【作者】 张小刚；

【导师】 安利国； 郑晓飞；

【作者基本信息】 山东师范大学 ， 细胞生物学， 2011， 硕士

【摘要】 H19/Igf2（胰岛素样生长因子2,insulin-like growth factor 2）印迹基因隶属于一个基因印迹群,分别位于人染色体11P15.5,在进化上具有高度的保守性。H19基因为母源性印迹基因,而Igf2基因为父源性印迹基因,两者相距90kb,表达调控相关,对个体的生长、发育及行为发展有重要的作用。H19/Igf2印迹基因在基因结构和类型上涉及到了编码基因、非编码基因、印迹基因、宿主基因、内含子基因、microRNA基因和反义基因,是研究基于RNA介导的遗传信息表达调控网络模式的最佳模型之一。首先,为研究H19/Igf2印迹基因模型中miR-483、miR-675及其宿主基因Igf2、H19之间在不同遗传背景、不同生理病理状态下共同的表达模式和表达调控规律,我们选取各种不同的细胞系,同时利用不同实验手段,检测分析上述各基因之间的表达调控关系,建立其表达调控模型。其次,本研究同时关注了miR-483的靶基因的预测和筛选,筛选获得了miR-483的一个靶基因-PDGFB （血小板来源的生长因子,platelet-derived growth factor beta polypeptide）,为下一步继续研究miR-483的功能以及调控奠定基础。取得的主要进展有:1.Igf2及H19基因除在少数细胞系中皆低表达外,在多数细胞系中其表达呈拮抗关系;而miR-483及miR-675在各细胞系中的表达与其宿主是协同一致的。结果显示,Igf2基因在HEK-293T、A549细胞中表达相对较高,而在HepG2、HeLa、HCC-Lm3及BEL-7402细胞中表达水平较低;H19基因在HepG2和HeLa、HCC-Lm3及BEL-7402细胞中表达相对较高,而在HEK-293T和A549细胞中表达水平较低;而在K562细胞中两者皆低表达;miR-483及miR-675在各细胞系中的表达与其宿主是协同一致的。2.miR-483很可能参与IGF2信号通路介导的肿瘤细胞的增殖和凋亡调控。结果显示,Chromeceptin处理HEK-293T、A549和HepG2细胞6h后,IGF2与miR-483在各细胞中表达变化不一致,IGF2在各细胞中的表达水平无明显变化,而miR-483则表达明显下调。Chromeceptin很可能通过抑制IGF2信号通路抑制了miR-483的生成过程,从而使成熟miR-483的表达下调。3.miR-483及miR-675表达会受其他因子的调控而与其宿主基因的表达不一致。结果显示,CoCl₂诱导HepG2、HeLa和A549细胞缺氧24h后,miR-483及miR-675在各细胞系中的表达与其宿主基因Igf2及H19的表达皆不一致。HepG2和HeLa细胞中H19表达无明显变化,A549细胞中H19的表达有所下调,而各细胞中miR-675的表达明显上调;各细胞中IGF2表达皆下调,而miR-483的表达明显上调。缺氧上调miR-483与miR-675的表达很可能是通过介导其生成,促进其表达水平上调。miR-483与miR-675具有其各自独特的加工机制。4.Igf2是miR-483的宿主基因,miR-483却能反馈下调Igf2基因的mRNA表达水平; miR-675能够抑制其宿主基因H19对IGF2的下调,而上调IGF2的表达。结果显示,过表达miR-483后IGF2的表达水平下调,而对H19的表达没有影响。同时,过表达miR-675后,H19表达无明显变化,而IGF2则表达上调。初步认为,miR-675可能负反馈调节其宿主基因H19的功能,促进IGF2的表达。5.筛选得到了miR-483的一个靶基因-PDGFB。过表达miR-483后PDGFB的mRNA水平下调初步认为PDGFB是miR-483的候选靶基因;双荧光素酶报告基因证实,miR-483能够直接作用于PDGFB的3’UTR区;过表达miR-483后可以显著抑制HepG2细胞中内源PDGFB蛋白的表达;Chromeceptin处理HepG2细胞6h及12h后,PDGFB的表达明显上调;而CoCl₂缺氧诱导处理HepG2细胞6h及24h后,PDGFB的表达明显下调。综上,本研究揭示了H19/Igf2印迹模型中,miR-483,miR-675及其宿主基因Igf2,H19在不同细胞系中的共同的表达规律以及调控关系,同时筛选获得了miR-483的一个靶基因-PDGFB。更多还原

