉林大学》2017年硕士论文

【摘要】：结直肠癌(CRC)在男性和女性肿瘤患者中的发病率分别居于第三和第二位,约30%的病人接受手术治疗后,仍会发生转移。由此,进一步研究结直肠癌发生发展的分子机制,提出更加有效的治疗方法十分必要。研究证明,HGF/c-Met通路与结直肠癌的发生与发展密切相关。HGF是一种在细胞增殖、存活、运动和形态中有重要作用的多效性生长因子,由间质细胞分泌,是酪氨酸激酶c-Met的特异性配体。c-Met表达于各种类型上皮细胞、内皮细胞、原始造血细胞等细胞的表面。HGF/c-Met通路主要与胚胎形成、器官形成、成人的组织修复以及肿瘤,尤其实体性肿瘤密切相关。在c-Met过表达的几种肿瘤细胞包括结直肠癌细胞中,c-Met与其特异性配体结合后促进信号级联,介导了细胞的增殖、抗凋亡功能,细胞的离散以及肿瘤细胞的迁移、侵袭和转移。相关临床研究证明,经单因素分析发现HGF和c-Met均为阳性的结直肠癌患者,平均生存期更短。因此这种特异性配体受体结合关系,使HGF/c-Met通路成为研究抗肿瘤治疗的理想靶点。SMAD4是TGF-信号通路的重要组成部分。研究表明,SMAD4在与免疫系统、基质、上皮的相互作用中起到重要而复杂的作用,而阻断SMAD4的表达,可促进结直肠癌的发生。并且SMAD4的缺失与晚期结直肠癌及其转移有关。临床研究证实,在家族性青少年息肉病(FJP)患者中已经鉴定出SMAD4和BMPR1A基因的突变,并且约占FJP病例的50%。SMAD4缺失除了增加发生结直肠癌的风险之外,在散发性CRC中也发现有高比例的SMAD4突变。且在具有微卫星不稳定性(MSI)和染色体不稳定性的肿瘤中,也有SMAD4突变的发现。因此,SMAD4是结直肠癌研究中的重要靶基因之一。据文献报道,HGF能转录介导抗凋亡蛋白Mcl-1的表达,相反,HGF不会改变Bcl-2及Bcl-x L的表达水平。Mcl-1属于Bcl-2抗凋亡蛋白家族的一员,可作为上游分子调控凋亡,通过线粒体释放细胞色素C等一系列级联事件来进行早期干预从而促进细胞存活。并且在几种类型肿瘤中,Bcl-2和Mcl-1蛋白的表达水平上调被视为发生抗失巢凋亡。新近研究表明,失巢凋亡的破坏对恶性乳腺癌和结直肠癌有显著的促进作用。并且,Mcl-1稳定表达和抑制Bim表达可作为抑制肿瘤细胞失巢凋亡的关键事件。此前的研究中,多为研究单一因素变化对肿瘤细胞的作用,本研究探讨在SMAD4突变的背景下,HGF诱导Mcl-1表达情况的分子机制,及Mcl-1的表达情况与失巢凋亡的相关性。研究证明,根据细胞类型的不同,HGF可能通过ERK、Akt或STAT信号通路调控Mcl-1的表达,且有文献报道,失巢凋亡与ERK和Akt信号通路有相关性。因此,我们在此研究基础上探讨HGF在SMAD4缺失的CRC细胞中调控Mcl-1表达的分子机制。基于以上研究和发现,本研究共计收集52例结直肠癌组织及40例癌旁组织,应用免疫组织化学方法检测肿瘤组织及癌旁组织中SAMD4与Mcl-1表达情况。并选取了四种结直肠癌细胞,即SMAD4野生型DLD-1、HCT15细胞,SMAD4突变的SW620、HT-29细胞,鉴定SMAD4的表达情况后,应用慢病毒转染的方法,使SMAD4在SW620、HT-29细胞中过表达,并获得稳定的细胞株。分别在蛋白及基因水平上对HGF诱导Mcl-1的表达水平进行检测,并对其分子机制进行探索。同时,应用24孔细胞失巢凋亡能力检测试剂盒检测HGF的刺激及SMAD4的表达情况对结直肠癌细胞失巢凋亡能力的影响。旨在为预防、延缓结直肠癌发生转移,提供新思路。目的本研究以SMAD4突变为背景,探究HGF在CRC细胞中诱导Mcl-1表达的作用和机制,及其对CRC细胞失巢凋亡能力的影响。方法1.应用免疫组织化学染色的方法,检测SMAD4、Mcl-1蛋白在结直肠癌及癌旁组织中的表达水平;2.应用Western Blot的方法检测,经HGF孵育后四种细胞Mcl-1的表达水平,应用慢病毒转染使SMAD4在SW620和HT29细胞过表达,Western Blot及RT-q PCR鉴定SMAD4表达水平,并检测经HGF刺激后,空载组及过表达组细胞Mcl-1的表达情况;3.应用CBA-080 Cyto Select 24-well Anoikis Assay试剂盒,检测各组细胞失巢凋亡率;4.应用Western Blot方法检测在四种野生型细胞(DLD-1、HCT15、SW620|、HT29)中,HGF诱导ERK、Akt蛋白的磷酸化水平;5.应用Western Blot方法检测转染前后SW620及HT29细胞ERK、Akt蛋白的磷酸化水平,并加入特异性抑制剂U0126、LY294002,检测加入通路抑制剂后Mcl-1的表达情况,由此确定HGF是通过ERK还是Akt信号通路来调控Mcl-1的表达。结果1.SMAD4在细胞胞浆、胞核呈阳性着色,且在癌旁组织中阳性率较高,Mcl-1在细胞胞浆呈阳性着色,且在结直肠癌组织中阳性率较高;2.HGF在表达SMAD4的DLD-1、HCT15细胞中对Mcl-1的表达没有显著影响,在不表达SMAD4的SW620、HT29细胞中能使Mcl-1的表达水平上调。经慢病毒载体转染获得了过表达SMAD4的SW620和HT29稳定细胞系。HGF可诱导空载SW620及HT29细胞中的Mcl-1表达水平上调,对SMAD4过表达的细胞则无显著作用;3.SMAD4缺失的SW620、HT29细胞失巢凋亡率明显降低;4.Western Blot检测发现,在表达SMAD4的DLD-1、HCT15的细胞中,HGF诱导ERK、Akt蛋白磷酸化水平低于SMAD4不表达的SW620、HT29细胞;5.Western Blot检测发现,HGF均能使空载及SMAD4过表达的SW620及HT29细胞的ERK、Akt蛋白磷酸化,且空载细胞的磷酸化水平明显高于SMAD4过表达细胞。野生型SW620和HT29细胞中加入ERK通路特异性抑制剂U0126后ERK通路磷酸化被特异性抑制,Mcl-1不表达,加入Akt通路抑制剂LY294002后Akt通路磷酸化被特异性抑制,而Mcl-1仍表达。结论1.Mcl-1在结直肠癌组织中高表达,癌旁组织中低表达;SMAD4在癌旁组织中高表达,肿瘤组织中低表达;HGF在CRC细胞中诱导Mcl-1的表达受SMAD4表达情况的影响;2.HGF可促进SMAD4缺失的结直肠癌细胞抗失巢凋亡能力增强;3.HGF在SMAD4缺失的结直肠癌细胞中是通过ERK信号通路而不是Akt信号通路来调控Mcl-1的表达。
[Abstract]:The incidence of colorectal cancer (CRC) in both male and female tumor patients is third and second respectively, and about 30% of the patients will still be transferred after surgical treatment. Therefore, it is necessary to further study the molecular mechanism of the development of colorectal cancer and to put forward more effective therapeutic methods. Research has proved that the HGF/c-Met pathway and colorectal cancer have been proved to be necessary. The development of cancer is closely related to the development of.HGF, a pleiotropic growth factor that plays an important role in cell proliferation, survival, motion and morphology. It is secreted by interstitial cells and is a specific ligand of tyrosine kinase c-Met,.C-Met expressed in the surface.HGF/c-Met pathway of various types of