【作者】 张腾飞；

【导师】 陆士新；

【作者基本信息】 北京协和医学院 ， 肿瘤学， 2012， 博士

【摘要】 食管癌是世界上常见的恶性肿瘤,其发病率居恶性肿瘤的第7位,死亡率居世界癌症死因的第6位。食管癌的分布有明显的地域聚集性,有50%的食管癌发生在中国,其中食管鳞状细胞癌(esophageal squamous cell carcinoma, ESCC)是主要的组织学类型。ESCC具有较强的侵袭转移能力,在早期就可以转移至周围淋巴结和邻近的组织器官,通常大多数食管鳞癌患者确诊时已为晚期,错过了手术的最佳时期,即使进行手术完全去除病灶,无复发生存期也仍然低于2年,再加上ESCC对放化疗一般不敏感,因此食管癌的预后较差。肿瘤的发生发展是一个多阶段、多基因参与的复杂过程,研究表明一些癌基因和抑癌基因的表达失调在肿瘤的发生发展、侵袭转移过程中发挥着重要的作用,对ESCC的分子病因学进行研究,尤其是对肿瘤发生发展和侵袭转移相关的分子机制的研究和寻找新的肿瘤分子标志物将有助于提高食管癌的诊治水平。MicroRNA是一类内源性的平均长度约为18～24个核苷酸的非编码小分子RNA,在表观遗传学和翻译后修饰调控过程中发挥着重要的作用。1nicroRNA能够识别并结合到靶基因的3’-UTR区的结合位点,通过诱导mRNA的降解降低靶基因的表达,在肿瘤中发挥着癌基因或者抑癌基因的功能,参与肿瘤的发生发展和侵袭转移等各个生命步骤。尤其是在外周血中能检测到稳定表达的microRNA,提示检测血清/血浆中的microRNA是发展非损伤性诊断和预后分子标志物的有效方法,也是本论文第一部分的研究重点。另外在一些mciroRNA靶基因的3’-UTR区结合位点存在等位基因多态性例如短小重复多态(short tandem repeat, STR)序列,microRNA能够对这些等位基因进行选择性调控,影响靶基因的表达从而产生疾病的易感性,在本论文第二部分中我们将对这一机制进行探讨。本文的第一部分是对ESCC中的microRNA-31进行研究。microRNA-31(miR-31)在多种肿瘤中发现异常表达,在多种鳞癌组织中被报道存在普遍的高表达,但在食管鳞癌中研究未见报道。本研究通过检测45对食管鳞癌癌与癌旁配对样本以及523例血清样本中miR-31的表达,发现miR-31在77.8%的肿瘤组织中呈现异常的高表达(P<0.001), ESCC血清中miR-31的表达明显高于健康对照(P<0.001)。随访结果显示,术后血清miR-31高表达的患者无复发生存率(P=0.001)和整体生存率(P=0.005)显著低于miR-31低表达的患者,ROC诊断曲线也表明血清miR-31的表达水平可以作为食管鳞癌的诊断、预后的标志物。体外实验证实,miR-31可以促进食管鳞癌细胞系的增殖、侵袭和转移能力,通过生物信息学预测、报告基因和western blot验证,发现miR-31可以显著调控三种抑癌基因EMP1, KSR2, RGS4。提示miR-31在食管鳞癌中的促癌作用可能是通过下调相关抑癌基因实现。其中对EMPl(上皮膜蛋白)的表达抑制,很可能是miR-31促进鳞癌发生发展的共同作用机制。本论文的第二部分是研究miR-1322对ECRG2基因的选择性调控。食管癌相关基因2(esophageal cancer-related gene2, ECRG2,又名serine peptidase inhibitor, Kazal type7, SPINK7)是本实验室在1998年从林县食管癌高发现场的食管鳞癌患者中克隆报告的新基因(Genebank Accession Number:AF268198),该基因在食管鳞癌组织中低表达外,被认为是ESCC的抑癌基因。在ECRG2基因的3’-UTR区发现了短串联重复(short tandem repeat, STR)序列,即3核苷酸TCA的重复。ECRG2基因中存在两种STR等位基因型：TCA3(TCATCATCA)和TCA4(TCATCATCA TCA),其中TCA3/TCA3基因型被国内外报道是ESCC的风险基因型,并且预后也较差。本研究旨在探讨microRNA对ECRG2基因短小重复序列多态的表达调控。首先生物信息学分析发现ECRG2基因STR序列附近存在1miR-580、miR-1182、 miR-1272、miR-1322的结合位点,通过报告基因的验证发现ECRG2基因TCA4基因型表达水平显著高于TCA3型,其3’-UTR区域的TCA多态序列主要受到miR-1322的调控。通过组织和血清表达水平的检测发现miR-1322在食管癌组织和血清中存在着异常高表达。提示miR-1322可能是的ESCC的潜在诊断标志物。综上所述,我们发现了miR-31和miR-1322在ESCC中的癌基因功能以及做为潜在诊断和预后标志物的价值,为肿瘤的早期诊断和治疗提供了新的理论思路。[摘要]目的microRNA-31(miR-31)在多种肿瘤中被报道异常表达,本论文旨在研究miR-31在食管鳞状细胞癌(oesophageal squamous cell carcinoma, ESCC)的功能。方法通过Real-time RT-PCR的方法检测45对配对的ESCC组织和523例血清样本中的miR-31的表达,523例血清标本包括120例ESCC和121例健康对照的发现阶段、81例ESCC和81例健康对照的验证阶段和120例直肠癌、肝癌、子宫癌、乳腺癌、胃癌和肺癌的其他肿瘤患者。Wilcoxon配对检验用于ESCC配对组织样本miR-31的表达差异检验,曼-惠特尼U检验用于血清样本miR-31的表达差异检验。结果miR-31在77.8%的ESCC组织中表达上调。ESCC血清中miR-31的表达明显高于健康对照(P<0.001)。发现组和验证组ESCC血清miR-31的ROC曲线下面积(AUC)分别为0.902(95%置信区间：0.857-0.936)和0.888(95%置信区间：0.819-0.939)。无复发生存分析(P=0.001)和肿瘤特异性生存分析(P=0.005)的结果都显示血清中高表达miR-31的ESCC患者预后较差。细胞系的实验表明miR-31能够增强细胞的克隆形成能力和侵袭转移能力。报告基因实验和Western blot的结果证实miR-31可调控3个抑癌基因的功能,分别是上皮膜蛋白1(epithelial membrane protein1, EMP1)、Ras2的激酶抑制蛋白(kinase suppressor of ras2, KSR2)和G蛋白信号调节蛋白4(regulator of G-protein signalling4, RGS4)。