【作者】 牛文洋；

【导师】 杨甲梅；

【作者基本信息】 第二军医大学 ， 外科学， 2011， 博士

【摘要】 研究背景及目的我国肝癌死亡率位居恶性肿瘤死亡率第二位,死于肝癌者占全球肝癌死亡人数的55%。几十年来,肝癌的临床诊治及基础研究均取得了丰硕成果,但其总体预后并未得到显著改善,难以早期诊断而失去手术时机是重要原因之一。AFP作为肝细胞癌特异的标志物,在临床工作中广泛应用,但其灵敏度及特异度尚不能令人满意。因此新的肝癌特异的肿瘤标志物亟待研究和发现。最近,一种非编码RNA——miRNAs(microRNAs)的发现为肿瘤的早期无创诊断提供了新思路。有研究表明,miRNAs在肿瘤组织及正常组织的表达谱存在差异,在不同肿瘤类型间也存在差异。miRNAs表达谱能够准确的将同一病理类型的肿瘤聚类。另外,肿瘤组织的miRNAs表达谱已经用于预后分析。近两年多个研究证明,非血细胞来源的miRNAs存在于血清和血清中,而且miRNAs在循环中能够非常稳定的存在。这为循环miRNAs作为潜在的肿瘤标记物提供了理论支持。本研究旨在筛选肝细胞癌血清中差异表达的miRNAs,并评估利用筛选出的血清miRNAs来无创诊断HCC的可行性。研究方法本研究共分三个部分。第一部分:收集10例HCC及10例健康对照的血清,将各组的血清等量混合,形成两个血清池,提取血清池总RNA(含有miRNAs),利用exqion的LNATM miRNA芯片对血清池的表达谱进行高通量的筛选。根据预设的条件,从中筛选出符合条件的miRNAs作为候选研究对象进行第二部分的研究。第二部分:收集来自18例健康对照(NC)、12例慢性乙型肝炎患者和18例肝细胞癌(HCC)患者的血清标本,采用SYBR green qRT-PCR的方法对筛选出的6种候选miRNAs进行定量研究和验证,比较各miRNAs的表达水平在各组间的差异。第三部分:在较大样本的临床病例中进行进一步的验证和应用。我们收集了72例肝细胞癌、26例胆管细胞癌、15例肝内良性占位、5例结肠癌肝转移、23例肝炎后肝硬化、20例健康对照的血清标本,其中10例肝细胞癌患者另外收集了术后第7天的血清样本。应用SYBR green qRT-PCR的方法对所有标本的miR-1915表达水平进行定量分析,比较各组间的表达水平差异。实验结果第一部分1、血清池中可提取出合格的RNA采取TRI BD试剂法分别从总体积为1.0ml的实验组及对照组血清池中提取约767.2ng、599.2ng总RNA,OD260/280 Ratio的比值均符合要求。2、总RNA可与miRNA芯片杂交从实验组及对照组提取出的总RNA与miRNA芯片杂交后,两张芯片均得到成像均匀、清晰的杂交图像,可证实总RNA中含有miRNAs。3、miRNAs在实验组及对照组血清中表达存在差异通过对芯片各探针点荧光强度的分析,并进行标准化及归一化处理后,可发现许多miRNAs在两组间的表达存在显著差异。以表达水平变化倍数R≥2为界,共有20种miRNA符合要求。4、筛选出六种候选miRNAs以表达水平变化倍数R≥2为界,并同时满足test-control值>1.0,最后筛选出hsa-miR-1915、hsa-miR-2116、hsa-miR-718、hsa-miR-1470、hsa-miR-193b*、hsa-miR-941等六种miRNA作为候选的血清学标志物。第二部分1、各血清标本中均可提取出合格的RNA 48份血清标本提取的总RNA浓度介于20.3到75.3 ng/ul,OD260/280的比值均符合要求。2、内参基因表达稳定、目标miRNAs扩增效率满意参考文献,初步选定U6 snRNA作为内参基因。通过检测其在各标本间的表达水平,结果显示,三组间U6 snRNA的Ct值未见显著性差异。U6 snRNA的扩增效率为102.8%,miR-1915的扩增效率为103.4%,认为内参基因与目标miRNA具有相同的扩增效率。3、候选miRNAs的初步验证与筛选U6 snRNA及目标miRNA均能有效扩增,空白对照不扩增;溶解曲线提示扩增产物具有特异性。以U6 snRNA为内参,对各miRNAs的表达水平进行标准化分析,通过统计学分析发现,第一部分实验中筛选出的miR-193b*、miR-718、hsa-miR-941、miR-1470等miRNAs在三组之间未见明显统计学差异。miR-1915在HCC组的表达水平分别是CHB组及NC组的15.9倍及16.1倍(P<0.01),而CHB及NC组之间未见显著性差异。miR-2116在HCC组的表达水平也显著高于NC组,但与CHB组的对比中,差异无显著性。4、miR-1915的检验效能以此次入组的血清样本为基础(N=48),利用ROC曲线分析miR-1915对HCC和非HCC的鉴别效能。结果显示,通过ROC分析得到的最佳cut-off值为2.02(△Ct,相对于U6 SnRNA),敏感性和特异性分别为88.89%和86.67%,得到的曲线下面积(AUC)为0.953(95%CI:0.8993-1.006)。第三部分1、结果进一步证实,HCC患者血清中的miR-1915表达水平显著高于肝脏内良性占位、其它病理类型的恶性占位、慢性肝脏疾病及健康对照。2、血清miR-1915的检验效能ROC曲线分析显示血清miR-1915对HCC(N=72)和非HCC对照(N=89)的鉴别效能。曲线下面积AUC=0.81,95% CI = 0.747–0.879。当界值取2.22(相对U6 snRNA的△Ct)时,灵敏度为63.9%,特异度86.5%。结论1、HCC患者及健康对照血清中均有miRNAs的表达;2、芯片检测发现,某些miRNAs的血清表达水平在HCC组及健康对照组之间存在显著性差异。3、筛选出的miR-1915表达水平在区分HCC及对照时有一定的敏感性及特异性,可作为潜在的肝细胞癌血清学标志物。更多还原

