本文選題：結(jié)直腸癌 + HGF��； 參考：《吉林大學》2017年碩士論文

【摘要】：結(jié)直腸癌(CRC)在男性和女性腫瘤患者中的發(fā)病率分別居于第三和第二位,約30%的病人接受手術(shù)治療后,仍會發(fā)生轉(zhuǎn)移。由此,進一步研究結(jié)直腸癌發(fā)生發(fā)展的分子機制,提出更加有效的治療方法十分必要。研究證明,HGF/c-Met通路與結(jié)直腸癌的發(fā)生與發(fā)展密切相關(guān)。HGF是一種在細胞增殖、存活、運動和形態(tài)中有重要作用的多效性生長因子,由間質(zhì)細胞分泌,是酪氨酸激酶c-Met的特異性配體。c-Met表達于各種類型上皮細胞、內(nèi)皮細胞、原始造血細胞等細胞的表面。HGF/c-Met通路主要與胚胎形成、器官形成、成人的組織修復(fù)以及腫瘤,尤其實體性腫瘤密切相關(guān)。在c-Met過表達的幾種腫瘤細胞包括結(jié)直腸癌細胞中,c-Met與其特異性配體結(jié)合后促進信號級聯(lián),介導(dǎo)了細胞的增殖、抗凋亡功能,細胞的離散以及腫瘤細胞的遷移、侵襲和轉(zhuǎn)移。相關(guān)臨床研究證明,經(jīng)單因素分析發(fā)現(xiàn)HGF和c-Met均為陽性的結(jié)直腸癌患者,平均生存期更短。因此這種特異性配體受體結(jié)合關(guān)系,使HGF/c-Met通路成為研究抗腫瘤治療的理想靶點。SMAD4是TGF-信號通路的重要組成部分。研究表明,SMAD4在與免疫系統(tǒng)、基質(zhì)、上皮的相互作用中起到重要而復(fù)雜的作用,而阻斷SMAD4的表達,可促進結(jié)直腸癌的發(fā)生。并且SMAD4的缺失與晚期結(jié)直腸癌及其轉(zhuǎn)移有關(guān)。臨床研究證實,在家族性青少年息肉病(FJP)患者中已經(jīng)鑒定出SMAD4和BMPR1A基因的突變,并且約占FJP病例的50%。SMAD4缺失除了增加發(fā)生結(jié)直腸癌的風險之外,在散發(fā)性CRC中也發(fā)現(xiàn)有高比例的SMAD4突變。且在具有微衛(wèi)星不穩(wěn)定性(MSI)和染色體不穩(wěn)定性的腫瘤中,也有SMAD4突變的發(fā)現(xiàn)。因此,SMAD4是結(jié)直腸癌研究中的重要靶基因之一。據(jù)文獻報道,HGF能轉(zhuǎn)錄介導(dǎo)抗凋亡蛋白Mcl-1的表達,相反,HGF不會改變Bcl-2及Bcl-x L的表達水平。Mcl-1屬于Bcl-2抗凋亡蛋白家族的一員,可作為上游分子調(diào)控凋亡,通過線粒體釋放細胞色素C等一系列級聯(lián)事件來進行早期干預(yù)從而促進細胞存活。并且在幾種類型腫瘤中,Bcl-2和Mcl-1蛋白的表達水平上調(diào)被視為發(fā)生抗失巢凋亡。新近研究表明,失巢凋亡的破壞對惡性乳腺癌和結(jié)直腸癌有顯著的促進作用。并且,Mcl-1穩(wěn)定表達和抑制Bim表達可作為抑制腫瘤細胞失巢凋亡的關(guān)鍵事件。此前的研究中,多為研究單一因素變化對腫瘤細胞的作用,本研究探討在SMAD4突變的背景下,HGF誘導(dǎo)Mcl-1表達情況的分子機制,及Mcl-1的表達情況與失巢凋亡的相關(guān)性。研究證明,根據(jù)細胞類型的不同,HGF可能通過ERK、Akt或STAT信號通路調(diào)控Mcl-1的表達,且有文獻報道,失巢凋亡與ERK和Akt信號通路有相關(guān)性。因此,我們在此研究基礎(chǔ)上探討HGF在SMAD4缺失的CRC細胞中調(diào)控Mcl-1表達的分子機制�；谝陨涎芯亢桶l(fā)現(xiàn),本研究共計收集52例結(jié)直腸癌組織及40例癌旁組織,應(yīng)用免疫組織化學方法檢測腫瘤組織及癌旁組織中SAMD4與Mcl-1表達情況。并選取了四種結(jié)直腸癌細胞,即SMAD4野生型DLD-1、HCT15細胞,SMAD4突變的SW620、HT-29細胞,鑒定SMAD4的表達情況后,應(yīng)用慢病毒轉(zhuǎn)染的方法,使SMAD4在SW620、HT-29細胞中過表達,并獲得穩(wěn)定的細胞株。