發(fā)布時(shí)間：2018-06-08 20:01

  本文選題：胃癌 + HoxC10　； 參考：《浙江大學(xué)》2017年博士論文

【摘要】：研究背景與目的同源盒(homeobox,Hox)基因家族分為A、B、C和D四簇,排列在一條同源DNA鏈上,其編碼的同源蛋白是轉(zhuǎn)錄因子,通常在轉(zhuǎn)錄水平調(diào)控靶基表達(dá)。若Hox基因異常表達(dá),會(huì)導(dǎo)致個(gè)體發(fā)育和組織器官形成過(guò)程中出現(xiàn)異常,并可能導(dǎo)致細(xì)胞惡性轉(zhuǎn)化形成腫瘤。目前HoxC10與腫瘤的相關(guān)研究報(bào)道較少。有研究發(fā)現(xiàn)HoxC10在乳腺癌原發(fā)灶中表達(dá)上調(diào),化療失敗后遠(yuǎn)處轉(zhuǎn)移灶中的HoxC10表達(dá)水平升高更明顯,HoxC10的高表達(dá)與接受化療的乳腺癌(雌激素受體陰性)患者的預(yù)后不良相關(guān)。另一項(xiàng)研究發(fā)現(xiàn)HoxC10在宮頸癌組織中呈現(xiàn)高表達(dá),并與宮頸癌細(xì)胞的侵襲轉(zhuǎn)移密切相關(guān)。迄今為止,HoxC10影響腫瘤生物學(xué)行為的具體分子機(jī)制尚未闡明,也無(wú)HoxC10在胃癌中作用的相關(guān)研究報(bào)道。胃癌是全球高發(fā)的惡性腫瘤之一。目前認(rèn)為胃癌的發(fā)生發(fā)展是多因素多階段的過(guò)程,涉及一系列癌基因激活和抑癌基因失活等分子變化。尋找胃癌發(fā)生發(fā)展的關(guān)鍵分子及其相互關(guān)系,對(duì)于進(jìn)一步闡明胃癌的發(fā)病機(jī)制,提供更加精確的臨床預(yù)測(cè)指標(biāo)和治療方案具有重要的意義。我們前期利用胃癌組織基因芯片篩選,首次發(fā)現(xiàn)HoxC10在胃癌組織中較癌旁組織高表達(dá)。為了闡明HoxC10在胃癌中的功能和分子作用機(jī)制,我們做了進(jìn)一步研究。研究?jī)?nèi)容和方法1、HoxC10在胃癌組織中的表達(dá)及臨床意義:通過(guò)對(duì)來(lái)自浙江大學(xué)附屬邵逸夫醫(yī)院和浙一醫(yī)院的2個(gè)胃癌臨床隊(duì)列進(jìn)行檢測(cè),分析HoxC10在胃癌和相應(yīng)癌旁非腫瘤組織中的表達(dá)水平差異及其與胃癌患者臨床病理參數(shù)間的相關(guān)性;運(yùn)用The Cancer Genome Atlas(TCGA)癌癥基因信息數(shù)據(jù)庫(kù)、KM-plotter生存分析數(shù)據(jù)庫(kù)進(jìn)行佐證,明確HoxC10在胃癌發(fā)生發(fā)展中的臨床意義。2、HoxC10對(duì)胃癌細(xì)胞生物學(xué)行為的影響:改變HoxC10的表達(dá)(過(guò)表達(dá)或敲減)并結(jié)合體外細(xì)胞功能試驗(yàn)和體內(nèi)裸鼠胃癌移植瘤動(dòng)物模型,研究HoxC10在胃癌惡性生物學(xué)行為(細(xì)胞增殖、周期調(diào)控、侵襲和遷移)中的作用。3、HoxC10潛在下游靶基因的篩選及驗(yàn)證:采用表達(dá)譜基因芯片技術(shù),篩選HoxC10基因潛在的下游靶基因并采用實(shí)時(shí)熒光定量PCR進(jìn)行驗(yàn)證;結(jié)合表達(dá)改變情況和轉(zhuǎn)錄因子結(jié)合位點(diǎn)信息學(xué)預(yù)測(cè),篩選出p21進(jìn)一步研究。4、HoxC10在p21轉(zhuǎn)錄調(diào)控中的作用及分子機(jī)制研究:通過(guò)改變HoxC10的表達(dá),結(jié)合熒光定量PCR、免疫印跡等技術(shù),在細(xì)胞、組織水平驗(yàn)證HoxC10和p21的表達(dá)相關(guān)性;免疫印跡技術(shù)檢測(cè)在敲減HoxC10后,p21下游周期相關(guān)信號(hào)通路關(guān)鍵分子的表達(dá)改變;利用應(yīng)用雙熒光素酶報(bào)告基因檢測(cè)、染色質(zhì)免疫共沉淀等技術(shù),探討并揭示胃癌HoxC10對(duì)p21的轉(zhuǎn)錄抑制分子調(diào)控機(jī)制。研究結(jié)果1、HoxC10在胃癌組織中的表達(dá)及臨床意義(1)新鮮冰凍胃癌及其配對(duì)癌旁非腫瘤組織樣本(n=70),通過(guò)實(shí)時(shí)熒光定量PCR技術(shù)發(fā)現(xiàn)HoxC10在胃癌組織中的mRNA表達(dá)水平顯著高于癌旁組織(高表達(dá)比例為 91.43%,64/70,P0.01)。免疫印跡法(Westernblotting)檢測(cè) HoxC10 的蛋白表達(dá),證實(shí)了較癌旁配對(duì)非腫瘤組織,胃癌組織中HoxC10的蛋白水平亦明顯增高。分析HoxC10的表達(dá)水平與患者臨床病理特征間的相關(guān)性,發(fā)現(xiàn)HoxC10的表達(dá)水平與腫瘤大小、浸潤(rùn)深度、淋巴結(jié)轉(zhuǎn)移及腫瘤分期顯著相關(guān)(P0.01)。(2)胃癌組織石蠟標(biāo)本,定制組織芯片。通過(guò)免疫組化分析發(fā)現(xiàn)HoxC10在胃癌組織中普遍高表達(dá)(91.3%,137/150,P0.01),HoxC10的蛋白表達(dá)與患者性別、腫瘤浸潤(rùn)深度及腫瘤分期顯著相關(guān)(n=195,P0.01)。Kaplan-Meier生存分析發(fā)現(xiàn),胃癌組織中HoxC 10高表達(dá)的患者預(yù)后較差,風(fēng)險(xiǎn)比為2.223(95%CI 1.361-4.186)。多因素COX回歸分析,發(fā)現(xiàn)HoxC10的高表達(dá)是胃癌預(yù)后差的一個(gè)獨(dú)立預(yù)測(cè)因素。(3)比對(duì)分析TCGA數(shù)據(jù)庫(kù)胃癌和癌旁組織中的差異基因,發(fā)現(xiàn)胃癌組織中HoxC10表達(dá)上調(diào)高達(dá)122倍(n=33,P0.01)。在KM-plotter胃癌數(shù)據(jù)庫(kù)(n=876)中,HoxC10高表達(dá)的患者預(yù)后差,風(fēng)險(xiǎn)比為1.8(95%C1 1.5-2.16)。2、HoxC10對(duì)胃癌細(xì)胞生物學(xué)行為的影響(1)CCK8、克隆形成等細(xì)胞增殖實(shí)驗(yàn),發(fā)現(xiàn)過(guò)表達(dá)HoxC10顯著促進(jìn)胃癌細(xì)胞的增殖,而干擾HoxC10則抑制細(xì)胞的增殖。