【Abstract】 Background and objectiveHepatocellular carcinoma(HCC) is the second cancer killer in China and there are about 55% of HCC associated deaths happened in China.Despite substantial progresses have been made in HCC clinical and basic research,the overall prognosis of HCC remains dismal because of the late diagnosis and low resection rate.A1though alpha fetoprotein(AFP) remains the best HCC biomarker,the specificity and sensitivity of which are still not satisfied.Thus,there is an urgent need for additional better diagnostic markers for HCC。Recently, the discovery of small non-protein-coding RNAs, so-called microRNAs (miRNAs) that play important roles in oncogenesis, has opened new opportunities of a non-invasive test for the early diagnosis of cancer. Studies have shown that profiles of miRNA expression differ between normal tissue and tumour tissues and vary among tumour types. Evidence suggests that miRNA expression profiles can cluster similar tumour types together more accurately. Furthermore, miRNA expression signatures have been used to predict prognosis. More recently, several reports also suggest that cell-free circulating miRNAs existed in serum and plasma. Accordingly, it raises the possibility of using miRNAs as novel non-invasive molecular markers for cancer detection. In the present study, we evaluated the feasibility of using serum miRNAs as a non-invasive diagnostic test for HCC.Methods and ResultsThis study was divided into three phases: phase I, marker discovery; phase II, marker selection and validation; and phase III, large-scale validation.Phase I: marker discoveryIn this phase, serum from ten patients with HCC and ten healthy controll were collected. The serum of each team were pooled together to generate a HCC serum pool and a healthy control serum pool. Then total RNA were isolated from each serum pool followed LNATM microarray scaning. By comparing miRNA probe intensity from the two pools,upregulated miRNAs in HCC serum were identified for further analysis in phase II. We found six miRNAs matching the criterior. Phase II: marker selection and validationSerum samples were collected from 18 patients with HCC before any therapy. Plasma from 18 healthy subjects and 12 patients with HBV infection was collected as control. SYBR green qRT-PCR assay was used for miRNA quantification in serum samples. We final selected miR-1915 as the HCC specific tumour marker.Phase III: large-scale validationSerum was collected from an independent group of 72 patients with HCC before any therapy. Serum from a set of 20 healthy subjects、15 patients with benign liver tumour、31 patients with other malignant liver tumour、23 patients with HBV induced liver cirrhosis was collected as the control. Serum from another 10 patients with HCC was collected before and 7 days after surgical resection. SYBR green qRT-PCR assay was used for miRNA quantification in serum samples .The miR-1915 expession level of each group was campared by△Ct method. The levels of miR-1915 were significantly high than the other groups. Furthermore,we also found that the levels of miR-1915 were significantly reduced in the postoperative samples when compared to the pre-operative samples.ROC curve analyses revealed that the serum levels of miR-1915 were useful biomarkers for differentiating patients with HCC from controls with ROC curve areas of 0.81(95% CI = 0.747–0.879).At the cut-off value of 2.22 for miR-1915 (relative expression in comparison to RNU6B snRNA), the sensitivity was 63.9% and the specificity was 86.5%.ConclusionsIn conclusion, differentially expressed miRNAs in serum of patients with HCC have been tested in this study.1、Some miRNAs express steadly in serum of HCC patients and healthy controls.2、By using microarray scaning, we found that the expression of certain miRNAs in serum was significantly different between HCC group and control group.3、Serum miRNA-1915 may serve as a potential biomarker for HCC detection.更多还原