Promoter Methylation Of Porcine Imprinted Gene NNAT And Its Expression Regulation

Imprinted gene plays a key regulatory role in the placentas ensuring the normal growth and development of embryos and placentas. The abnormal expression of imprinted genes often lead to the abnormal placental development, phenotypic defects and other diseases, even worse, tumorigenesis and embryonic death. Imprinted genes are divided into maternal imprinting and paternal imprinting. Maternally imprinted gene expressing paternal allele often promote cell proliferation, embryo growth and nutrition transportation. While, paternal imprinted gene expressing maternal allele tend to inhibit embryo growth, cell division and tissue development.In this study, we focused on the paternally expressing maternally imprinted genes NNAT to do some associated expression mechanism and regulation research. Bioinformatic analysis revealed that there are 3 methylation islands in NNAT promoter. We researched the NNAT promoter methylation level between the macroglossia pigs and normal pigs, as well as the foreign breed of Large White and domestic breed of Erhualian. Besides, we studied the relationship of the downstream regulatory pathway of NNAT and glucose transportation associated pathway, the expression changes of NNAT and associated genes have been detected between Large White and Erhualian at 75 days and 90 days of gestation. In addition, through high expression of NNAT at the cellular level, study of expression changes of GLUT gene suggesting that this series genes have different bioligical mechanism in different breeds. All in all, we obtain the following results:a) Using the Pyrosequencing and Real-Time PCR technology, the relationship between expression and promoter methylation level of NNAT was investigated in nuclear transferred macroglossia pigs, nuclear transfered and in vitro fertilized normal pigs, the results showed that the expression differences between macroglossia pigs and normal pigs are related to the promoter methylation level. Demethylation of gene promoter can result in increased expression. Using the same way to detect the relationship in the placentas of Large White and Erhualian at 75 days and 90 days of gestation, the results discovered the expression change of NNAT does not due to the methylation level, the changes of methylation level were not detected in normal pigs.b) The speculation and analysis of the downstream regulation pathway of NNAT Using IPA software based on the literature revealed that NNAT regulate GLUT maybe through the PI3K signaling pathway. c) On the tissue lecel, the expression differences of NNAT and glucose tranportation associated gene in the placentas of Large White and Erhualian pigs at 75 days and 90 days of gestation were detected by Real-Time PCR. It was found that the expression regulation of glucose transportation associated genes, in vivo, are not directly up regulated by the high expression of NNAT, but rather in a more complex biological mechanism to ensure the normal development of embryos.d) On cell level, Real-Time PCR was utilized to detect the expression changes of the GLUT gene in JEG3 cell which transfected pcNDA3.1-NNAT vector. The results demostrated that overexpression of NNAT gene can cause up-regulated expression of GLUT1.