分別在蛋白及基因水平上對HGF誘導(dǎo)Mcl-1的表達水平進行檢測,并對其分子機制進行探索。同時,應(yīng)用24孔細胞失巢凋亡能力檢測試劑盒檢測HGF的刺激及SMAD4的表達情況對結(jié)直腸癌細胞失巢凋亡能力的影響。旨在為預(yù)防、延緩結(jié)直腸癌發(fā)生轉(zhuǎn)移,提供新思路。目的本研究以SMAD4突變?yōu)楸尘?探究HGF在CRC細胞中誘導(dǎo)Mcl-1表達的作用和機制,及其對CRC細胞失巢凋亡能力的影響。方法1.應(yīng)用免疫組織化學染色的方法,檢測SMAD4、Mcl-1蛋白在結(jié)直腸癌及癌旁組織中的表達水平;2.應(yīng)用Western Blot的方法檢測,經(jīng)HGF孵育后四種細胞Mcl-1的表達水平,應(yīng)用慢病毒轉(zhuǎn)染使SMAD4在SW620和HT29細胞過表達,Western Blot及RT-q PCR鑒定SMAD4表達水平,并檢測經(jīng)HGF刺激后,空載組及過表達組細胞Mcl-1的表達情況;3.應(yīng)用CBA-080 Cyto Select 24-well Anoikis Assay試劑盒,檢測各組細胞失巢凋亡率;4.應(yīng)用Western Blot方法檢測在四種野生型細胞(DLD-1、HCT15、SW620|、HT29)中,HGF誘導(dǎo)ERK、Akt蛋白的磷酸化水平;5.應(yīng)用Western Blot方法檢測轉(zhuǎn)染前后SW620及HT29細胞ERK、Akt蛋白的磷酸化水平,并加入特異性抑制劑U0126、LY294002,檢測加入通路抑制劑后Mcl-1的表達情況,由此確定HGF是通過ERK還是Akt信號通路來調(diào)控Mcl-1的表達。結(jié)果1.SMAD4在細胞胞漿、胞核呈陽性著色,且在癌旁組織中陽性率較高,Mcl-1在細胞胞漿呈陽性著色,且在結(jié)直腸癌組織中陽性率較高;2.HGF在表達SMAD4的DLD-1、HCT15細胞中對Mcl-1的表達沒有顯著影響,在不表達SMAD4的SW620、HT29細胞中能使Mcl-1的表達水平上調(diào)。經(jīng)慢病毒載體轉(zhuǎn)染獲得了過表達SMAD4的SW620和HT29穩(wěn)定細胞系。HGF可誘導(dǎo)空載SW620及HT29細胞中的Mcl-1表達水平上調(diào),對SMAD4過表達的細胞則無顯著作用;3.SMAD4缺失的SW620、HT29細胞失巢凋亡率明顯降低;4.Western Blot檢測發(fā)現(xiàn),在表達SMAD4的DLD-1、HCT15的細胞中,HGF誘導(dǎo)ERK、Akt蛋白磷酸化水平低于SMAD4不表達的SW620、HT29細胞;5.Western Blot檢測發(fā)現(xiàn),HGF均能使空載及SMAD4過表達的SW620及HT29細胞的ERK、Akt蛋白磷酸化,且空載細胞的磷酸化水平明顯高于SMAD4過表達細胞。野生型SW620和HT29細胞中加入ERK通路特異性抑制劑U0126后ERK通路磷酸化被特異性抑制,Mcl-1不表達,加入Akt通路抑制劑LY294002后Akt通路磷酸化被特異性抑制,而Mcl-1仍表達。結(jié)論1.Mcl-1在結(jié)直腸癌組織中高表達,癌旁組織中低表達;SMAD4在癌旁組織中高表達,腫瘤組織中低表達;HGF在CRC細胞中誘導(dǎo)Mcl-1的表達受SMAD4表達情況的影響;2.HGF可促進SMAD4缺失的結(jié)直腸癌細胞抗失巢凋亡能力增強;3.HGF在SMAD4缺失的結(jié)直腸癌細胞中是通過ERK信號通路而不是Akt信號通路來調(diào)控Mcl-1的表達。
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.34

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