Functional Studies Of MiR-1188 On Mouse Embryo Development And Hepatoma Cells

Mi R-1188 is located in transcripts of Meg8, which has been identified as a maternally expressed gene in the mouse Dlk1-Dio3 imprinted cluster. In mammals, the Dlk1-Dio3 genomic region, located on mouse chromosome 12(12q F1) contains the paternally expressed imprinted genes Dlk1, Rtl1, and Dio3 and the maternally expressed imprinted genes Meg3(Gtl2), anti-sense Rtl1(as-Rtl1), Meg8. This region hosts, in addition to two long intergenic RNAs, the Meg3 and Meg8, one of the largest micro RNA clusters in the genome, with 52 mi RNAs in mouse and 54 mi RNAs in human, in the forward strand and one(mi R-1247) in the reverse strand. Previous research has shown that major regulatory components and mi RNAs in this region are involved in embryo development and pathogenesis of different diseases. And, the deletion of the Meg3 gene could interfere embryo development and contribute to hepatoma cells proliferation; mi R-127 and mi R-370, located in upstream and downstream of mi R-1188, also could involve embryo development and hepatocarcinogenesis. We assumed that there might be the same functions between mi R-1188 and other genes in Dlk1-Dio3 genomic region. In this study, we focus on mi R-1188 and its functions, investigate the dynamic expression patterns of mi R-1188 in embryogenesis. Combined with embryo development related target genes, the research on overexpression of mi R-1188 influences on embryo development. Meanwhile, combined with tumor related target genes, the research on the specific lower expression of mi R-1188 in Hepa1-6 cells is carried out the role of mi R-1188 in liver cancer.Firstly, we analyzed the expression patterns of mature mi R-1188 in different stages of embryo development and different kinds of mouse cancer cells. mi R-1188 expression in embryo was studied by real-time PCR(q RT-PCR) and RNA in situ hybridization(ISH). In early embryonic stage, q RT-PCR showed gradually decreased expression level of mi R-1188(4-cell to morula) and significanly increased expression in 4-cell, increases expression level mi R-1188 in mid-late embryonic stage(E12.5-E18.5), and mi R-1188 is high expression in liver, medium expression in brain, lung and heart, and low expression in kidney. mi R-1188 and host Meg8 share similar expression patterns. With the transfer of function of liver development, the expressions levels of mi R-1188 were gradually reduced, and high during embryo liver hematopoietic period, the highest in E15.5, lower after the birth. Then we used q RT-PCR to describe the expression pattern of mi R-1188 in the various cancer cell lines, the results showed that mi R-1188 in Hepa1-6 cells was the lowest than other cell lines. Meanwhile, comparision of differently expression of mi R-1188 in normal adult liver tissue and Hepa1-6 cells.To get further know about the function of mi R-1188 about embryo development, model of E15.5 with tail vein injection by over-expressed mi R-1188 lentiviral vector to pregant mice, observed that mi R-1188 caused embryo weight loss, and placenta weight increase accordingly. And q RT-PCR results show that mature mi R-1188 in embryo, placenta and amniotic sac were excessive enrichment. Among them, the expressions of mi R-1188 in lung and liver were high. But HE and in situ hybridization showed that mi R-1188 could not cause abnormal changes of major organs. mi RNA are conserved short RNAs that suppress protein expression through base pairing with the 3′-untranslated region(UTR) of target m RNAs. Through bioinformatics prediction, we postulated that Smad3 involved in embry development might represent targets of mi R-1188, combined with luciferase reporter assays and western blot identified that mi R-1188 targets Smad3. Correlation analysis indicated that there was a significant inverse correlation between mi R-1188 and Smad3 m RNA expression in mouse embryo development periods. The results showed that the expression of Smad3 in 2-cells is higher than it in 4-cells in early embryonic stage, the lowest in E15.5 than it in other mid-late embryonic stage, and Smad3 was lower expression in E15.5 liver. Then in the model of E15.5 with over-expressed mi R-1188, there was an inverse correlation between mi R-1188 and Smad3 m RNA in placenta, heart, liver and kidney. All these suggested that mi R-1188 could involve in embryo development though regulating Smad3.At last, in order to identify the function of mi R-1188 about tumorigenesis, the expressions of endogenous mi R-1188 were changed in Hepa1-6 cells. The results revealed that mi R-1188 significantly inhibited Hepa1-6 cells proliferation and colony formation, triggered morphological changes, increased caspase-3 activation and induced hepatoma cells apoptosis. Meanwhile, we postulated that Bcl-2 and Sp1 involved in tumorigenesis are targets of mi R-1188, identified that mi R-1188 could regulate their expressions at the post-transcription level, and correlation analysis indicated that there was a significant inverse correlation between mi R-1188 and Bcl-2 or Sp1 protein levels between normal liver tissues and Hepa1-6 cells, respectively. Besides, to explore the molecules involved in apoptosis, we measured the expressions of Bax, Bcl-XL and p53. Overexpression of mi R-1188 significantly suppressed the expression of Bcl-XL, and the expression of Bax and p53 was up-regulated. To investigate further mi R-1188 mediated apoptosis and invasion by modulating Bcl-2 and Sp1 levels, mi R-1188 analogs and targets si RNAs were co-transfected into Hepa1-6 cells. Finally, cell transplant assay in vivo showed that overexpression of mi R-1188 could impaire the proliferation of Hepa1-6 cells. Therefore, mi R-1188 might serve an important function in apoptosis and invasion pathways.Taken together, mi R-1188 is a widely expressed mi RNA in the embryo development and cancer cell lines. mi R-1188 caused embryo weight reduction and increased placenta weight, as well as inhibited the growth of liver cancer cells in vitro and vivo, and involved in the organism development and carcinogenesis though regulating the exptessions of target genes. The results will contribute to the further investigation of regulatory mechanism and functions of mi RNA in the Dlk1-Dio3 domain.