(2)采用流式細(xì)胞周期檢測(cè),發(fā)現(xiàn)過(guò)表達(dá)HoxC10后胃癌細(xì)胞周期G1-S期轉(zhuǎn)換明顯加快,干擾HoxC10后細(xì)胞阻滯在G1期。應(yīng)用細(xì)胞周期同步化藥物諾考達(dá)唑(Nocodazole)處理細(xì)胞,進(jìn)一步證明敲減HoxC10使胃癌細(xì)胞周期阻滯在G1期。(3)采用雙重染色流式細(xì)胞凋亡檢測(cè),發(fā)現(xiàn)過(guò)表達(dá)HoxC10抑制胃癌細(xì)胞的凋亡,而敲減HoxC10的表達(dá)則促進(jìn)細(xì)胞凋亡。(4)Transwell試驗(yàn)表明過(guò)表達(dá)HoxC10能夠顯著促進(jìn)胃癌細(xì)胞的遷移、侵襲能力,而HoxC10表達(dá)下調(diào)則抑制細(xì)胞的遷移、侵襲。(5)胃癌皮下移植瘤模型,發(fā)現(xiàn)過(guò)表達(dá)HoxC10后裸鼠瘤體的生長(zhǎng)較對(duì)照組明顯增快,而干擾HoxC10可減慢瘤體的生長(zhǎng)速度。3、HoxC10潛在下游靶基因的篩選及驗(yàn)證(1)在胃癌細(xì)胞BGC中敲減HoxC10并做表達(dá)譜基因芯片分析,以差異倍數(shù)1.3為界,并結(jié)合差異基因的功能分析,我們篩選出一系列差異表達(dá)的mRNA,如ELF5、PRDM2、AKT3、CDKN1A(p21)、SLC39A10、ZEB1、NRP2 等。(2)采用實(shí)時(shí)熒光定量PCR技術(shù)在胃癌細(xì)胞中進(jìn)行驗(yàn)證。其中PRDM2、AKT3和p21等在敲減HoxC10的胃癌細(xì)胞株中顯著上調(diào),ZEB1、NRP2等顯著下調(diào)(P0.05)。(3)借助Genomatix和JASPAR兩個(gè)在線轉(zhuǎn)錄因子結(jié)合位點(diǎn)預(yù)測(cè)數(shù)據(jù)庫(kù),分析發(fā)現(xiàn)HoxC10與p21啟動(dòng)子區(qū)存在多個(gè)可能的結(jié)合位點(diǎn)。4、HoxC10在p21轉(zhuǎn)錄調(diào)控中的作用及分子機(jī)制研究(1)在多株胃癌細(xì)胞中改變(過(guò)表達(dá)和敲減)HoxC10的表達(dá),發(fā)現(xiàn)過(guò)表達(dá)HoxC10,p21的表達(dá)水平相對(duì)下調(diào),而敲減HoxC10后,p21的表達(dá)相對(duì)上調(diào);在裸鼠胃癌移植瘤組織中,免疫組化分析HoxC10和p21的表達(dá)情況,發(fā)現(xiàn)二者呈負(fù)相關(guān);在穩(wěn)定敲減HoxC10的胃癌細(xì)胞中,p21及其下游周期相關(guān)信號(hào)通路的關(guān)鍵分子CDK2、CDK4、c-Myc、CyclinD1、pRb 等的表達(dá)發(fā)生改變。(2)采用雙熒光素酶報(bào)告基因技術(shù),分析HoxC10表達(dá)改變對(duì)p21轉(zhuǎn)錄活性的影響。結(jié)果發(fā)現(xiàn)過(guò)表達(dá)HoxC10明顯抑制p21的轉(zhuǎn)錄活性,而干擾HoxC10的表達(dá),p21的轉(zhuǎn)錄活性增強(qiáng)。(3)設(shè)計(jì)針對(duì)p21基因啟動(dòng)子區(qū)的引物,采用染色質(zhì)免疫共沉淀技術(shù),進(jìn)一步鑒定發(fā)現(xiàn)HoxC10與P21啟動(dòng)子區(qū)存在結(jié)合位點(diǎn)。研究結(jié)論1、在胃癌組織中HoxC10普遍高表達(dá),且與腫瘤浸潤(rùn)深度、腫瘤分期及臨床預(yù)后相關(guān),HoxC10高表達(dá)是胃癌預(yù)后差的一個(gè)獨(dú)立預(yù)測(cè)因素。2、HoxC10可調(diào)控G1細(xì)胞周期、抑制胃癌細(xì)胞凋亡,促進(jìn)胃癌細(xì)胞增殖和裸鼠移植瘤形成,促進(jìn)胃癌細(xì)胞遷移和侵襲能力,從而發(fā)揮促癌基因的作用。3、篩選發(fā)現(xiàn)系列HoxC1O可能調(diào)控的下游重要腫瘤相關(guān)基因,其中HoxC10能與p21的啟動(dòng)子區(qū)結(jié)合并抑制其轉(zhuǎn)錄活性,影響其下游周期相關(guān)信號(hào)分子的表達(dá),從而影響胃癌細(xì)胞的增殖和周期調(diào)控。
[Abstract]:Background and purpose homeobox (Hox) gene family is divided into four clusters of A, B, C and D, arranged on a homologous DNA chain. The encoded homologous protein is a transcription factor, which usually regulates the target expression at the transcriptional level. If the Hox gene is abnormal expression, it can lead to abnormal development and tissue formation, and may lead to cells. The malignant transformation forms tumor. There are few reports about HoxC10 and tumor. Some studies have found that the expression of HoxC10 in the primary breast cancer is up to up, and the level of HoxC10 expression in the distant metastasis is more obvious after the failure of chemotherapy, and the high expression of HoxC10 is associated with the poor prognosis of the patients with breast cancer receiving chemotherapy (estrogen receptor negative). Another study found that HoxC10 is highly expressed in cervical cancer and is closely related to the invasion and metastasis of cervical cancer