【作者】 宋宇；

【导师】 田野；

【作者基本信息】 苏州大学 ， 肿瘤学， 2014， 博士

【摘要】 研究背景和目的：胃癌是严重危害人类健康的最常见的恶性肿瘤之一，发病率位居所有恶性肿瘤的第二位，死亡率列所有恶性肿瘤的第三位。目前根治性手术仍然是胃癌的主要治疗方式，也是唯一有希望治愈胃癌的手段。但临床上仍有大多数患者确诊时已是中晚期，手术效果不佳，直接影响预后。因此深入研究胃癌发生、发展的分子机制，可为胃癌早期诊断提供依据，同时也为临床治疗提供有效的靶点治疗，具有重要的临床意义。近年来有研究证明Hedgehog(HH)信号传导通路在胃的胚胎发育过程中起非常重要的调控作用，该通路的过度激活可导致胃癌的发生，因此HH通路与胃癌的发生、发展密切相关，针对该通路的特异性靶向治疗有可能成为临床上治疗胃癌的有效的新措施。HH通路主要由HH配体，膜受体Patched (Ptch)、Smoothened (Smo)及下游的转录因子Gli组成。近年来有研究发现Hedgehog通路中还包括Hedgehog-interactingprotein (HHIP)基因，HHIP能和Patched竞争性结合Hh蛋白，从而阻断HH信号转导。对HH通路来说，HHIP基因与Patched基因是该通路的两个负反馈因子，可直接抑制Hedgehog通路，具有极其重要的意义。因此本课题选用HHIP和Patched为目的基因，进行深入研究。表观遗传学是指DNA序列不发生变化，但基因表达却发生了可遗传的改变，这种改变是细胞内除了遗传信息以外的其他可遗传物质发生的改变，即基因型未发生变化而表型却发生了改变，且这种改变在发育和细胞增殖构成中能稳定传播。其中DNA甲基化，组蛋白修饰改变，非编码RNA等是表观遗传修饰的主要方式，也是研究的热点。目前研究发现：基因组的广泛低甲基化是肿瘤细胞的一个普遍特征；肿瘤抑制基因启动子区的高甲基化可导致抑癌基因沉默、失活致表达产物减少或丧失。DNA甲基化与胃癌之间关系密切，研究发现在胃癌的各个阶段都能检测有基因甲基化的存在，甚至癌前病变阶段也能发现。本文对胃癌中HHIP、Patched基因的表达及甲基化水平进行检测和分析，并通过随访，观察HHIP、Patched基因的表达与三年总生存率的相关性，试图发现HHIP、Patched基因在胃癌发生、发展的作用机制及与胃癌预后的相关性。目前有报道Patched基因在胃癌中启动子区呈高甲基化，而研究主要集中在胃癌细胞株，对HHIP和Patched基因在胃癌组织中的甲基化状态分析未见相关报道，因此本文以HHIP和Patched为研究基因，分别检测两个基因在胃癌组织、癌旁组织中的表达和启动子区CpG岛甲基化状态，分析其与临床病理特征的关系。并通过随访，观察HHIP、Patched基因的表达与三年总生存率的相关性。对人胃癌细胞株AGS在体外予去甲基化药物5-aza-dc干预，观察干预前后HHIP mRNA表达差异，启动子区CpG岛甲基化状态的影响。构建稳定的HHIP过表达慢病毒载体，转染胃癌细胞株AGS，观察转染前后HHIP mRNA表达和启动子区CpG岛甲基化状态的差异。从而揭示HHIP、Patched基因在胃癌发生、发展的作用机制及与胃癌预后的相关性，为胃癌的早期诊断、判断预后和靶向治疗提供新的思路。研究方法和材料：1.研究材料胃癌标本取自2009年6月-2013年12月在张家港市第一人民医院接受手术治疗的病人，术前均未经化疗和放疗，术后病理检查确定组织类型均为胃腺癌，无其他部位原发肿瘤。共选取60例胃癌组织和60例癌旁正常组织，每例标本的癌组织和癌旁正常组织均常规病理切片证实，并液氮保存。其中男性患者34例，女性患者26例，年龄36-72岁之间，平均年龄60.82岁，Ⅱ期胃癌患者40例，Ⅲ期患者20例，高中分化32例，低分化患者28例，有淋巴结转移24例，无淋巴结转移36例。胃癌细胞株AGS购自中国科学院上海细胞库，来源于一个未经治疗的切除的胃肿瘤碎块，为腺癌细胞，具有局部浸袭转移能力。2.研究方法主要分为对胃癌组织及癌旁组织研究；对胃癌细胞株AGS研究。2.1.对胃癌组织及癌旁组织的研究2.1.1.半定量RT-PCR取人胃癌组织和癌旁组织，使用Trizol试剂抽提总RNA，由逆转录试剂盒转录成cDNA后，采用RT-PCR法检测HHIP、Patched mRNA表达水平。2.1.2.免疫组化法取人胃癌组织和癌旁组织蜡块切片，按照APC法进行HHIP和Patched蛋白测定。2.1.3. bisulfite sequencing PCR（BSP）法常规抽提胃癌组织和癌旁组织DNA，亚硫酸盐处理后，进行BSP检测并测序，分析HHIP、Patched启动子区CpG岛每个位点甲基化状态。2.1.4. methylation-specific PCR (MSP)法常规抽提胃癌组织和癌旁组织DNA，亚硫酸盐处理后，应用Methyl Primer Express v1.0软件设计MSP引物，予MSP法检测HHIP启动子区CpG岛甲基化状态。2.1.5.随访采用Kaplan-Meier法进行三年总生存率的统计。2.2.对胃癌细胞株AGS的研究2.2.1予去甲基化药物5-aza-dc干预AGS细胞株，进行相关研究。2.2.1.1MTT法用0、0.5、1.0、2.0、5.0、10uM的5-aza-dc药物处理生长良好的AGS细胞株24h、48h、72h。用MTT法检测不同浓度下AGS细胞株的生长情况。2.2.1.2Annexin V／PI法检测凋亡用0、0.5、1.0、2.0、5.0、10uM的5-aza-dc对AGS细胞株进行干预，采用流式细胞仪检测细胞凋亡。2.2.1.3半定量RT-PCR使用Trizol试剂抽提AGS细胞株干预前后的RNA，由逆转录试剂盒转录成cDNA，采用RT-PCR法检测干预前后HHIP基因mRNA的差异。2.2.1.4BSP法常规抽提干预前后AGS细胞株DNA，亚硫酸盐处理后，予BSP法检测干预前后HHIP启动子区CpG岛每个位点甲基化状态变化。2.2.1.5MSP法常规抽提干预前后AGS细胞株DNA，亚硫酸盐处理后，予MSP法检测干预前后HHIP启动子区CpG岛甲基化状态变化。2.2.2构建稳定的HHIP过表达慢病毒载体，转染AGS，进行相关研究2.2.2.1筛选和构建稳定的HHIP过表达慢病毒载体2.2.2.2MSP法常规抽提转染前后AGS细胞株DNA，亚硫酸盐处理后，予MSP法检测干预前后HHIP启动子区CpG岛甲基化状态变化。2.2.2.3研究HHIP过表达对AGS细胞株生长、增殖的影响。3.统计分析采用SPSS16.0统计软件进行统计处理。计量资料以均数±标准差（x s）表示，计数资料以百分比（%）表示，采用x2检验、t检验、Kaplan-Meier法、Spearman相关分析等，p＜0.05为有统计学差异。研究结果：1.对胃癌组织及癌旁组织的研究1.1.