结论miR-31在ESCC中起到原癌基因的功能,是ESCC可能的诊断及预后标志物。[摘要]目的食管癌相关基因2(esophageal cancer-related gene2, ECRG2)的3’UTR区存在短串联重复序列多态性(short tandem repeat, STR),并且与食管鳞癌的发病和预后相关。本研究旨在探讨microRNA通过STR序列对ECRG2基因的调控。方法通过生物信息学预测ECRG2基因STR序列可能结合的microRNA,通过报告基因实验对候选microRNA与STR序列的结合进行验证。对证实与ECRG2基因STR有调控作用的microRNA进行标志物研究,在44对食管鳞癌的配对组织标本中通过Real-time PCR进行表达水平的检测,接着在120例ESCC(?)(?)120例健康对照的发现组、81例ESCC和81例健康对照的验证组进行血清中microRNA表达水平的检测。结果通过生物信息学预测到miR-580、miR-1182、miR-1272和miR-1322与ECRG2基因STR序列有结合,通过报告基因检测发现只有miR-1322能够显著下调TCA3基因型的表达(p<0.01),同时对TCA4基因型没有显著的下调功能(p>0.05)。通过Real-tine PCR发现miR-1322在ESCC组织和血清中都有高表达(p<0.01)。ROC曲线分析发现240例样本的发现组AUC值为0.847(95%置信区间：0.795-0.890),160例样本的验证组也得到类似的结果AUC值为0.845(95%置信区间：0.780-0.897),miR-1322能够用于ESCC的诊断。结论miR-1322通过3’UTR区的SRT序列调控ECRG2基因的表达,miR-1322是可能的ESCC诊断标志物。更多还原

【Abstract】 Oesophageal cancer ranks seventh and sixth in cancer incidence and mortality rate worldwide, respectively. A total of50%of all oesophageal cancer worldwide occurs in China. Esophageal squamous cell carcinoma (ESCC) accounts for approx.90%of all oesophageal carcinomas diagnosed at an advanced stage. The difficulty in managing ESCC is due to its aggressive invasion and early metastasis to lymph nodes, adjacent tissue and organs. The majority of cases are diagnosed at a relatively late stage of the disease, when the chances of surgical intervention are lost. Even with surgery, the median survival rate of ESCC patients after RO resection (the complete removal of all tumour with microscopic examination of margins showing no tumour cells) is less than2years. Additionally, ESCC is relatively resistant to both chemotherapy and radiotherapy. Many studies have shown that the development and metastasis of ESCC relates to the dysregulation of several oncogenes and tumour suppressor genes in multiple pathways. Understanding the molecular pathogenesis of ESCC, and especially the mechanisms of tumorigenesis and metastasis, is extremely important for developing novel biomarkers and treatment strategies.microRNAs (miRNAs), which are endogenous small single-stranded non-coding RNAs ranging from19to25nt, play an important role in epigenetic and post-transcriptional regulation networks. Their roles in cancer development and metastasis lead to an extensive exploration of oncogenetic or tumour-suppressivemiRs inmultiple cancers, ofwhich some were potential diagnostic and prognostic markers for certain cancers. Since miRs have been shown to be stable in serum and an increasing number reports have revealed that circulating miRs could serve as a diagnostic marker for various cancers.The expression changes of miR-31are frequently reported in multiple cancers. Interesting, miR-31is widely up-regulated inmost SCCs. In the first part the expression of miR-31was detected in45paired ESCC tissues and523serum