Studies On Mechanisms Of Imprinted Genes In The Development Of NDFMS

Macrosomia was defined as those neonates whose birth weights(BW) were ? 4000 g. A rapid increase in the rate of macrosomia has been reported. Fetal macrosomia has been associated with a range of maternal and perinatal complications. Furthermore, macrosomic infants are at higher risks of type 2 diabetes mellitus, hypertension, and obesity in adulthood. Diabetes mellitus is a well-established risk factor for fetal macrosomia. In recent years, with screening for gestational diabetes, the incidence of diabetic fetal macrosomia declined, while that of the non-diabetic fetal macrosomia(NDFMS) incidence has been rising. However, the detailed mechanisms remain unclear. Therefore, it is very important to investigate the molecular mechanisms of NDFMS for promoting maternal and child health.Placenta is of paramount importance for the intrauterine development and growth of the fetus. A significant factor in placental development and function is epigenetic regulation. Imprinted genes, for instance, which are epigenetically regulated, are almost abundantly expressed in the placenta. Imprinted genes play critical roles in regulation of fetal and placental development, and disruption of this critical process has been associated with abnormal placental development and function with possible consequences for a wide range of pregnancy complications. Herein, we quantified the expression levels of 95 experimentally postulated imprinted genes of human in placentas of NDFMS and normal pregnancies. Then, in vitro assay was employed to investigate the epigenetic regulation of the disturbed imprinted genes involved in the development of NDFMS. The study implicated the pathogenesis of gestational trophoblastic disease from the point of imprinted genes, and provided new sights into prevention and treatment of NDFMS.Part I Expression of imprinted genes in placentas of NDFMS and normalObjectiveImprinted genes play critical roles in regulation of fetal and placental development. Disruption of this critical process has been associated with abnormal placental development and function. It remains unclear whether the aberrant imprinted genes contribute to the development of NDFMS.MethodsQuantitative RT-PCR(q RT-PCR) analysis was performed to evaluate the expressions of 95 “experimentally validated” imprinted genes of humans in 10 NDFMS and 10 normal placentas, and then a replication was conducted in a larger population containing 20 NDFMS and 20 controls.ResultsOf the 95 genes tested, 79 were expressed in the placental tissues we examined. A total of 22 genes were identified significantly different between 10 NDFMS and 10 controls. Among the six genes identified in the validation stage, four(DLGAP2, IGF2, SGK2, and WT1) were paternally expressed(maternally imprinted), two(H19 and KCNQ1DN) were maternally expressed(paternally imprinted).ConclusionWe presented a quantitative survey of 95 imprinted genes in NDFMS and normal human placentas. The observation of differential expression in a subset of imprinted genes lends support to the notion that regulation of imprinted genes in placental development plays an important role in the development in NDFMS. Further investigations in functional characterizations are needed to validate these results.Part II The role of mi RNA in imprinted genes participating in the development of NDFMSChapter I Function and mechanisms of IGF2 and its intronic micro RNAsObjectiveIntronic micro RNAs have been reported to play antagonistic or synergetic roles as enemies or partners of their host genes. Insulin-like growth factor 2(IGF2), a member of the insulin family, is an important polypeptide regulator in placenta and fetal development. mi R-483 was identified as an intronic mi RNA encoded in intron 2 of the IGF2 transcript. Our aim was to study the expressions of mi R-483-5p and mi R-483-3p in NDFMS, and determine the relationships between these mi R-483 and IGF2 at the level of expression and function.MethodsExpression of mi RNA and m RNA was measured by q RT-PCR. Pyrosequencing was used to detect the methylation status of mi R-483 on the DNA level. The enhanced or knocked down expression of mi RNAs was accomplished by transient transfection with the mimics or inhibitors. The enhanced or knocked down expression of IGF2 was transfected with plasmid encoding IGF2 or si RNA duplex. In addition, transwell was used to measure the ability of cell migration; CCK-8 was used to measure the cell proliferation; and flow cytometry assays were used to measure cell cycle and apoptosis. Besides, bioinformatics softwares including Target Scan?Pic Tar and DIANA LAB, were used to predict the target genes of mi RNA. Western blot was conducted to detect the protein expression level of the potential target genes. Finally, dual-luciferase reporter assay was conducted to confirm mi RNA binding with the target gene.Results1. We observed up-regulation of mi R-483-5p and mi R-483-3p in the placentas of NDFMS. The hypo methylation of Cp G2 and Cp G6 of mi R-483 may contribute to the excessive expression of mi R-483. 2. It was found that IGF2 could transcriptionally regulate the expression of mi R-483-5p and mi R-483-3p,and promoted proliferation and migration of HTR-8/SVneo cells, without directly influencing the cell cycle and apoptosis of trophoblast cells. 3. It was presented that mi R-483-5p promoted migration of HTR-8/SVneo cells by up-regulating expression of IGF2 P0 transcripts. 4. mi R-483-3p did not influence the expression of IGF2 m RNA. Instead, it may promote cell proliferation of HTR-8/SVneo cells by regulating expression of RB1CC1.ConclusionThese data suggest that mi R-483-5p and mi R-483-3p can be coexpressed together with its host gene IGF2 and revealed the synergetic role of them in promoting migration and proliferation of trophoblast cells. In addition, these observations not only provide supporting evidence for the codependent expression of intronic mi RNAs and their host gene in vitro, but also give insight into pathogenesis of NDFMS.Chapter II Function and mechanisms of H19 and its intronic micro RNAsObjectiveThe imprinted gene H19, which was known as one of the first imprinted genes, was paternal imprinted and maternal expressed. H19 is highly expressed in the developing embryo and several embryonic tissues, such as placenta. It has been found to be deregulated in many cancers and fetal growth syndromes in humans. Indeed, exon 1 of H19 harbors a mi RNA-containing hairpin that has been found to serve as the template for two distinct mi RNAs, mi R-675-5p and mi R-675-3p, and it has been suggested that it may be these mi RNAs that confer functionality on H19. However, the functions remain unknown.Methodsq RT-PCR was used to detect the expression level of mi RNA and m RNA. The knocked down expression of H19 was transfected with si RNA duplex. In addition, transwell was used to measure the ability of cell migration; CCK-8 was used to measure the cell proliferation; and flow cytometry assays were used to measure cell cycle and apoptosis.Results1. It was presented that mi R-675-5p was up-regulated in the placentas of NDFMS. 2. Knockdown in H19 RNA by si RNA treatment results in reduction of H19, while ectopic expression of mi R-675-5p or mi R-675-3p had also no effect to their host gene. 3. Results showed that ectopic expression of mi R-675-5p could regulate cell proliferation as its host gene H19.ConclusionH19 long noncoding RNA has a critical trans-regulatory function in cell proliferation, which is mediated by the mi RNAs encoded within H19. It provides the experimental basis for H19 gene of the regulation of the trophoblast cellular function, which suggested that H19-mi R-675 module lead to the identification of new pathogenic process of NDFMS.