miR-26a作为胃癌预后生物标志物初探

发布时间：2018-08-27 11:36

【摘要】：背景:胃癌(gastric cancer,GC)是目前世界上发病率和死亡率分居第五位和第三位的常见恶性肿瘤。近年来,胃癌的全球发病率和死亡率都呈下降趋势,但胃癌仍是发展中国家癌症相关死亡的主要原因。在世界范围内,胃癌的男性发病率是女性的两倍,且发病率与死亡率都有显著的地域差别。在我国,胃癌是排名第三的致死性肿瘤,仅次于肺癌和肝癌。早期胃癌并无明显症状,临床确诊时多为中晚期,从而失去最佳治疗时机。尽管胃癌的诊断手段和治疗方案日渐先进,但是胃癌患者的预后仍然不佳。微小RNA(micro RNA,mi RNA)是在真核生物中发现的一类内源性的具有调控功能的非编码蛋白质小RNA,参与肿瘤细胞的生物调控过程,在肿瘤的诊断和预后中扮演着重要的角色。大量研究报道mi RNAs与包括胃癌在内的很多种恶性肿瘤的预后密切相关。选择合适的mi RNAs作为胃癌的预后生物标志物具有巨大的临床价值。本实验室前期通过Taq Man低密度芯片(Taq Man low density array,TLDA)筛选出四个在胃癌血浆中表达差异明显的mi RNAs(mi R-148a、mi R-142-3p、mi R-26a和mi R-195),本研究拟在胃癌患者的组织中检测出特异性表达的mi RNAs,并将其作为胃癌预后的生物学标志。方法:本研究通过RT-PCR(实时定量聚合酶链式反应)实验在一阶段南通胃癌石蜡样本中检测mi R-148a、mi R-142-3p、mi R-26a和mi R-195的表达水平,筛选出与胃癌预后相关的mi RNAs,在宜兴石蜡样本中继续验证一阶段研究结果。在Pubmed中搜索关于候选mi RNAs、gastric cancer和survival或prognosis有关的文献,运用STATA11.0等软件进行Meta分析后,再确定进行下一步的进行功能研究的mi RNA。在胃癌细胞株MGC 803、BGC 823、SGC 7901、MKN 28和正常细胞株GES-1检测mi RNAs本底表达,选择表达最低的细胞株进行功能实验探索。经RT-PCR证实转染成功后,通过CCK-8(cell counting kit-8,细胞计数试剂盒)细胞增殖试验分析细胞增殖能力的改变,运用平板克隆试验检测单个细胞的恶性程度,采用Transwell细胞迁移侵袭试验检验细胞迁移和侵袭能力的变化,应用流式细胞仪检测细胞周期凋亡的变化。结果:在一阶段南通387例石蜡样本结果显示,mi R-26a和mi R-148a的表达水平与预后显著相关,P值分别为0.009和0.005;mi R-142-3p和mi R-195的表达水平于预后无关,P值分别为0.320和0.119。因此在二阶段宜兴石蜡样本中不在验证mi R-142-3p和mi R-195。mi R-26a和mi R-148a的表达水平、浸润深度、淋巴结转移、TNM分期、分化程度与不良预后均有关系。多因素Cox回归模型显示mi R-26a和mi R-148a能改善胃癌的预后(mi R-26a:HR=0.76,95%CI=0.61-0.94;mi R-148a:HR=0.73,95%CI=0.58-0.91);另外,TNM分期和化疗也与胃癌预后显著相关,P值均0.05。鉴于前期已有多篇有关mi R-148a与胃癌研究报道,本研究主要侧重于mi R-26a与胃癌预后相关的生物学机制探讨。在胃癌细胞株MGC 803中mi R-26a显著低于正常细胞GES-1(P0.01)。在MGC 803细胞中过表达mi R-26a 48h后,增殖能力有明显下降(P0.001);克隆形成率下降了69%(P=0.004);癌细胞通过小室的迁移率下降了48%(P=0.028);侵袭能力显著下降(P=0.031)。流式细胞术显示增加细胞内mi R-26a表达,细胞周期无明显改变,但细胞凋亡率明显增高(P=0.004)。结论:癌组织中的mi R-26a、mi R-148a的表达水平可能与胃癌预后相关。肿瘤的TNM分期、浸润深度、淋巴结转移等均能影响胃癌生存时间。过表达mi R-26a可抑制胃癌的发生和发展。我们的研究提示mi R-26a的表达水平可以用来预测胃癌患者手术预后的情况,是潜在的预后预测生物标志物,为其今后的临床治疗提供重要参考。
[Abstract]:BACKGROUND: Gastric cancer (GC) is the fifth and third most common malignant tumor in the world. In recent years, the incidence and mortality of gastric cancer have been declining globally, but gastric cancer is still the main cause of cancer-related death in developing countries. In the world, the incidence of male gastric cancer is female. Gastric cancer is the third most lethal tumor in China, after lung cancer and liver cancer. Early gastric cancer has no obvious symptoms, and most of the clinical diagnosis is in the middle and advanced stages, thus losing the best opportunity for treatment. The prognosis of cancer patients is still poor. MicroRNAs (micro RNAs) are a class of endogenous non-coding protein small RNAs found in eukaryotes, which are involved in the biological regulation of tumor cells and play an important role in the diagnosis and prognosis of cancer. The prognosis of various malignant tumors is closely related. Selection of appropriate mi RNAs as prognostic biomarkers of gastric cancer is of great