半定量RT-PCR HHIP、Patched基因在人胃癌组织及癌旁组织中均可见表达，HHIP mRNA在胃癌中的表达强度0.8181±0.381，低于癌旁组织中的表达强度1.6033±0.262，而Patched mRNA在胃癌中的表达强度1.7537±1.150，低于癌旁组织中的2.7993±1.458。HHIP、Patched mRNA表达与性别、年龄、TNM分期、淋巴结转移等临床病理特征无明显统计学差异。但Patched mRNA在低分化胃癌中表达明显低于中高分化，HHIP mRNA表达与分化程度未见统计学差异。1.2.免疫组化法HHIP蛋白在胃癌组织中的阳性率为18/60（30%），在癌旁正常组织中的阳性率为36/60（60%），Patched蛋白在胃癌组织中为20/60(33.33%)，癌旁组织中阳性率为48/60(80%)。HHIP、Patched蛋白表达与性别、年龄、TNM分期、分化程度、淋巴结转移等临床病理特征无明显统计学差异。1.3. BSP法60例胃癌组织中表现为HHIP、Patched CpG位点完全甲基化或部分甲基化，而癌旁组织中CpG位点大部分非甲基化，少数甲基化。将HHIP和PatchedmRNA表达与甲基化程度进行Spearman相关分析，发现HHIP和Patched mRNA表达与甲基化程度呈负相关。1.4. MSP法60例胃癌组织中HHIP启动子区CpG岛甲基化有28例，甲基化率达46.67%，而癌旁组织中甲基化率为20%，HHIP基因启动子区CpG岛甲基化率明显高于相应的癌旁组织.1.5.随访通过随访，发现HHIP和Patched阳性表达胃癌患者三年总生存率优于阴性表达患者，因此HHIP和Patched可作为判断胃癌预后的预测因子。2.对胃癌细胞株AGS的研究2.1.予去甲基化药物5-aza-dc干预AGS细胞株，进行相关研究。2.1.1MTT法5-aza-dc对AGS细胞株增殖的抑制作用随着药物浓度的增加和时间的延长逐渐增加，呈时间和剂量依赖性。2.1.2Annexin V／PI法检测凋亡在一定剂量范围内随着5-aza-dc药物浓度的增加，细胞凋亡率增加。但达到一定浓度后，细胞凋亡率随着药物浓度的增加而下降。2.1.3半定量RT-PCR AGS细胞株在5-aza-dc干预后HHIP基因被激活，表达明显增高。2.1.4BSP法5-aza-dc干预后HHIP基因启动子区CpG位点甲基化明显减少。2.1.5MSP法胃癌细胞株AGS甲基化程度为99.7%±0.67%，经去甲基化干预后，甲基化程度明显下降，甚至表现为无甲基化。2.2构建稳定的HHIP过表达慢病毒载体，转染AGS，进行相关研究2.2.1成功构建稳定的HHIP过表达慢病毒载体。2.2.2MSP法胃癌细胞株AGS甲基化程度为99.67%，过表达慢病毒载体转染后，甲基化程度明显下降，甚至表现为无甲基化。2.2.3HHIP基因过表达后可抑制胃癌细胞株生长、增殖。结论：1、胃癌和癌旁组织中可见HHIP和Patched mRNA和蛋白表达，但在胃癌组织中HHIP和Patched基因低表达。2、HHIP和Patched mRNA和蛋白表达与性别、年龄、TNM分期、淋巴结转移等临床病理特征无关。但Patched mRNA在低分化胃癌中表达明显低于中高分化，HHIP mRNA表达与分化程度未见统计学差异。3、HHIP和Patched可能作为判断胃癌预后的预测因子。4、胃癌组织和胃癌细胞株中均存在HHIP和Patched启动子区CpG岛高甲基化，是胃癌区别于正常胃组织的分子事件，与HHIP和Patched抑癌机制有关，可能与胃癌的发生有关，有可能成为胃癌早期诊断的肿瘤标志物。5、5-aza-dc可以诱导细胞凋亡，抑制肿瘤增殖，在一定浓度范围呈时间和剂量依赖性。去甲基化药物有可能成为一种胃癌治疗途径。6、HHIP基因过表达后可抑制胃癌细胞株生长、增殖，有可能成为胃癌的新的生物学标记，针对HHIP形成的药靶，有可能成为胃癌治疗的一个新的途径。更多还原

【Abstract】 Background and objects：Gastric cancer is one of the most common cancers with serious hazard to humanhealth，accounting for the second most frequent malignancy and the third cause ofcancer-related death. Now radical surgery is still the main treatment and the only mean tocure gastric cancer. But most gastric cancer patients are usually diagnosed in the laterstages of the disease in clinical and has very poor prognosis due to patients withunresectable therapeutic options. Therefore, there is clinical significance to extensivelyinvestigate the molecular mechanisms of gastric carcinogenesis, to Provide the basis for theearly diagnosis of gastric cancer, and also provide an effective target in the treatment ofclinical treatment.In recent years, studies have shown that the hedgehog pathway plays an importantrole in gastral embryonic development and carcinogenesis. Gastric cancer development hasbeen intimate associated with hedgehog signaling pathways. To aim pathway-specifictargeted therapy may become a effective new measure in the clinical treatment ofgastric cancer. The HH pathway mainly includes HH ligand，membrane receptor ofPatched (Ptch)、Smoothened (Smo) and downstream of the transcription factor Gli. Humanhedgehog-interacting