epithelial cells, endothelial cells, primitive hematopoietic cells and other cells. It is closely related to embryo formation, organ formation, adult tissue repair and tumor, especially solid tumors. In c-Met overexpressed tumor cells including colorectal cancer cells, c-Met promotes signal cascade after combining with its specific ligands and mediates cell proliferation, anti apoptosis, cell dispersion and tumor cells Migration, invasion and metastasis. Related clinical studies have shown that the average survival time of HGF and c-Met patients with positive colorectal cancer is shorter by single factor analysis. Therefore, this specific ligand receptor binding relationship makes the HGF/c-Met pathway an ideal target for the study of anti-tumor therapy,.SMAD4 is an important part of the TGF- signaling pathway. It shows that SMAD4 plays an important and complex role in the interaction with the immune system, matrix and epithelium, and blocking the expression of SMAD4 can promote the occurrence of colorectal cancer. And the absence of SMAD4 is associated with advanced colorectal cancer and its metastasis. Clinical studies have confirmed that SMAD4 and BMPR1 have been identified in familial juvenile polyposis (FJP) patients. The mutation of the A gene and the 50%.SMAD4 deletion in FJP cases, in addition to increasing the risk of colorectal cancer, also found a high proportion of SMAD4 mutations in sporadic CRC. And in tumors with microsatellite instability (MSI) and chromosome instability, there are also the discoveries of SMAD4 mutagenesis. Therefore, SMAD4 is a study of colorectal cancer. One of the important target genes. According to the literature, HGF can transcribe and mediate the expression of anti apoptotic protein Mcl-1. On the contrary, HGF does not change the expression level of Bcl-2 and Bcl-x L,.Mcl-1 belongs to a member of the anti apoptotic protein family of Bcl-2, and can be used as an upstream molecule to regulate apoptosis and to intervene early through a series of cascading events such as mitochondria release of fine cytochrome C. In several types of tumors, the up regulation of Bcl-2 and Mcl-1 protein expression is considered to be the occurrence of anti nesting apoptosis. Recent studies have shown that the destruction of anoikis has a significant promoting effect on malignant breast cancer and colorectal cancer. And the stable expression of Mcl-1 and the inhibition of Bim expression can be used to inhibit the loss of tumor cells. The key events of nesting apoptosis. In previous studies, most of the effects of single factor changes on tumor cells were studied. This study explored the molecular mechanism of HGF induced Mcl-1 expression in the background of SMAD4 mutation, and the correlation between the expression of Mcl-1 and the loss of nesting apoptosis. The study shows that, according to the different cell types, HGF may pass ERK, Akt The STAT signaling pathway regulates the expression of Mcl-1 and reports that anoikis is associated with the ERK and Akt signaling pathways. Therefore, on the basis of this study, we discuss the molecular mechanism of HGF in the regulation of Mcl-1 expression in SMAD4 deleted CRC cells. Based on the above study and discovery, 52 cases of colorectal cancer and 40 cases of para cancer were collected in this study. Tissue, immunohistochemical method was used to detect the expression of SAMD4 and Mcl-1 in tumor tissues and adjacent tissues, and four kinds of colorectal cancer cells, that is, SMAD4 wild type DLD-1, HCT15 cells, SMAD4 mutation SW620, HT-29 