【作者】 朱娟娟；

【导师】 张胜权； 郑晓飞；

【作者基本信息】 安徽医科大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 目的:研究人类长链非编码RNA母系印记基因3(MEG3)在肝癌发生发展过程中的作用,并探讨其分子机制。方法:通过高通量测序比较肝癌组织和癌旁组织非编码RNA的表达变化,筛选出若干肝癌相关长链非编码RNA基因(long noncoding RNAs, lncRNAs)。构建出这些lncRNAs的真核表达载体,并筛选出高表达的稳定细胞株株,运用qRT-PCR、Western Blots、RNA-pulldown和表达谱芯片等技术探索MEG3的相互作用蛋白及作用机理。结果:qRT-PCR方法在多组肝癌组织及其癌旁组织中检测若干个lncRNAs的表达,结果显示多数肝癌组织中多个lncRNA异常表达,其中MEG3表达显著下调。成功构建了若干个lncRNAs的真核表达质粒,筛选获得了稳定高表达MEG3基因的SK-Hep-1肝癌细胞株,表达谱芯片结果经生物信息学分析得到若干可能与MEG3发挥抑制细胞增殖有影响的基因。结论:MEG3在多数肝癌组织中表达下调,MEG3能显著地抑制细胞增殖,但对细胞周期和细胞凋亡可能无显著作用,本研究还获得了稳定高表达MEG3基因的SK-Hep-1肝癌细胞株,并应用生物信息学对其表达谱分析,筛选获得了MEG3功能相关基因。结合文献报道和初步验证,发现有以下一些因子可能是MEG3调节的靶基因,如IL-8,IL-24,IL-1A,CPEB4,CHAC1,BACT1等。这些蛋白均与细胞生长和增殖相关。更多还原

【Abstract】 Objective:Study of human long noncoding RNA maternal imprinted genes 3(MEG3) on cell cycle and apoptosis, and exploring its molecular mechanisms. Methods:Comparison of the high-throughput sequencing of hepatocellular carcinoma and liver tissues, screen out several long noncoding RNAs. Constructed expression vector of these lncRNAs, and selected stable lines with high expression, using qRT-PCR,western blot,RNA-pulldown and microarray techniques to explore the interacting proteins with MEG3 and its mechanisms. Results: By using qRT-PCR to validate screened several lncRNAs in 23 groups of HCC and its adjacent tissues, we found that the expression of MEG3 decreases in most HCC tissues. Finally constructed the eukaryotic expression plasmid of several lncRNAs, screened stable cell lines with high expression of MEG3, acquired several genes which may be relavent to inhibition of cell prolifiration of MEG3 using bioinformation to analyse the microarray. Conclusions: The expression of MEG3 decreases in most HCC tissues; MEG3 could significantly inhibit cell prolifiration, without affect cell cycle and apoptosis, we also acquired stable hepatoma cells strains with high expression of MEG3, and bioinformatic analysis of their expression profiles, selected genes related to MEG3.Combination of literature and preliminary validation, the following factors may be target genes which were regulated by MEG3,such as IL-8,IL-24,IL-1A,CPEB4,CHAC1,BACT1 and so on. These proteins are associated with cell growth and proliferation.更多还原