protein (HHIP) has been discovered recently. HHIP binds all threeHh proteins with an affinity equal to that of Patched, and functions to negatively regulatethe hedgehog pathway. Indeed, both HHIP and Patched genes, two negative regulators ofhedgehog signaling, can directly inhibit the pathway, has a very important significance.Therefore this topics chosen HHIP and Patched gene in-depth study.Epigenetics refers to the sequence of DNA does not change, but the gene expressionwas heritable changed, which were changed to the genetic information in cells other than genetic material change, namely the phenotype changed but genotype has not changed, andthis change can propagate stably in development and cell proliferation.DNA methylation，histone modification, non coding RNA were included inepigenetic modification, and also a research hotspot. The current study found: widehypomethylation of the genome is a common feature of the tumor cells; hypermethylationof the tumor suppressor gene promoter region silencing of tumor suppressor genes, canlead to reduction or loss of the inactivation induced expression product. DNA methylationis closely related with gastric cancer, the study found methylation existed in each stage ofgastric cancer，even precancerous lesions. The article willdetect and analyze the expressionand methylation levels of HHIP、Patched gene in gastric carcinoma, and through thefollow-up, to observe the correlation between the expression of HHIP、Patched gene andthree year overall survival rate, trying to find HHIP、Patched gene mechanism in gastriccarcinogenesis、development and their relationship with prognosis of gastric cancer.It was said Patched gene promoter methylation in gastric cancer. However, thesestudies focus on gastric cancer cell lines，no study has precisely analyzed the methylationof Patched and HHIP gene promoters in gastric cancer to date. In this study, we focus onHHIP and Patched gene，detect the expression of HHIP and Patched in gastric cancertissues and adjacent normal tissues，and then detect the promoter methylation status ingastric cancer，analyze its relationship with clinical pathological features. Throughfollow-up, to observe the correlation between the expression of HHIP、Patched gene andthree year overall survival rate. Then to discover the HHIPmRNA expression differencesand the change of CpG island methylation status before and after demethylation drug5-aza-dc intervention. To build a stable HHIP overexpression of lentivirus vector,transfected into gastric cancer cell lines AGS, then observe the difference about mRNAexpression and methylation status of CpG islands in promoter region before and aftertransfection. To reveal the development mechanism of HHIP, Patched gene in gastriccarcinogenesis, and their relationship with prognosis of gastric cancer. Which can providenew ideas for the early diagnosis、prognosis and targeted therapy of gastric.Methods