clinical value. MiR-195. We intend to detect the expression of specific mi RNAs in gastric cancer tissues and use them as biological markers for prognosis of gastric cancer. Methods: The expression of MI R-148a, mi R-142-3p, mi R-26a and MI R-195 in paraffin samples of one-stage Nantong gastric cancer was detected by RT-PCR (real-time quantitative polymerase chain reaction). In Pubmed, we searched for the literature about candidate mi RNAs, gastric cancer, survival or prognosis, and then used STATA 11.0 software for meta-analysis to determine the next step of functional research in the stomach. The expression of MIRNAs in cancer cell lines MGC 803, BGC 823, SGC 7901, MKN 28 and normal cell lines GES-1 were detected. The cell lines with the lowest expression were selected for functional experiment. After the transfection was confirmed by RT-PCR, the cell proliferation test of CCK-8 (cell counting kit-8, cell counting kit-8) was used to analyze the change of cell proliferation ability. Prolongation test was used to detect the degree of malignancy of single cell, Transwell cell migration and invasion test was used to examine the changes of cell migration and invasion ability, and flow cytometry was used to detect the changes of cell cycle apoptosis. The expression levels of MIR-142-3p and MIR-195 were 0.009 and 0.005, respectively. The expression levels of MIR-142-3p and MIR-195 were not correlated with prognosis, P values were 0.320 and 0.119, respectively. Cox regression model showed that MIR-26a and MIR-148a could improve the prognosis of gastric cancer (MIR-26a: HR = 0.76, 95% CI = 0.61-0.94; MIR-148a: HR = 0.73, 95% CI = 0.58-0.91); in addition, TNM staging and chemotherapy were also significantly correlated with the prognosis of gastric cancer, P values were 0.05. Biological mechanisms associated with prognosis of gastric cancer were investigated. MiR-26a in gastric cancer cell line MGC 803 was significantly lower than that in normal cell GES-1 (P 0.01). Overexpression of MIR-26a in MGC 803 cells significantly decreased the proliferation ability (P 0.001), the clone formation rate (P = 0.004), the migration rate of cancer cells through compartments (P = 0.028), and the invasion rate (P = 0.028). The expression of MIR-26a and MIR-148a in cancer tissues may be related to the prognosis of gastric cancer. TNM stage, depth of invasion and lymph node metastasis can all affect the prognosis of gastric cancer. Overexpression of MIR-26a can inhibit the occurrence and development of gastric cancer. Our study suggests that the expression of MIR-26a can be used to predict the prognosis of gastric cancer patients after surgery. It is a potential biomarker for predicting prognosis and provides an important reference for future clinical treatment.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.2

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