胃癌发生相关的miRNA的筛选及miR-196a的功能学研究
发布时间：2018-09-12 15:42
【摘要】：胃癌(Gastric Cancer, GC)是较为常见的消化道系统恶性肿瘤。近年来,在欧美等发达国家,GC的发病率逐年下降,但在东亚地区尤其是中国,GC的发病率并未见显著下降,同时,GC的死亡率也居高不下。GC患者的5年生存率(5-year survival rate)徘徊在20%～25%,中位生存时间(median survival duration)仅为24个月。导致胃癌患者生存率低的主要原因是早诊率低、转移率高、无敏感有效的治疗方法及研究基础薄弱等。深入研究胃癌的发生发展机理,寻找能够早期发现、监测疾病进展和预测疗效的分子标志物,有助于提高胃癌患者的生活质量及延长生存期。MicroRNA(MiRNA)是一种调控癌基因和抑癌基因表达非编码单链小分子RNA,研究显示,其进化保守,长度约22个核苷酸,在肿瘤增殖(proliferation)、侵袭(invasion)和转移(metastasis)过程起到重要作用。研究表明miRNAs在胃癌的诊断、疗效监测和预后评估中具有重要价值,但是miRNA在胃癌发生发展中的具体作用机制及其临床意义还有待于进一步研究,因此,本研究将筛选与胃癌发生相关的miRNAs表达谱,并对其中的部分关键的miRNA进行功能学和分子生物学研究。第一部分 胃癌发生相关的microRNA表达谱系筛选目的：筛选与人胃癌发生高度相关的miRNAs,期望为胃癌的诊断提供新的工具和方法。方法：选取胃癌癌旁非肿瘤粘膜上皮组织66例和原发灶胃癌组织162例。利用RT-PCR检测两者之间1920个miRNAs差异,利用生物信息学技术分析找出能区分胃癌和癌旁非肿瘤上皮组织中的miRNAs表达模型。结果：筛选出12个与胃癌的发生高度相关的miRNAs (hsa-miR-196a, has-miR-196b, hsa-miR-204, hsa-miR-133b, hsa-miR-148a, hsa-miR-375, hsa-miR-551b, hsa-miR-31#, hsa-miR-224, hsa-miR-31, hsa-miR-378d和hsa-miR-37),其中2个上调,10个下调,能区分胃癌与正常粘膜,其有效性95.08%,特异性97.56%(P0.005)。结论：胃癌发生过程中伴有niRNAs的表达异常,我们筛选出与胃癌发生高度相关的miRNA表达谱能够较好的区分胃癌和非癌组织,为胃癌的诊断提供了新的工具。第二部分：hsa-miR-196a在胃癌细胞系中功能学及作用靶点的研究目的：探讨hsa-miR-196a促进胃癌细胞增殖、侵袭和转移的功能学研究,以及潜在的分子生物学机制。方法：构建hsa-miR-196a-shRNA下调胃癌细胞SGC7901和BGC823中miR-196a的表达,利用增殖实验(MTT)、克隆形成(Colony formation)、划痕实验(Wound healing)、侵袭转移实验(Transwell)检测miR-196a表达下调后胃癌细胞的功能学改变,并利用RT-PCR技术检测miR-196a以及可能的作用靶点HOXA7、HOXC8、SLC9A6、ZMYND11和CCDC47的mRNA的表达。结果:①RT-PCR结果显示hsa-miR-196a-shRNA能够成功干扰SGC7901和BGC823中miR-196a的表达：②SGC7901和BGC823中miR-196a的表达下调后,其细胞的增殖能力、克隆形成能力、爬行及侵袭能力均明显下降(P0.05)；③SGC7901和BGC823中miR-196a的表达下调后,HOXA7、HOXC8和CCDC47表达在上述两种细胞中均明显下调,然而SLC9A6、ZMYND11在SGC7901实验组中表达上调,在BGC823实验组中表达下调。结论：miR-196a可能通过调控HOXA7、HOXC8和CCDC47等基因mRNA水平的改变,从而促进胃癌细胞的增殖、侵袭和转移。第三部分：探讨HOXA7和HOXC8在人胃癌中的表达及其临床价值目的：研究HOXA7和HOXC8在人胃癌组织中的表达及临床意义、预后价值。方法：利用免疫组化(IHC)和组织芯片(TMA)检测HOXA7和HOXC8在人胃癌组织标本中的表达,并统计分析其表达与胃癌生物学行为及预后的关系。结果：①胃癌组织中HOXA7和HOXC8的表达明显高于癌旁正常粘膜,两者的表达率分别为：55.6%(134/241)和60.6%(146/241)。② HOXA7高表达与浸润深度(tumor invasion)呈正相关(P=0.02), HOXC8高表达与肿瘤大小(tumor size)、浸润深度(tumor invasion)、淋巴结转移(regional lymph node metastasis)、TNM (TNM stage)有相关性(P0.05),③胃癌组织中HOXA7和HOXC8的表达与预后的相关性：单因素生存分析(Kaplan-Meier analysis)显示HOXA7过度表达的GC患者总生存期(overall survival)为56个月,明显低于HOXA7低表达的GC患者[75.6月(P=0.003)]。HOXC8过度表达的GC患者预后也明显较HOXC8低表达的GC患者差[中位生存时间(median survival duration):24.9月V.S 90.0月, P0.001], COX多因素回归分析(Cox regression model)显示HOXC8过度表达是判断GC患者重要的独立预后因素之一。结论：HOXA7和HOXC8的过度表达在GC的发生和发展过程中起到一定的促进作用,有成为潜在的胃癌分子治疗靶点和评估GC患者预后的分子标志物(tumor biomarkers)的可能。
[Abstract]:Gastric cancer (GC) is a common malignant tumor of the digestive tract. In recent years, the incidence of GC has declined year by year in developed countries such as Europe and the United States, but in East Asia, especially in China, the incidence of GC has not decreased significantly. At the same time, the mortality rate of GC is still high. The 5-year survival rate of GC patients hovers at 20%. The main reasons for the low survival rate of gastric cancer patients are low early diagnosis rate, high metastasis rate, insensitive and effective treatment methods and weak research foundation. MicroRNA is a small non-coding single-stranded RNA that regulates the expression of oncogenes and tumor suppressor genes. Studies have shown that it is evolutionarily conserved and has a length of about 22 nucleotides, and plays an important role in tumor proliferation, invasion and metastasis. Studies have shown that microRNAs play an important role in the diagnosis, efficacy monitoring and prognosis evaluation of gastric cancer. However, the specific mechanism and clinical significance of microRNAs in the development of gastric cancer need to be further studied. Therefore, this study will screen the expression profiles of microRNAs associated with gastric cancer and some of the key microRNAs. Methods: 66 cases of non-tumor mucosal epithelium adjacent to gastric cancer and 162 cases of primary gastric cancer were selected. Results: Twelve highly correlated microRNAs (hsa-microRNAs-196a, has-microRNAs-196b, hsa-microRNAs-204, hsa-microRNAs-133b, hsa-microRNAs-148a, hsa-microRNAs-148a, hsa-microRNAs-microRNAs) were screened for gastric cancer. - 