Expression And Mechanism Analysis Of MiR-370during Mouse Development And Its Impact On Development

microRNA (miRNA) is highly conserved non-coding RNA involved in cellproliferation, differentiation, apoptosis, metabolism and signal transduction, tumorcells transformation in life process by negatively regulating multiple target genes.Research shows miR-370is a paternal imprinted gene, and locates on Dlk1-Dio3imprint region on mouse distal region of chromosome12and human chromosome14. It exists in the third intron of a long non-coding RNA Rian (AB063319), andmature miR-370is100%conserved in the mammals. Although abundant evidenceproved that Dlk1-Dio3played an important role in pluripotent stem cells, and wasinvolved in a variety of genetic defects in human and mice, like growth retardation,metabolic disorders, and defection of non-coding RNA in this region closelyassociate with tumor, miR-370, as a imprinted miRNA in this region, its expressionpattern has never been studied. Neither do we know much about miR-370functionin embryonic development. Therefore, in this study we focused on miR-370geneand designed a serial experiment to screen and identify its target genes andexplained its potential function in deployment by bioinformatics, cell and moleculartechnique, morphological analysis and immunohistochemistry. In addition, weanalyzed its expression patterns during normal mice embryonic development. Bybuilding gene knock-in mouse model and analyzing phenotypic abnormalities, wetried to explain the potential function of miR-370enrichment in transgenic mice,and elaborated potential role of miRNA in Dlk1-Dio3imprint area during mousedevelopment.First, we used quantitative RT-PCR technique to describe the expressionpattern of miR-370on cell level. In14murine cell lines, miR-370had relativelyhigh expression in3tumor cells, but it was lowly expressed in normal immortalizedcell lines TM3, MS5, NIH3T3and3T3L1. In18human cell lines, two cancer celllines K562, PANC-1and normal bladder cell line SV-HUC-1exhibited relativelyhigh expression of miR-370, while other cells exhibited relatively low expression.By treatment of demethylating drug, epigenetic regulation of miR-370waspreliminarily analyzed. It showed that low expression of miR-370in normal TM3cell lines closely related with high methylation status of upstream methylatedGtl2-DMR region, while high expression of miR-370in MLTC-1had no directrelationship with tumor cell proliferation.Then by bioinformatic prediction and pathway analysis, we succeed inidentifying an effective binding site in3’UTR of Dnmt3a. Then the target effect wasproved in AtT20and NIH3T3cells by overexpressing and inhibiting miR-370experiment. Both endogenous and ectopic expressed miR-370can specifically recognize the3’UTR of Dnmt3a in luciferase vectors and affect protein encoding.Further, with the same experimental design, western blot demonstrated thatmiR-370could also affect endogenous Dnmt3a protein level. These result indicatedthe expression of miR-370might relate with multiple cell progresses, and mighthave important and complex function.Second, we further elaborated the spatiotemporal expression pattern ofmiR-370. Real-time quantitative RT-PCR analysis of preimplantation mouseembryos showed that mature miR-370couldn’t be detected before blastocyst stage.Its expression significantly activated from implantation to E9.5. Activatedexpression status maintained from E9.5to birth, and it decreased a little in late stageof embryos. Whole mount in situ hybridization exhibited expression pattern ofmiR-370in E9.5-E11.5mouse embryos, and showed that miR-370highly expressedin brain tissue and neural tube indicating miR-370probably involved in thedevelopment of central nervous system during this period. Further more,quantitative real-time RT-PCR of E15.5mouse embryo and tissue slide in situhybridization proved miR-370was a tissue-specific expressed gene whoseexpression level was much higher in embryos than in adult. miR-370mainlyexpressed in most part of the brain, adrenal gland, dorsal root ganglia, tongue,olfactory cortex and bone primordia, while the expression level was low insubstantive organs such as liver, lung, heart, kidney, etc. The expression patternduring mouse embryonic development suggested that miR-370might play multipleroles in various organ generation and development.To further clarify function of miR-370, miR-370knock-in transgenic mousemodel was built. By conducting several statistical analysis of phenotypies, we foundthat miR-370transgenic male mice gained more weight than wide type both beforeand after birth, female mice and placental weight did not change significantly.miR-370transgenic mice had less embryos, more unusual deaths than wide type,and accompanied by teratoma. Sex ratio of female to male in transgenic mice wassignificantly decreased compared with the wild type. Quantitative RT-PCR analysisof miR-370in transgenic mice showed there was stable enrichment of miR-370inthe testis. This accumulation was accompanied by atrophy of seminiferous tubules.These result indicated miR-370enrichment might lead to seminiferous tubulesatrophy syndrome, and affect spermatogenesis, releasing and maturation process inthe epididymis.Finally, we verified the targeting effect of miR-370to Dnmt3a ontranscriptional level in E18.5and adult testis. By fluorescence in situ hybridizationand fluorescence immunohistochemistry, we proved that miR-370enrichment andDnmt3a protein presented in similar cells and they exhibited a certain degree ofnegative correlation. Unfortunately, DNA methylation status was stablet, and methylation of the IG-DMR couldn’t accurately reflect the impact of miR-370targeting Dnmt3a;leaving us a clue for further study of miR-370’s function.In summary;the imprinted non-coding miR-370is a temporal specificityexpression miRNA in mouse embryo development. It can target methyltransferaseDnmt3a and negatively regulate it on both cellular and in vivo level. In addition;miR-370enrichment have a significant impact on male reproductive organs. Theseresults provide important clues for the study of miR-370function in mouseembryonic development;and provide data support for further illustration of themammalian non-coding RNA regulation and function.