cells, identified the expression of SMAD4, used the method of lentivirus transfection, so that SMAD4 in SW620, HT-29 cells over. The expression level of HGF induced Mcl-1 was detected at the protein and gene level, and its molecular mechanism was explored. At the same time, the effect of HGF stimulation and the expression of SMAD4 on the aniapoptotic ability of colorectal cancer cells was detected by the detection kit of 24 hole cell apoptosis ability. In order to prevent and delay the metastasis of colorectal cancer, a new idea is provided. The purpose of this study is to explore the role and mechanism of HGF in inducing the expression of Mcl-1 in CRC cells with the background of SMAD4 mutation and its effect on the ability of CRC cell apoptosis. Method 1. the immunohistochemical staining method was used to detect SMAD4, Mcl-1 protein in colorectal cancer and cancer. Expression level in para tissue; 2. Western Blot was used to detect the expression level of four cells after HGF incubation. SMAD4 was overexpressed in SW620 and HT29 cells by lentivirus transfection. Western Blot and RT-q PCR were used to identify the expression level of SMAD4, and the expression of empty and overexpressed groups was detected; 3. CBA-080 Cyto Select 24-well Anoikis Assay kit was used to detect the apoptosis rate of the cells in each group. 4. the phosphorylation level of four kinds of wild type cells (DLD-1, HCT15, SW620|, HT29) were detected by Western Blot method. The level of acidification and the addition of specific inhibitor U0126, LY294002, to detect the expression of Mcl-1 after the entry inhibitor, thus determine that HGF is regulated by ERK or Akt signaling pathway to regulate the expression of Mcl-1. As a result, 1.SMAD4 is positive in cytoplasm, and the positive rate in the paracellal tissues is higher, and Mcl-1 is positive in cytoplasm. The positive rate in colorectal cancer tissues is high, and 2.HGF has no significant influence on the expression of Mcl-1 in the expression of SMAD4 DLD-1 and HCT15 cells. In HT29 cells that do not express SMAD4 SW620, the expression of Mcl-1 can be up-regulated. The SW620 and HT29 stable cell lines expressing SMAD4 are obtained by transfection of the lentivirus vector. The expression level of Mcl-1 in HT29 cells was up, and there was no significant effect on SMAD4 overexpressed cells; 3.SMAD4 missing SW620 and HT29 cells decreased the rate of nesting apoptosis; 4.Western Blot detection found that HGF induced the SMAD4 DLD-1 and HCT15 cells. Ern Blot detection showed that HGF could make ERK and Akt protein phosphorylation of SW620 and HT29 cells expressed in SW620 and HT29 cells, and that the phosphorylation level of the empty cells was significantly higher than that of the SMAD4 overexpressed cells. The phosphorylation of Akt pathway was specifically inhibited after the KT pathway inhibitor LY294002, while Mcl-1 was still expressed. Conclusion 1.Mcl-1 is highly expressed in the colorectal cancer tissue and low expression in the para cancer tissues; SMAD4 is highly expressed in the para cancer tissue and low expression in the tumor tissue; HGF in CRC cells is influenced by the expression of SMAD4 in CRC cells; 2.HGF can promote SMAD. 4 of the missing colorectal cancer cells increased the ability to resist anoikic apoptosis, and 3.HGF regulated the expression of Mcl-1 through the ERK signaling pathway instead of the Akt signaling pathway in the SMAD4 missing colorectal cancer cells.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34

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