HoxC10在胃癌中的作用及其转录调控p21的分子机制研究

发布时间：2018-06-08 20:01

  本文选题：胃癌 + HoxC10　； 参考：《浙江大学》2017年博士论文

【摘要】：研究背景与目的同源盒(homeobox,Hox)基因家族分为A、B、C和D四簇,排列在一条同源DNA链上,其编码的同源蛋白是转录因子,通常在转录水平调控靶基表达。若Hox基因异常表达,会导致个体发育和组织器官形成过程中出现异常,并可能导致细胞恶性转化形成肿瘤。目前HoxC10与肿瘤的相关研究报道较少。有研究发现HoxC10在乳腺癌原发灶中表达上调,化疗失败后远处转移灶中的HoxC10表达水平升高更明显,HoxC10的高表达与接受化疗的乳腺癌(雌激素受体阴性)患者的预后不良相关。另一项研究发现HoxC10在宫颈癌组织中呈现高表达,并与宫颈癌细胞的侵袭转移密切相关。迄今为止,HoxC10影响肿瘤生物学行为的具体分子机制尚未阐明,也无HoxC10在胃癌中作用的相关研究报道。胃癌是全球高发的恶性肿瘤之一。目前认为胃癌的发生发展是多因素多阶段的过程,涉及一系列癌基因激活和抑癌基因失活等分子变化。寻找胃癌发生发展的关键分子及其相互关系,对于进一步阐明胃癌的发病机制,提供更加精确的临床预测指标和治疗方案具有重要的意义。我们前期利用胃癌组织基因芯片筛选,首次发现HoxC10在胃癌组织中较癌旁组织高表达。为了阐明HoxC10在胃癌中的功能和分子作用机制,我们做了进一步研究。研究内容和方法1、HoxC10在胃癌组织中的表达及临床意义:通过对来自浙江大学附属邵逸夫医院和浙一医院的2个胃癌临床队列进行检测,分析HoxC10在胃癌和相应癌旁非肿瘤组织中的表达水平差异及其与胃癌患者临床病理参数间的相关性;运用The Cancer Genome Atlas(TCGA)癌症基因信息数据库、KM-plotter生存分析数据库进行佐证,明确HoxC10在胃癌发生发展中的临床意义。2、HoxC10对胃癌细胞生物学行为的影响:改变HoxC10的表达(过表达或敲减)并结合体外细胞功能试验和体内裸鼠胃癌移植瘤动物模型,研究HoxC10在胃癌恶性生物学行为(细胞增殖、周期调控、侵袭和迁移)中的作用。3、HoxC10潜在下游靶基因的筛选及验证:采用表达谱基因芯片技术,筛选HoxC10基因潜在的下游靶基因并采用实时荧光定量PCR进行验证;结合表达改变情况和转录因子结合位点信息学预测,筛选出p21进一步研究。4、HoxC10在p21转录调控中的作用及分子机制研究:通过改变HoxC10的表达,结合荧光定量PCR、免疫印迹等技术,在细胞、组织水平验证HoxC10和p21的表达相关性;免疫印迹技术检测在敲减HoxC10后,p21下游周期相关信号通路关键分子的表达改变;利用应用双荧光素酶报告基因检测、染色质免疫共沉淀等技术,探讨并揭示胃癌HoxC10对p21的转录抑制分子调控机制。研究结果1、HoxC10在胃癌组织中的表达及临床意义(1)新鲜冰冻胃癌及其配对癌旁非肿瘤组织样本(n=70),通过实时荧光定量PCR技术发现HoxC10在胃癌组织中的mRNA表达水平显著高于癌旁组织(高表达比例为 91.43%,64/70,P0.01)。免疫印迹法(Westernblotting)检测 HoxC10 的蛋白表达,证实了较癌旁配对非肿瘤组织,胃癌组织中HoxC10的蛋白水平亦明显增高。分析HoxC10的表达水平与患者临床病理特征间的相关性,发现HoxC10的表达水平与肿瘤大小、浸润深度、淋巴结转移及肿瘤分期显著相关(P0.01)。(2)胃癌组织石蜡标本,定制组织芯片。通过免疫组化分析发现HoxC10在胃癌组织中普遍高表达(91.3%,137/150,P0.01),HoxC10的蛋白表达与患者性别、肿瘤浸润深度及肿瘤分期显著相关(n=195,P0.01)。