samples using real-time RT (reverse transcription)-PCR. The result showed that miR-31was up-regulated in77.8%of the ESCC tissues. Serum miR-31levels in ESCC patients were significantly higher than in normal controls (P<0.001). Patients with high-levels of serum miR-31also had a poorer prognosis in relapse-free survival (P=0.001) and tumour-specific survival (P=0.005). ROC analysis suggested that miR-31can serve as a potential diagnostic and prognostic biomarker for ESCC. In vitro studies showed that miR-31promoted ESCC colony formation, migration and invasion. Luciferase reporter and Western blot assays confirmed that three tumour suppressor genes, namely EMP1(epithelial membrane protein1), KSR2(kinase suppressor of ras2) and RGS4(regulator of G-protein signalling4), were targeted by miR-31. The oncogene role of miR-31in ESCC mainly is dued to the down-regulation of several tumor suppresion genes especially EMP1.Esophageal cancer-related gene2(ECRG2), also known as SPINK7) is regarded as a tumor suppressor gene in esophageal cancer. It was first cloned and identified by our laboratory (Genbank Accession Number AF268198). There is a triplet TCA short tandem repeat (STR) polymorphism located in the3’-UTR of ECRG2with two specific alleles, TCA3(TCATCATCA) and TCA4(TCATCATCATCA). The TCA3allele is proved to be a risk factor for ESCC by that subjects who carried the TCA3/TCA3genotype were at an increased risk of ESCC and a significantly poorer prognosis. In the second part we investigated all the microRNAs predicted to bind at the ECRG2STR region and regulation the function of ECRG2. miR-580、miR-1182、miR-1272、miR-1322were predicted to bind at the STR region. The expression of TCA4is higher than TCA3because that miR-1322could significantly down-regulate the with TCA3allele (<0.01), but it could not down-regulate the with TCA4allele significantly (>0.05). MiR-1322was also expressed significantly higher in ESCC tissue and serum samples than in controls (both<0.01). ROC analysis suggested that miR-1322can serve as a potential diagnostic biomarker for ESCC. MiR-1322can regulate in an allele-specific manner and that serum levels of miR-1322can serve as a potential diagnostic biomarker for patients with ESCC.In conlusion, we verified the oncogene function and potential diagnosis biomarker of miR-31and miR-1322in ESCC, which shed light on the early detection and target therapy for ESCC. [Abstract] MicroRNA-31(miR-31) is frequently altered in numerous cancers. The aim of the present study was is to investigate the role of miR-31in oesophageal squamous cell carcinoma (ESCC). Method We measured miR-31in45paired ESCC tissues and523serum samples using real-time RT (reverse transcription)-PCR. The serum samples were divided into a discovery group (120ESCCs and121normal controls), a validation