【作者】 韦源；

【导师】 王家祥；

【作者基本信息】 郑州大学 ， 小儿外科， 2010， 硕士

【摘要】 背景和目的MicroRNA(miRNA)是一类广泛分布在动植物中的高度保守的非编码单链小RNA (nod-coding small RNA),长度大约有19nt～23nt,它的功能是负调控基因表达。目前,随着对miRNA研究的不断深入,发现了其许多未知的功能,包括调节细胞增殖、分化和凋亡,主要是靠调控信号分子的表达实现,这些信号分子包括细胞因子、生长因子、转录因子、促凋亡和抗凋亡因子。通过激活原癌基因的表达以及抑癌基因的突变缺失,从而引起肿瘤的发生发展。因而：miRNA与恶性肿瘤的发生、发展、预后有着密切的关联。而肝细胞癌(hepatocellular carcinoma, HCC)又是我国很常见的恶性肿瘤,其死亡率在各类肿瘤致死率中排名第二。肝细胞癌的发生、发展是多因素、多步骤的一系列复杂过程。所以,寻找肝细胞癌发生、发展的相关基因,了解肝细胞癌的分子遗传学机制从而为肝细胞癌的诊断、治疗提供理论基础为目前的研究热点。miRNA的研究为肝癌的研究提供了一个新的思路和希望。但是迄今为止,肝癌的发生,发展的miRNA相关的分子机制仍不是很清楚,进一步探索肝癌的miRNA分子发生机制对提高肝癌的诊断与治疗水平有很重要的意义,因此本研究拟采用miRNA芯片技术筛选肝癌细胞株SMMC-7721和人胚肝细胞株CCC-HEL-1差异表达的miRNAs,建立其miRNA表达谱,筛选感兴趣的miRNA,为以后的研究提供依据。并探讨其与肝癌的发生发展的关系,以期为阐明肝癌的发生的分子机制提供新的依据。方法体外培养人肝癌细胞株SMMC-7721和人胚肝细胞株CCC-HEL-1, Trizol法抽取总的RNA后,紫外吸收测定法在260nm和280nm以及变性琼脂糖凝胶电泳鉴定RNA的质量和浓度,用的miRCURYTM Array Power Labeling kit分离并Hy3荧光标记miRNA,将Hy3荧光标记的miRNA进行与miRNA芯片杂交反应,洗片后,利用生物芯片扫描仪扫描芯片,将图像信号转换为数字信号,进行数据分析和统计学处理,筛选有统计学意义的差异miRNA,导入Cluster3.0进行聚类分析,并挑选感兴趣的miRNA应用Real time PCR方法进行验证。结果芯片结果显示,以两组细胞株差异表达的miRNAs (P<0.05)以上调或者下调两倍(fold change>2)作为筛选标准。发现SMMC-7721较CCC-HEL-1细胞株差异表达的miRNAs共238个。上调的miRNAs154个,下调的miRNAs有84个。上调4倍(fold change>4)的miRNAs共64个,上调10倍(fold change >10)的miRNAs共24个,由高到低依次为hsa-miR-205,hsa-miR-16,hsa-miR-1 01,hsa-miR-195,hsa-miR-30b,hsa-miR-30e,hsa-miRPlus-E1209,hsa-miR-200a,hsa-m iR-378,hsa-miR-15a,hsa-let-7f,hsa-miR-141,hsa-miR-424,hsa-miR-181a-2*,hsa-miR-24,hsa-miR-30a,hsa-let-7a,hsa-miR-26a,hsa-miR-26a-2*,hsa-miR-130a,hsa-miR-15b, hsa-miR-23a,hsa-miR-27a,hsa-miR-193b,hsa-miRPlus-C1005,其中上调最高的hsa-miR-205上调52倍,下调的miRNAs共84个,下调4倍(fold change>4)的有22个,下调5倍(fold change>5)的有10个,为下调倍数由高到低依次为hsa-miRPlus-E1120, hsa-miRPlus-A1027, hsa-miRPlus-E1012, hsa-miR-608, hsa-miRPlus-F1208, hsa-miRPlus-F1205, hsa-miR-767-3p, hsa-miR-373*, hsa-mi R-769-3p, hsa-miRPlus-E1245,其中的hsa-miRPlus-E1120下调13倍应用Realtime PCR方法检测let-7f在SMMC-7721-1和CCC-HEL-1-4中表达情况,溶解曲线呈单峰,显示引物的特异性良好,结果显示,与CCC-HEL-1细胞株相比,SMMC-7721细胞株中的let-7f表达上调,为上调2.3倍。应用Realtime PCR方法检测miR-205在SMMC-7721-1和CCC-HEL-1-4由表达情况,溶解曲线呈单峰,显示引物的特异性良好,结果显示,与CCC-HEL.1细胞株相比,SMMC-7721细胞株中的miR-205表达上调,为上调2.7倍。结论1、实验结果提示人肝癌细胞株SMMC-7721与人胚肝细胞株CCC-HEL-1存在差异性的,miRNA。建立了其miRNA差异表达谱。为进一步研究差异miRNA提供依据。2、经过Realtime PCR验证,miR-205和let-7f在肝癌细胞株中明显上调。为其功能学检测提供依据。3、hsa-miRPlus-E1120, hsa-miRPlus-A1027, hsa-miRPlus-E1012, hsa-miR-608,等miRNA在肝癌中下调。更多还原