375, hsa-Mi-551b, hsa-Mi-31#, hsa-Mi-224, hsa-Mi-Mi-31, hsa-Mi-378d and hsa-Mi-37, two of which were up-regulated and 10 down-regulated, were able to distinguish gastric cancer from normal mucosa. The efficacy was 95.08% and specificity was 97.56% (P 0.005). Conclusion: Abnormal expression of niRNAs was associated with gastric cancer. We screened the expression of microRNA highly associated with gastric cancer. Duplex spectroscopy provides a new tool for the diagnosis of gastric cancer. Part II: Functional and functional targets of hsa-microRNA-196a in gastric cancer cell lines. Objective: To investigate the role of hsa-microRNA-196a in promoting the proliferation, invasion and metastasis of gastric cancer cells and its potential molecular biological mechanisms. HSa-microRNA-196a-shRNA was constructed to down-regulate the expression of microRNA-196a in gastric cancer cells SGC7901 and BGC823. Colony formation, Wound healing, invasion and metastasis were used to detect the functional changes of gastric cancer cells after down-regulated expression of microRNA-196a. RT-PCR was used to detect the expression of microRNA-196a and microRNA-196a. Results: The results of RT-PCR showed that hsa-miR-196a-shRNA could successfully interfere with the expression of Mi-196a in SGC7901 and BGC823. After down-regulation of the expression of Mi-196a in SGC7901 and BGC823, the proliferation, cloning, crawling and invasiveness of SGC7901 and BGC823 cells were significantly decreased. The expression of HOXA7, HOXC8 and CCC47 was significantly down-regulated in SGC7901 and BGC823, but the expression of SLC9A6 and ZMYND11 was up-regulated in SGC7901 and down-regulated in BGC823. Conclusion: Mi-196a may regulate the mRNA levels of HOXA7, HOXC8 and CCC47. Objective: To investigate the expression and clinical value of HOXA7 and HOXC8 in human gastric cancer. To study the expression and prognostic value of HOXA7 and HOXC8 in human gastric cancer. Methods: Immunohistochemistry (IHC) and tissue microarray (TMA) were used to detect HOXA7 and HOXC8 in human stomach. Results: The expression of HOXA7 and HOXC8 in gastric cancer tissues was significantly higher than that in adjacent normal mucosa. The expression rates of HOXA7 and HOXC8 were 55.6% (134/241) and 60.6% (146/241), respectively. The high expression of HOXC8 was correlated with tumor size, tumor invasion, regional lymph node metastasis, and TNM stage (P 0.05). The correlation between the expression of HOXA7 and HOXC8 and prognosis in gastric cancer was analyzed by Kaplan-Meier analysis. The overall survival was 56 months, significantly lower than that of GC patients with low HOXA7 expression [75.6 months (P = 0.003)]. The prognosis of GC patients with overexpression of HOXC8 was also significantly worse than that of GC patients with low HOXC8 expression [median survival time: 24.9 months V.S 90.10 months, P.001], COX multivariate regression model CONCLUSION: Overexpression of HOXC8 and HOXA7 may play an important role in the occurrence and development of GC, and may be a potential therapeutic target for gastric cancer and a molecular marker for evaluating the prognosis of GC patients.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.2
本文编号：2239482
[Abstract]:Gastric cancer (GC) is a common malignant tumor of the digestive tract. In recent years, the incidence of GC has declined year by year in developed countries such as Europe and the United States, but in East Asia, especially in China, the incidence of GC has not decreased significantly. At the same time, the mortality rate of GC is still high. The 5-year survival rate of GC patients hovers at 20%. The main reasons for the low survival rate of gastric cancer patients are low early diagnosis rate, high metastasis rate, insensitive and effective treatment methods and weak research foundation. MicroRNA is a small non-coding single-stranded RNA that regulates the expression of oncogenes and tumor suppressor genes. Studies have shown that it is evolutionarily conserved and has a length of about 22 nucleotides, and plays an important role in tumor proliferation, invasion and metastasis. Studies have shown that microRNAs play an important