The Effect Of Imprinting Gene H19 On Behaviour Of Trophoblast And Research Of Correspongding Signal Transduction Molecules

Background and Objective:Pre-eclampsia is a life-threatening, pregnancy-related disease and leading cause of global maternal and neonatal mortality, affecting 2-7% of pregnancies worldwide. Many risk factors contribute to the development and severity of pre-eclampsia, which include primiparity, exposure to sperm, multifetal gestation, chronic hypertension, previous pre-eclampsia, pregestational diabetes mellitus, maternal low birth weight, and others. Clinical symptoms and disease severities vary from mild to life-threatening, including seizure, hemolysis, liver dysfunction, low platelet count, fetal hypoxia, and growth retardation, even with disseminated intravascular coagulation, placental abruption, cerebral hemorrhage, acute renal failure, circulation collapse and death. Although pre-eclampsia is commonly associated with hypertension and proteinuria, the etiology and pathogenic process of pre-eclampsia remain unclear. Several maternal genetic factors and their trophoblast effects are important to the susceptibility of individual pregnant women to develop pre-eclampsia. Continuous approaches to identify genetic factors and their function will be of great significance in understanding the pathogenesis of pre-eclampsia.Genomic imprinting, unlike the majority of genes where expression is from both inherited alleles of a gene, leads to paternal and maternal alleles of some genes having different levels of activity, one of the paternal alleles silenced, another expressed. The silencing mechanism is known to allel specific DNA methylation and/or histone modifications. The imprinted H19 gene, as one of the first genes showed to be paternal imprinted and maternal expressed, is expressed abundantly in the human placenta and in several embryonic tissues,and is only marginally expressed in nearly all normal adult tissues. In adulthood a basal H19 gene expression has been detected only in mammary gland, cardiac and skeletal muscles and to a lesser extent in kidney, adrenal gland and lung.Previous studies have shown that the H19 gene is transcripted into a non-coding RNA, and acts mainly or exclusively at the RNA level. However, the function of this non-coding RNA remains to be elucidated. The H19 gene is highly expressed in human embryonic tissue, regulating the growth and development of the embryo and the differentiation of placental cytotrophoblasts. However, its precise role in the regulation of reproduction and fetal development is poorly understood. It has been suggested that H19 functions as a tumor suppressor, on the basis of loss of heterozygosity at 11p15.5 with the retention of the paternal chromosome in some Wilms'tumor cases, embryonal rhabdomyosarcoma, and Beckwith-Wiedemann cancer predisposing syndrome. Studies published by others show that H19 was an oncogene. Interestingly, the development of pre-eclampsia is associated with a decreased invasive capacity of trophoblasts. Given that a remarkable reduction in the expression of the H19 gene is associated with the development of choriocarcinoma and its highly invasive capacity, we reasonably hypothesized that the imprinting status of the H19 gene may contribute to the pathogenesis of pre-eclampsia. Our study also showed that the biallelic expression of H19 gene existed in some cases at the early stage of normal pregnancy and changed into monoallelic expression near at 10 weeks of gestation. The dynamic alternations in the patterns of H19 gene imprinting may regulate the maintenance of normal pregnancy and be related with the trophoblast regulative invasion.To investigate the possible function of H19 on human trophoblast and study the effect of H19 on the corresponding signal conduction gene, we transiently transfected human choriocarcinomal cell line JEG-3 cells with pRC/CMV–H19 containing a sense full-length H19 cDNA under the control of the cytomegalovirus promoter and pRC/CMV and a lentiviral expression vector containing H19 small interfering RNA was constructed. It provides the experimental basis for the regulation of the trophoblast cellular function with H19 gene.The study elucidate the pathogenesis of trophoblastic correlated diseases (gestational trophoblastic diseases, preeclampsia, abortion and fetal growth retardation) from the point of the gene imprinting and offer a new way for prevention and cure of preeclampsia.The results and conclusions were as follow:1. A lentiviral expression vector containing H19 small interfering RNA didn't influence the cellular morphology, and was better than pRC/CMV–H19 containing a sense full-length H19 cDNA. It is better for study that adopt the method of RNA interfering because of the high expression of H19 in the placental trophoblast, human choriocarcinomal cell line JEG-3 cells and JAR cells. The successful construction of a lentiviral expression vector containing H19 small interfering RNA establish the basis for the continued function study.2. After the knockdown of H19 expression, the invasion ability and the expression of MMP-9 gene had never changed. The apoptosis of JAR cell were repressed and the cell cycle was holded up at the stage of G1 after the interferece of H19 RNA expression of JAR cells. It is suggested that H19 could regulate the proliferation and apoptosis of trophoblast cell and couldn't direct influence the invasion of trophoblast cell.3. It was confirmed that H19 couldn't regulate directly the expression of IGF2 in the human choriocarcinomal cell line by the two ways of RNA interfering and the transfection of pRC/CMV–H19 containing a sense full-length H19 cDNA . The excessive expression of H19 inhibited the expression of HES1 and DUSP5 in JEG-3 cells, and after the knockdown of H19 expression, the expression of HES1 and DUSP5 in JAR cells were up-regulated.It is suggested that HES1 and DUSP5 could have the important role in signal transduction in the regulation of biological function of trophoblast.4. The expression of IGF2 mRNA and HES1 mRNA decreased in the patients with severe pre-eclampsia than that in the normal pregnancy and pre-eclampsia patients showed biallelic expression of H19 gene in the previous experiment. It is suggested that the loss of H19 gene imprinting in the early stage of pregnancy could induce the excessive expression of H19, and impede the expression of HES1 and DUSP5, contributing to trophoblast differentiation obstruction, apoptosis of trophoblast cell and the pathogenic process of pre-eclampsia.5. The expression of H19 didn't influence the expression of EPS15 and there was no difference in the expression of EGFR and EPS15 between the patients with severe pre-eclampsia and normal pregnancy. It is suggested that EGFR and EPS15 could not be important signaling molecules to regulate the expression of EGF and take part in the pathogenesis of pre-eclampsia.