group (81ESCCs and81controls), and a final group comprising six other common tumours (colorectal, liver, cervical, breast, gastric and lung cancers; total n=120). A Mann-Whitney U test and Wilcoxon matched-pairs test were used for the statistics. Result miR-31was up-regulated in77.8%of the ESCC tissues. Serum miR-31levels in ESCC patients were significantly higher than in normal controls (P<0.001). It yielded an ROC (receiver operating characteristic) AUC (area under the curve) of0.902[95%CI (confidence interval),0.857-0.936] in the discovery group and a similar result in the validation group [ROC AUC,0.888(95%CI,0.819-0.939)]. Patients with high-levels of serum miR-31also had a poorer prognosis in relapse-free survival (P=0.001) and tumour-specific survival (P=0.005). In vitro studies showed that miR-31promoted ESCC colony formation, migration and invasion. Luciferase reporter and Western blot assays confirmed that three tumour suppressor genes, namely EMP1(epithelial membrane protein1), KSR2(kinase suppressor of ras2) and RGS4(regulator of G-protein signalling4), were targeted by miR-31. Conclusion We conclude that miR-31plays oncogenetic functions and can serve as a potential diagnostic and prognostic biomarker for ESCC. [Abstract] Aims A short tandem repeat (STR) polymorphism in the3’UTR region of esophageal cancer-related gene2(ECRG2, also known as) has been widely reported to be associated with the incidence and the prognosis of esophageal squamous cell carcinoma (ESCC). This study explores how the microRNA binding to the STR. region affects expression in ESCC. Method Dual-luciferase reporter assays were used to verify the effects of the four microRNAs (miR-580, miR-1182, miR-1272, and miR-1322) predicted to bind the STR region of the3’untranslated region (UTR). The expression of identified effective microRNA was then analyzed in44paired ESCC and adjacent normal tissues and402case-controlled serum samples (divided into a discovery group and an independent validation group) by real-time RT-PCR assay. Result We found that only miR-1322could significantly down-regulate the with TCA3allele (<0.01), but it could not down-regulate the with TCA4allele significantly (>0.05). MiR-1322was also expressed significantly higher in ESCC tissue and serum samples than in controls (both<0.01). Additionally, serum levels of miR-1322yielded an under receiver operating characteristic (ROC) curve area of0.847(95%CI,0.795-0.890) for discriminating ESCCs from healthy controls in the discovery group and a similar result was obtained in the validation group (under ROC area is0.845;95%CI,0.780-0.897). Conclusion We conclude that miR-1322can regulate in an allele-specific manner and that serum levels of miR-1322can serve as a potential diagnostic biomarker for patients with ESCC.更多还原