and material： 1. Research material Surgical specimens from60patients with gastric cancer andadjacent normal tissues were collected from the Department of Surgery, Zhangjiagang FirstHospital between June2009and December2013，without preoperative chemotherapy andradiotherapy, pathologic examination to determine tissue types are gastric adenocarcinoma.All surgically resected tissue specimens were snap frozen in liquid nitrogen until use.Patients included34males and26females with an age range from36to72years (mean60.82years). According to the TNM staging system,40cases were stage II patients and20cases were stage III patients.32cases were well and moderately differentiated, while28were poorly differentiated.24cases had lymph node metastasis, but36cases were withoutlymph node metastasis. The gastric cancer AGS cell line was purchased from ShanghaiInstitute of Cell Bank, which come from a resected gastric tumor fragments untreatedadeno-carcinoma cell, have the capability of the local Baptist passage of transfer.2. Research Methods: mainly focus on gastric carcinoma and adjacent normaltissues，focus on gastric cancer cell line AGS.2.1Research about gastric tissues2.1.1reverse transcription-PCR RNA was isolated from the gastric cancer andnormal tissues by Trizol agent. cDNA was then converted from RNA using a reversetranscription kit. Detect the expression levels of HHIP, Patched mRNA by RT-PCRmethod.2.1.2Immunohistochemistry Take paraffin-embedded tissue sections to detectthe HHIP and Patched protein according with APC method.2.1.3bisulfite sequencing PCR（BSP） DNA was extracted per our standardprotocol. After bisulfite treatment, to analyse HHIP and Patched promoter CpG islandmethylation status of each locus by detecting and sequencing with BSP method.2.1.4methylation-specific PCR (MSP) DNA was extracted per our standardprotocol. After bisulfite treatment, to design MSP primers by Methyl Primer Express v1.0software, then to detect HHIP promoter CpG island methylation status by MSP.2.1.5follow up Statistics of three year overall survival rate by Kaplan-Meiermethod.2.2Research about gastric cancer cell lineAGS2.2.1To interfere AGS cell line by demethylation drug5-aza-dC, undertake relatedresearch. 2.2.1.1MTT assay AGS cell line treated with0、0.5、1.0、2.0、5.0、10uM5-aza-dcafter24h,48h,72h, to detect AGS cell growth on different concentrations using the MTTassay.2.2.1.2Annexin V/PI-flow cytometry assay AGS cell line treated with0、0.5、1.0、2.0、5.0、10uM5-aza-dc, then detect apoptosis by flow cytometry.2.2.1.3reverse transcription-PCR RNAwas isolated from AGS cell line by Trizolagent before and after intervention. cDNA was then converted from RNA using a reversetranscription kit, then to detect the expression differences of HHIP mRNA by RT-PCRmethod.2.2.1.4BSP method AGS cell line DNA was extracted per our standard protocol.After bisulfite treatment, to analyse HHIP promoter CpG island