Study On Function Of MiR-483 And MiR-675 In H19/IGF2

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.