cells. So far, the specific molecular mechanism of HoxC10's effects on tumor biological behavior has not been elucidated, and there is no related research on the role of HoxC10 in gastric cancer. The development of gastric cancer is a multi factor and multi stage process, involving a series of molecular changes, such as oncogene activation and inactivation of tumor suppressor genes. It is of great significance to find out the key molecules and their relationship in the development of gastric cancer. It is of great significance to further clarify the pathogenesis of gastric cancer and to provide more accurate predictors and treatment schemes for the pathogenesis of gastric cancer. We first found the high expression of HoxC10 in gastric cancer tissue by using the gene chip of gastric cancer tissue. In order to clarify the function and molecular mechanism of HoxC10 in gastric cancer, we have done further research. 1, the expression and clinical significance of HoxC10 in gastric cancer tissue: through the Zhejiang University 2 clinical cohort of gastric cancer in the affiliated Sir Run Run Shaw Hospital and Zhejiang Yi hospital were tested to analyze the difference in the expression level of HoxC10 in gastric cancer and the non cancer tissue adjacent to the corresponding cancer and the correlation with the clinicopathological parameters of the patients with gastric cancer; the database of The Cancer Genome Atlas (TCGA) cancer gene information and the KM-plotter survival analysis database To confirm the clinical significance of HoxC10 in the development of gastric cancer,.2, the effect of HoxC10 on the biological behavior of gastric cancer cells: to change the expression of HoxC10 (over expression or knock down) and to combine the cell function test in vitro and the animal model of nude mice with gastric cancer transplantation in vivo to study the malignant biological behavior (cell proliferation and cycle regulation) of HoxC10 in gastric cancer. The screening and validation of.3, HoxC10 potential downstream target genes in invasion and migration: using gene chip technology, screening potential downstream target genes of HoxC10 gene and using real-time fluorescence quantitative PCR to verify the target gene, combined with the expression change and the information prediction of the transcription factor binding site, and screening the p21 to further study.4, HoxC10 Study on the role and molecular mechanism of p21 transcriptional regulation: by changing the expression of HoxC10, combining fluorescence quantitative PCR, Western blot, and other techniques, the expression of HoxC10 and p21 is verified at the cell and tissue level; the expression of key molecules of the cycle related signaling pathway in the downstream of p21 is altered by immunoblotting technology. Double luciferase reporter gene detection, chromatin immunoprecipitation and other techniques to explore and reveal the regulatory mechanism of HoxC10 on p21 in gastric cancer. Results 