【Abstract】 The H19/Igf2（insulin-like growth factor 2）genes belong to an imprinted cluster, conserved on human chromosome 11p15.5. H19 is maternally expressed and Igf2 gene is transcribed from the paternal allele. These genes are 90kb apart and play important role during embryonic growth, development and behavior development. This locus is one of best paradigms to study and research the expression regulation networks by RNA, which contains coding gene, non-coding gene, imprinted gene, host gene, sequence gene, microRNA and antisense gene.First of all, to study a common mechanism and mode of regulation between miR-483, miR-675 and their host genes Igf2, H19 in different backgrounds of heredity and physiological, pathologic status, we chose different types cell lines and used different experimental methods to identify the expression of these genes for finding a common mode of regulation. Secondly, our study focused on predicting and screening target genes of miR-483. We identified PDGFB （platelet-derived growth factor beta polypeptide） as one of miR-483 target genes to establish foundation for identifying miR-483 function.The main progresses are listed:1. Igf2 and H19 genes were expressed antergically in most of cell lines except that these genes are all low expression in a few cell lines;miR-483,miR-675 and their host genes Igf2,H19 were coordinate expressed in all cell lines we chose. Results showed that IGF2 was high expression in HEK-293T and A549 but low expression in HepG2, HeLa, HCC-Lm3 and BEL-7402; H19 was high expression in HepG2, HeLa, HCC-Lm3 and BEL-7402 but low expression in HEK-293T and A549; IGF2 and H19 were all low expression in K562; miR-483, miR-675 and their host genes Igf2, H19 were coordinate expressed in all cell lines.2. miR-483 involved probably in the pathway of IGF2 signals mediated tumor cell proliferation and apoptosis regulation. Results showed that miR-483 and its host gene Igf2 were not coordinate expressed in HEK-293T, A549 and HepG2 after 6h using Chromeceptin.IGF2 was detected no visible change but miR-483 was detected down regulation in HEK-293T, A549 and HepG2. It was probable that Chromeceptin inhibited miR-483 generating process by inhibiting the pathway of IGF2 signals so that make mature miR-483 down regulation.3. miR-483 and miR-675 regulated by other factors so that they were not completely coordinate expressed and their host genes. Results showed that miR-483 and miR-675 were not coordinate expressed in HepG2, HeLa and A549 after 24h under hypoxic conditions by CoCl₂.H19 was detected no visible change in HepG2 and HeLa, down regulation in A549 but miR-675 was obvious up-regulation in all cell lines we chose; IGF2 was detected down regulation in all cell lines but miR-483 was obvious up-regulation. These results indicated that miR-483 and miR-675 generating processes were all promoted under hypoxic conditions so that their expression levels were all up-regulated; miR-483 and miR-675 had their respective generating process regulated by other factors.4. Igf2 gene was miR-483 host gene but Igf2 mRNA was feedback down regulated by miR-483; H19 could down regulated IGF2 but miR-675 inhibited H19 function to up-regulate IGF2. Results showed that ectopic expression of miR-483 could down regulate Igf2 expression level and had no effect to H19 .Meanwhile, ectopic expression of miR-675 had also no effect to its host gene H19 but could up-regulate Igf2 gene. It is probable that miR-675 was feedback its host gene H19 to up-regulate Igf2 gene.5. We gained one target gene of miR-483, PDGFB. Ectopic expression of miR-483 down regulated PDGFB mRNA level; Dual luciferase reporter experiments showed that miR-483 directly targeted the 3’-untranslated region of PDGFB. Ectopic expression of miR-483 inhibited PDGFB protein level in HepG2; PDGFB was detected up-regulation after 6 h and 12h using Chromeceptin in HepG2; PDGFB was detected down regulation after 6h and 24h under hypoxic conditions by CoCl₂.On the whole, our investigation revealed a common mechanism and mode of regulation between miR-483, miR-675 and their host genes Igf2, H19 in different cell lines. Meanwhile, we identified PDGFB as one of miR-483 target genes.更多还原