methylation status of eachlocus by detecting and sequencing with BSP method.2.2.1.5MSP method AGS cell line DNAwas extracted per our standard protocol.After bisulfite treatment, to detect HHIP promoter CpG island methylation status by MSPbefore and after intervention.2.2.2To build a stable HHIP overexpression of lentivirus vector, to transfect AGS,undertake related research.2.2.2.1To build a stable HHIP overexpression of lentivirus vector.2.2.2.2MSP method AGS cell line DNAwas extracted per our standard protocol.After bisulfite treatment, to detect HHIP promoter CpG island methylation status by MSPbefore and after intervention.2.2.2.3To observe the effect overexpression HHIP gene onAGS cell line.3. Statistical Analysis All of the measurement data were expressed as mean±SD,count data are expressed as a percentage (%) by SPSS16.0software. Apply X2test, t test,Kaplan-Meier mean，Spearman correlation analysis, et al. A value of P<0.05wasconsidered statistically significant.Results1. Research about gastric tissues1.1reverse transcription-PCR HHIP and Patched express in gastric cancer andadjacent normal tissues. HHIP mRNA intensity of expression0.8181±0.380in gastriccancer lower than1.6033±0.263in adjacent normal tissues. PatchedmRNA intensity ofexpression1.7537±1.150in gastric cancer lower than2.7993±1.458in adjacent normal tissues. HHIP, Patched mRNA expression have no statistically significant differences withclinical pathological features: gender, age, TNM stage, lymph node metastasis. ButPatched mRNA in poorly differentiated gastric carcinoma expression was significantlylower than in highly differentiated, difference of HHIP mRNA expression and the degreeof differentiation was observed in the control.1.2Immunohistochemistry HHIP protein positive rate was18/60(30%) ingastric cancer, while36/60(60%) in adjacent normal tissues, the positive rate ofPatched protein in gastric cancer20/60(33.33%), adjacent tissues was48/60(80%). HHIP,Patched protein expression have no statistically significant differences with clinicalpathological features: gender, age, TNM stage, degree of differentiation, lymph nodemetastasis.1.3BSP60cases of gastric cancer tissues showed HHIP and Patched CpG sitescompletely or partially methylated, while unmethylated or minority methylation inadjacent tissues. Through Spearman correlation analysis we found the HHIP and PatchedmRNAexpression was negatively correlated with the degree of methylation.1.4MSP There were28cases HHIP promoter CpG island methylation in60casesof gastric cancer, the methylation rate of46.67%, while methylation rate of20%inadjacent tissues, HHIP gene promoter CpG island methylation was significantly higherthan the corresponding adjacent tissues (p <0.05).1.5follow up Through follow-up, found that the patients of positive expression ofHHIP and Patched in the three year overall survival rate surpass the negative expression, soHHIP and Patched can