Kaplan-Meier生存分析发现,胃癌组织中HoxC 10高表达的患者预后较差,风险比为2.223(95%CI 1.361-4.186)。多因素COX回归分析,发现HoxC10的高表达是胃癌预后差的一个独立预测因素。(3)比对分析TCGA数据库胃癌和癌旁组织中的差异基因,发现胃癌组织中HoxC10表达上调高达122倍(n=33,P0.01)。在KM-plotter胃癌数据库(n=876)中,HoxC10高表达的患者预后差,风险比为1.8(95%C1 1.5-2.16)。2、HoxC10对胃癌细胞生物学行为的影响(1)CCK8、克隆形成等细胞增殖实验,发现过表达HoxC10显著促进胃癌细胞的增殖,而干扰HoxC10则抑制细胞的增殖。(2)采用流式细胞周期检测,发现过表达HoxC10后胃癌细胞周期G1-S期转换明显加快,干扰HoxC10后细胞阻滞在G1期。应用细胞周期同步化药物诺考达唑(Nocodazole)处理细胞,进一步证明敲减HoxC10使胃癌细胞周期阻滞在G1期。(3)采用双重染色流式细胞凋亡检测,发现过表达HoxC10抑制胃癌细胞的凋亡,而敲减HoxC10的表达则促进细胞凋亡。(4)Transwell试验表明过表达HoxC10能够显著促进胃癌细胞的迁移、侵袭能力,而HoxC10表达下调则抑制细胞的迁移、侵袭。(5)胃癌皮下移植瘤模型,发现过表达HoxC10后裸鼠瘤体的生长较对照组明显增快,而干扰HoxC10可减慢瘤体的生长速度。3、HoxC10潜在下游靶基因的筛选及验证(1)在胃癌细胞BGC中敲减HoxC10并做表达谱基因芯片分析,以差异倍数1.3为界,并结合差异基因的功能分析,我们筛选出一系列差异表达的mRNA,如ELF5、PRDM2、AKT3、CDKN1A(p21)、SLC39A10、ZEB1、NRP2 等。(2)采用实时荧光定量PCR技术在胃癌细胞中进行验证。其中PRDM2、AKT3和p21等在敲减HoxC10的胃癌细胞株中显著上调,ZEB1、NRP2等显著下调(P0.05)。(3)借助Genomatix和JASPAR两个在线转录因子结合位点预测数据库,分析发现HoxC10与p21启动子区存在多个可能的结合位点。4、HoxC10在p21转录调控中的作用及分子机制研究(1)在多株胃癌细胞中改变(过表达和敲减)HoxC10的表达,发现过表达HoxC10,p21的表达水平相对下调,而敲减HoxC10后,p21的表达相对上调;在裸鼠胃癌移植瘤组织中,免疫组化分析HoxC10和p21的表达情况,发现二者呈负相关;在稳定敲减HoxC10的胃癌细胞中,p21及其下游周期相关信号通路的关键分子CDK2、CDK4、c-Myc、CyclinD1、pRb 等的表达发生改变。(2)采用双荧光素酶报告基因技术,分析HoxC10表达改变对p21转录活性的影响。结果发现过表达HoxC10明显抑制p21的转录活性,而干扰HoxC10的表达,p21的转录活性增强。(3)设计针对p21基因启动子区的引物,采用染色质免疫共沉淀技术,进一步鉴定发现HoxC10与P21启动子区存在结合位点。研究结论1、在胃癌组织中HoxC10普遍高表达,且与肿瘤浸润深度、肿瘤分期及临床预后相关,HoxC10高表达是胃癌预后差的一个独立预测因素。2、HoxC10可调控G1细胞周期、抑制胃癌细胞凋亡,促进胃癌细胞增殖和裸鼠移植瘤形成,促进胃癌细胞迁移和侵袭能力,从而发挥促癌基因的作用。3、筛选发现系列HoxC1O可能调控的下游重要肿瘤相关基因,其中HoxC10能与p21的启动子区结合并抑制其转录活性,影响其下游周期相关信号分子的表达,从而影响胃癌细胞的增殖和周期调控。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.2

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