Study Of The Role Of MicroRNAs And Their Implications In Inorganic Arsenic Related Toxicology Research

The population epidemiological studies has been demonstrated that chronic arsenic exposure may induce the embryo heart or nervous development and embryo toxicity. In this study, we have taken advantage of chick embryos as experimental model systems to screen the differential expression of mRNA and miRNA in arsenic treated group compared to control group using microarray. Microarray analyses revealed that the transcription of a set of miRNAs and mRNAs have been changed upon arsenite administration. The relationship of the microRNA and genes were counted by their differential expression values, and according to the interactions of microRNA and genes in Sanger microRNA database to build the microRNA-Gene-Network. The microarray network analysis indicated several key genes including Nrp1,stmn1,HDAC4,Nrcam etc were significantly upregulated in chick embryo, corresponding with the down-regulation of miRNAs in chick embryo. Nrpl is centrally located in miRNA-mRNA networks, integrative analysis implicated Nrp1 as a downstream target of miR-9 and mir-181b. Neuropilin-1 (Nrpl) is a 120-130kDa single spanning transmembrane glycoprotein which previously identified as a neuronal receptor that mediates repulsive growth cone guidance, has been shown recently to function also in endothelial cells as a coreceptor in vitro of VEGF receptorConsidering the crucial role of Nrp1 in angiogenesis, we further investigates the role of mir-9 and mir-181b in sodium arsenite-mediated angiogenesis in the human umbilical vein endothelial cell line EA.hy926. The expression of miR-9 and mir-181b were decreased but the expression of Nrpl is increased in the arsenic-treated cells compared to control in a time and dose dependent manner. To screen the targets regulated by the miR-mediated RNA silencing pathway, We transiently transfected EAhy.926 cells with miR-9 or miR-181b, the western blot analysis revealed that the protein level of Nrpl was reduced in the cells transfected miR-9 or miR-181b compared with the control cells. However, the protein level of Nrpl was markedly increased in the cells transfected anti-miR-9 or anti-miR-181b compared with the control cells. To further examine whether miR-9 and miR-181b direct targets Nrp1, the luciferase reporter containing the Nrpl 3'UTR fragment with the miR-9 or miR-181b a binding site was transfected into EA.hy926 cells. When the miR-9 or miR-181b was cotransfected into the cell with Nrpl reporter constructs, luciferase activity was repressed significantly compared to cotransfection with positive and negative control of pre-miR miRNA precursors. However, when the miR-9 or miR-181b was cotransfected into the cell with deleted seed sequence of 3'UTR, the repression of luciferase activity was completely abolished. Taken together, these results suggested that miR-9 and miR-181b repressed Nrpl through a specific 3'-UTR binding site.Since the transwell migration assay and the tube forming assay are commonly used tests to study the angiogenesis response of endothelial cells to angiogenic inducers or inhibitors. To determine the role of these miRNA, cells were transfected with miR-9 or mir-181b in a 6-well plate according to manufacturer's protocol before NaAsO2 exposure. The results indicated that arsenite increased cell migration relative to the control. However, the cell migration increased by NaAsO2 was repressed following transfection with miR-9 and mir-181b in EA.hy.926 cells. The functional effect of arsenite on angiogenic potential was tested by tube forming assays. Consistent with the migration assay, the tube forming assay results revealed that over-expression miR-9 or mir-181b inhibits arsenic-induced angiogenesis. Results from our study suggested that arsenite induced increase in cell migration and angiogenesis was mediated by miRNA via increasing the expression of Nrp1. Our study demonstrated that environmental toxins sodium arsenite induced angiogenesis via changing the miRNAs and their cognate mRNA target sequences.In summary, the present study identifies the expression of miRNAs, like many of the genes important in toxicology, can be regulated by environmental toxicants. To our knowledge, this represents the first observation of microRNA could mediate the arsenic-induced angiogenesis in vitro and in vivo, represents a new frontier toward our understanding of the mechanisms of toxicology of NaAsO2.