be used as a predictor of prognosis of gastric cancer.2. Research about gastric cancer cell lineAGS2.1. To interfere AGS cell line by demethylation drug5-aza-dC, undertake relatedresearch.2.1.1MTT assay The effect of proliferation inhibition was gradually increasewith the increase of the concentration of the drug and the extension of time.,which was atime-and dose-dependent manner.2.1.2Annexin V/PI-flow cytometry assay In a certain dose range rate ofapoptosis increased with the increase of the concentration of5-aza-dc. But after reached acertain concentration, the apoptotic rate decreased with the increase of the concentration.2.1.3reverse transcription-PCR After5-aza-dc intervention, HHIP gene is activated. The expression of HHIP was significantly higher.2.1.4BSP method After5-aza-dc intervention, HHIP gene promoter CpG sitesmethylation was significantly reduced.2.1.5MSP method The methylation degree in gastric cancer cell line AGS was99.7%±0.67%. After5-aza-dc intervention. the degree of methylation was significantlydecreased, and even showed unmethylation.2.2. To build a stable HHIP overexpression of lentivirus vector, to transfect AGS,undertake related research.2.2.1Astable HHIP overexpression of lentivirus vector was build successful.2.2.2The methylation degree in gastric cancer cell line AGS was99.67%. Aftertransfect by overexpression of lentivirus vector, the degree of methylation was significantlydecreased, and even showed unmethylation.2.2.3Overexpression HHIP gene can inhibit the growth and proliferation of gastriccancer cell line.Conclusion:1、There were HHIP、Patched mRNA and protein expression，the intensity ofexpression in gastric cancer lower than in adjacent normal tissues.2、HHIP、Patched mRNA and protein expression have no statistically significantdifferences with clinical pathological features: gender, age, TNM stage, lymph nodemetastasis. But Patched mRNA expression was significantly lower in poorly differentiatedgastric carcinoma than in highly differentiated, difference of HHIP mRNA expression ofthe degree of differentiation was observed in the control.3、HHIP and Patched can be used as a predictor of prognosis of gastric cancer.4、There were HHIP and Patched gene low expression and promoter CpG islandmethylation in gastric cancer, which was a molecular event distinguished gastric cancerfrom normal gastric tissue, maybe concerned with the incidence of gastric cancer. It maybecome a tumor marker for early diagnosis of gastric cancer.5、5-aza-dc can induce apoptosis, Inhibit tumor proliferation,in a certain concentrationrange it appeared In a time-and dose-dependent manner. Demethylation may become atumor therapeutic approach.6、Overexpression HHIP gene can inhibit the growth and proliferation of gastric cancer cell line, may be a new biological marker in gastric cancer, the drug targets HHIPformation, may become a new way to the treatment of gastric cancer.更多还原