role in the diagnosis, efficacy monitoring and prognosis evaluation of gastric cancer. However, the specific mechanism and clinical significance of microRNAs in the development of gastric cancer need to be further studied. Therefore, this study will screen the expression profiles of microRNAs associated with gastric cancer and some of the key microRNAs. Methods: 66 cases of non-tumor mucosal epithelium adjacent to gastric cancer and 162 cases of primary gastric cancer were selected. Results: Twelve highly correlated microRNAs (hsa-microRNAs-196a, has-microRNAs-196b, hsa-microRNAs-204, hsa-microRNAs-133b, hsa-microRNAs-148a, hsa-microRNAs-148a, hsa-microRNAs-microRNAs) were screened for gastric cancer. - 375, hsa-Mi-551b, hsa-Mi-31#, hsa-Mi-224, hsa-Mi-Mi-31, hsa-Mi-378d and hsa-Mi-37, two of which were up-regulated and 10 down-regulated, were able to distinguish gastric cancer from normal mucosa. The efficacy was 95.08% and specificity was 97.56% (P 0.005). Conclusion: Abnormal expression of niRNAs was associated with gastric cancer. We screened the expression of microRNA highly associated with gastric cancer. Duplex spectroscopy provides a new tool for the diagnosis of gastric cancer. Part II: Functional and functional targets of hsa-microRNA-196a in gastric cancer cell lines. Objective: To investigate the role of hsa-microRNA-196a in promoting the proliferation, invasion and metastasis of gastric cancer cells and its potential molecular biological mechanisms. HSa-microRNA-196a-shRNA was constructed to down-regulate the expression of microRNA-196a in gastric cancer cells SGC7901 and BGC823. Colony formation, Wound healing, invasion and metastasis were used to detect the functional changes of gastric cancer cells after down-regulated expression of microRNA-196a. RT-PCR was used to detect the expression of microRNA-196a and microRNA-196a. Results: The results of RT-PCR showed that hsa-miR-196a-shRNA could successfully interfere with the expression of Mi-196a in SGC7901 and BGC823. After down-regulation of the expression of Mi-196a in SGC7901 and BGC823, the proliferation, cloning, crawling and invasiveness of SGC7901 and BGC823 cells were significantly decreased. The expression of HOXA7, HOXC8 and CCC47 was significantly down-regulated in SGC7901 and BGC823, but the expression of SLC9A6 and ZMYND11 was up-regulated in SGC7901 and down-regulated in BGC823. Conclusion: Mi-196a may regulate the mRNA levels of HOXA7, HOXC8 and CCC47. Objective: To investigate the expression and clinical value of HOXA7 and HOXC8 in human gastric cancer. To study the expression and prognostic value of HOXA7 and HOXC8 in human gastric cancer. Methods: Immunohistochemistry (IHC) and tissue microarray (TMA) were used to detect HOXA7 and HOXC8 in human stomach. Results: The expression of HOXA7 and HOXC8 in gastric cancer tissues was significantly higher than that in adjacent normal mucosa. The expression rates of HOXA7 and HOXC8 were 55.6% (134/241) and 60.6% (146/241), respectively. The high expression of HOXC8 was correlated with tumor size, tumor invasion, regional lymph node metastasis, and TNM stage (P 0.05). The correlation between the expression of HOXA7 and HOXC8 and prognosis in gastric cancer was analyzed by Kaplan-Meier analysis. The overall survival was 56 months, significantly lower than that of GC patients with low HOXA7 expression [75.6 months (P = 0.003)]. The prognosis of GC patients with overexpression of HOXC8 was also significantly worse than that of GC patients with low HOXC8 expression [median survival time: 24.9 months V.S 90.10 months, P.001], COX multivariate regression model CONCLUSION: Overexpression of HOXC8 and HOXA7 may play an important role in the occurrence and development of GC, and may be a potential therapeutic target for gastric cancer and a molecular marker for evaluating the prognosis of GC patients.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.2
【参考文献】
相关期刊论文 前1条
1 Vivian Yvonne Shin;Kent-Man Chu;;MiRNA as potential biomarkers and therapeutic targets for gastric cancer[J];World Journal of Gastroenterology;2014年30期
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