【Abstract】 Background and objectiveMicroRNA (miRNA) are a class of non-coding small RNA (nod-coding small RNA), They are widely distributed in plants and animals with length of about 19nt～23nt single-stranded RNA, its function is a negative regulator of gene expression. Now, with the deepening research of miRNA and many of its un known functions were found, including regulation of cell proliferation, differen tiation and apoptosis, mainly by regulating the expression of signaling molecul es to achieve. These signaling molecules including cytokines, growth factors, tr anscription factor, pro-apoptotic and anti-apoptotic factor. By activating the exp ression of proto-oncogenes and tumor suppressor gene mutation deletionWhich led to the development of tumors. MiRNA are closely related to t he occurrence, development, and prognosis of cancerHCC (hepatocellular carcinoma, HCC) is a common malignant tumor in C hina, it’s mortality rate is second highest in the malignant tumor .The occurre nce and development of HCC is a multi-factor, multi-step complex process. Lo oking for occurrence and development related genes of HCC, in order to un derstand the molecular genetic mechanism of hepatocellular carcinoma so as to HCC diagnosis, treatment and providing a theoretical foundation is the current research focus. The research of miRNA provides a new idea and hope for ca ncer researchBut so far, the occurrence of liver cancer and development related to the molecular mechanism of miRNA is still not very clear, and further explore m olecules mechanism of the miRNA in liver cancer will improving the diagnos is and treatment of liver cancer and this is very important, the present study was screening differentially expressed miRNAs between hepatoma cell line SM MC-7721 and human embryo liver cell line CCC-HEL-1 by miRNA microarra y, and to explore the relationship between the occurrence and development of liver cancer.It will clarify the molecular mechanisms of liver cancer.the present study was screening differentially expressed miRNAs between hepatoma cell line SMMC-7721 and human embryo liver cell line CCC-HEL-1 by miRNA microarray, and to explore the relationship between the occurrenc e and development of liver cancer.It will clarify the molecular mechanisms of liver cancer.validate differential expression of MicroRNA to explore the relationship be tween differential expression of miRNA and liver cancer by Real-time quantitat ive PCRMethodsHuman hepatoma cell line SMMC-7721 and human embryo liver cell line CC C-HEL-1 were purchased from Chinese Academy of Medical Sciences, Institute of Basic Medical Cell Center of Basic Medicine,Total RNA extraction by Tr izol method, Identify the quality and concentration of RNA by NanoDro p ND-1000 at 260nm and 280nm and denaturing Agarose Gel Electrophoresi s .Isolating and labeling miRNA by Hy3 and Exiqon’s miRCURYTM Array Power Labeling kit, the miRNA was hybridized on miRNAarray, Scanning is performed with the Axon GenePix 4000B microarray scanner.The result of miRNA array was verified by real time PCRResultsBy means of up-regulation or down-regulation up to twice (fold change> 2) a s a screening criteria, This study showed that total 238 differential expressed miRNAs were found, including 154 over-expression and 84 low-expression mi RNAs. Four-fold increase (fold change> 4) of the miRNAs were 64,10-fold i ncrease (fold change>10) of the miRNAs were24,ranked as hsa-miR-205, hsa-m iR-16, hsa-miR-101, hsa-miR-195, hsa-miR-30b, hsa-miR-30e,hsa-miRPlus-E1209, hsa-miR-200a,hsa-miR-378,hsa-miR-15a,hsa-let-7f,hsa-miR-141,hsa-miR-424,hsa-m iR-181 a-2*,hsa-miR-24,hsa-miR-30a,hsa-let-7a,hsa-miR-26a,hsa-miR-26a-2*,hsa-mi R-130a,hsa-miR-15b,hsa-miR-23a,hsa-miR-27a,hsa-miR-193b,hsa-miRPlus-C1005, The highest fold change was up to 52 (P< 0.05). A total of 84 of the miRN As down-regulate, there were 22 miRNAs are up to 4 times (fold change> 4) , there were 10 are up to 5 times (fold change> 5), ranked as hsa-miRPl us-E1120, hsa-miRPlus-A1027, hsa-miRPlus-E1012, hsa-miR-608, hsa-miRPlus-F 1208, hsa-miRPlus-F1205, hsa-miR-767-3p, hsa-miR-373*, hsa-miR-769 -3p, hs a-miRPlus-E1245, which hsa-miRPlus-E1120 reduced 13-fold.Application Realtime PCR to detect the expression of let-7f between S MMC-7721-1 and CCC-HEL-1-4.The dissolution curve has a single peak, This indicating a good specificity of the primers .Compare to CCC-HEL-1 cell line , The expression of let-7f is increase 2.3 times in SMMC-7721 cell line.Application Realtime PCR to detect the expression of miR-205 betweenSMMC-7721-1 and CCC-HEL-1-4.The dissolution curve has a single peak. This indicating a good specificity of the primers .Compare to CCC-HEL-1 cell line. The expression of miR-25 is increase 2.7times in SMMC-7721 cell lineCouclusion 1, The result suggests that there are different miRNA between the human hepatoma cell line SMMC-7721 and human fetal liver cell line CCC-HEL-1 Established between the miRNA expression profiles.2, After validation of Realtime PCR, miR-205 and let-7f were up-regulatio n in liver cancer cell lines3, Hsa-miRPlus-E1120, hsa-miRPlus-A1027, hsa-miRPlus-E1012, hsa-miR-6 08, and other miRNA are down-regulation in liver cancer cell line.更多还原