Preliminarily Study On Expression And Function Of MicroRNA-145in Mouse Pre-implantation Embryo

Micro-RNA is a class of non-coding single-stranded RNA of about22-25nucleotides, which is an important transcript control factor and involved in organism growth and cell differentiation. In pluripotent cells, microRNA-145can influence cell pluripotent and differentiation by influencing the expression of Oct4and Sox2. Whether the same regulation mechanism of micro RNA-145exists in the early mammalian embryo differentiation process remains unclear. In this study, we selected mouse as model, and obtained the over-expressed microRNA-145embryos by micro-injecting microRNA-145over-expression vector. Then we detected the trend of the expression of microRNA-145、Oct4and Sox2in embryos and over-expressed microRNA-145embryos by real-time PCR and analyzed the consistency relationship between this change and the development of embryo. Data accumulated from this research may help to preliminarily reveal the possible function of microRNA-145in differentiation process of pre-implantation embryo. The thesis includes three parts, the results were as follows:1The superovulation protocol of Kun-ming mouse was optimized. The results showed:①After intraperitoneally injecting different doses of hormone to mouse(5IU PMSG+5IU HCG、5IU PMSG+10IU HCG,10IU PMSG+5IU HCG and10IU PMSG+10IU HCG), the results of superovulation showed significant differences among the four groups (27.50per mouse、19.57per mouse、20.81per mouse、21.71per mouse, respectively, P<0.05) and the blastocyst rates were also significantly different among the four groups (77.27%、63.99%、65.68%、67.08%,respectively, P<0.05).②Mice were superovulated in different estrus periods and the result showed that there was no significant difference between diestrus group (29.52per mouse) and estrus group (29.12per mouse),(P>0.05), and the blastocyst rates also showed no significant differences between diestrus group (65.82%) and estrus group (64.70%),(P>0.05).2The changes of the expression of microRNA-145、Oct4and Sox2in embryos were detected by real-time PCR. The results showed that the expression of microRNA-145in mouse2-cell embryo、4-cell embryo、8-cell embryo and blastocyst had significant differences (P<0.05), the expression of microRNA-145showed a gradually increased trend during the embryonic development. The expression of Oct4and Sox2in mouse2-cell embryo and blastocyst had significant differences (P<0.05), the expression were down-regulated during the embryonic differentiation.3After micro-injecting microRNA-145over-expression vector into zygote, the development of embryos and the expression of pluripotency transcription factors Oct4and Sox2were investigated. The results showed that the division rate and blastocyst were both significantly different between experiment group(74.72%;46.23%) and control group (100%;76.44%)(P<0.05). Meanwhile, the expression of microRNA-145, Oct4and Sox2gene were detected by real-time PCR. Data showed that the expression of microRNA-145was significantly up-regulated in experimental group (P<0.05). the expression of Sox2was significantly different (P<0.05) between experimental group and control group, however the expression of Oct4showed no significant difference (P>0.05) between experimental group and control group.In conclusion, a combination of hormones with5IU PMSG+5IU HCG was a suitable treatment for mouse superovulation and subsequent embryo development, and the different estrus status did not significantly influence superovulation. During the development of pre-implantation embryo in mouse, the up-regulated expression of microRNA-145and down-regulated expression of Oct4, Sox2and the pre-implantation embryonic differentiation happened simultaneously. In mouse pre-implantation embryo, over-expression of microRNA-145reduced the ability of development and differentiation of embryo, and significantly down-regulated the expression of Sox2on mRNA level.