1, the expression and clinical significance of HoxC10 in gastric cancer tissue and its clinical significance (1) fresh frozen gastric cancer and its paired non tumor tissue samples (n=70), by real-time fluorescence quantitative PCR Technology The expression of mRNA in gastric cancer tissues was significantly higher than that of para cancerous tissue (high expression ratio was 91.43%, 64/70, P0.01). The protein expression of HoxC10 was detected by immunoblotting (Westernblotting), which proved that the level of the egg white of HoxC10 in gastric cancer tissues was also significantly higher than that of non cancerous tissues. The expression level of HoxC10 was analyzed and the expression level of HoxC10 was analyzed. The correlation between the clinicopathological features of the patients was found to be significantly associated with the size of the tumor, depth of invasion, lymph node metastasis and tumor staging (P0.01). (2) the paraffin specimens of the gastric carcinoma and the custom tissue microarray. The high expression of HoxC10 in gastric cancer tissues (91.3%, 137/150, P0.01), and HoxC10 protein was found by immunohistochemical analysis. N=195, P0.01.Kaplan-Meier survival analysis showed that the prognosis of patients with high expression of HoxC 10 in gastric carcinoma was poor and the risk ratio was 2.223 (95%CI 1.361-4.186). The high expression of HoxC10 was an independent predictor of poor prognosis of gastric cancer (3). (3 Compared with the differential genes in the gastric cancer and adjacent tissues of the TCGA database, it was found that the expression of HoxC10 in gastric cancer tissues was up to 122 times (n=33, P0.01). In the KM-plotter gastric cancer database (n=876), the prognosis of the patients with high expression of HoxC10 was poor, the risk ratio was 1.8 (95%C1 1.5-2.16).2, and HoxC10 on the biological behavior of gastric cancer cells (1) CCK8, cloned It was found that the expression of HoxC10 significantly promoted the proliferation of gastric cancer cells, while interference with HoxC10 inhibited the proliferation of cells. (2) the flow cytometry was used to detect the G1-S phase transition of gastric cancer cell cycle after HoxC10 expression, and HoxC10 cells were blocked at G1 phase after HoxC10. Nocodazole treated cells further proved that knockout HoxC10 prevented the cell cycle of gastric cancer in G1 phase. (3) the expression of HoxC10 inhibited the apoptosis of gastric cancer cells by double staining flow cytometry, and the expression of knock down HoxC10 promoted apoptosis. (4) Transwell test showed that overexpression of HoxC10 could significantly promote gastric cancer. Cell migration, invasion ability, and HoxC10 expression downregulation inhibited cell migration and invasion. (5) subcutaneous transplantation of gastric cancer model, found that the growth of nude mice after expression of HoxC10 was significantly faster than the control group, while interference HoxC10 slowed down the growth rate of the tumor body.3, HoxC10 potential downstream target gene screening and verification (1) in gastric cancer cell BGC By subtraction HoxC10 and gene chip analysis, the difference multiplier 1.3 was bounded, and the differential gene functional analysis was combined. We screened a series of differentially expressed mRNA, such as ELF5, PRDM2, AKT3, CDKN1A (p21), SLC39A10, ZEB1, NRP2 and so on. (2) the real time fluorescein quantitative PCR technique was used to verify the gastric cancer cells. ZEB1, NRP2 and so on were significantly up-regulated in the gastric cancer cell line of HoxC10. (3) with the use of two on-line transcription factor binding sites of Genomatix and JASPAR to predict the database, it was found that there were several possible binding sites.4 in HoxC10 and p21 promoter region, and the role and molecular mechanism of HoxC10 in p21 transcription regulation (1) The expression of overexpression and subtracting HoxC10 in multiple gastric cancer cells showed that the expression of HoxC10, the expression level of p21 was relatively down, and the expression of p21 was relatively up-regulated after the knockout of HoxC10. In the tumor tissues of nude mice, immunohistochemical analysis of the expression of HoxC10 and p21 found that the two were negatively correlated, and the gastric cancer was stably subtracting HoxC10. In the cell, the expression of CDK2, CDK4, c-Myc, CyclinD1, pRb, and other key molecules of the p21 and its downstream related signaling pathways changed. (2) the effect of HoxC10 expression changes on the transcriptional activity of p21 was analyzed by double luciferase reporter gene technique. The results showed that the expression of HoxC10 clearly inhibited the transcriptional activity of p21, but interfered with HoxC10 expression, p21. Transcriptional activity enhancement. (3) design primers for the promoter region of p21 gene, use chromatin immunoprecipitation technique to further identify the binding site of HoxC10 and P21 promoter region. Conclusion 1, high expression of HoxC10 in gastric cancer tissues, and related to tumor invasion depth, tumor staging and clinical prognosis, HoxC10 high expression An independent predictor of poor prognosis of gastric cancer,.2, HoxC10 can regulate the cell cycle of G1 cells, inhibit the apoptosis of gastric cancer cells, promote the proliferation of gastric cancer cells and the formation of nude mice, promote the migration and invasion of gastric cancer cells, thus play the role of the cancer promoting gene.3, and screen the important tumor related genes that may be regulated by a series of HoxC1O, which may be regulated by HoxC1O. HoxC10 can bind to the promoter region of p21 and inhibit its transcriptional activity, and affect the expression of signal molecules in its downstream cycle, thus affecting the proliferation and cycle regulation of gastric cancer cells.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.2

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