| There are variety of pathological reasons of preeclampsia (PE) and intrauterine growth restriction (IUGR) and the clinical phynotype is different from case to case. But it can be confirmed that both of these pregnancy-associated diseases are due to placental inefficiency. By now, some placental specific biochemistry markers are available for the prediction of preeclampsia, but there is still a scarcity in this field, especially for the clinical diagnositic biomarkers. And due to the high heterogeneity of IUGR, and its parallel occurance with other diseases, it’s hard to unveil the real molecular mechanism of IUGR by analyzing the transctiptom data with classical classification from clinic. On the other hand, as an important gene regulatory element, microRNA involved in placental development processes, such as cell differentiation, cell proliferation, apoptosis, cell invation and angiogenesis. And microRNA can also be used as biomarkers that are stable in maternal circulation system. Thus, the main purpose of this study is to identify differential genes in preeclampsia and IUGR by analyzing placental transcriptom, and get candidate microRNAs as biomarkers by the prediction of differential genes in preeclampsia and IUGR. To accomplish that, we utilized Illumina Human6.0Bead Array to study the transcriptom from86human placentas, and predict affected microRNAs by using Modulated Modularity Clustering (MMC) and Gene Sets Enrichment Analysis (GSEA). Then we confirmed the differential expression of candidate microRNAs by qRT-PCR in hman placenas, and constructed decision trees based on the actual expression level of candidate microRNAs. Besides that, the stability of microRNA in maternal plasma was confirmed. The main results are listed below:(1) Based on the classical clinic classification,128genes were found to be differentially expressed in preeclampsia at a Bonferroni p<0.05significant level and2109genes at FDR<0.05level; But only1differentially expressed gene was found in IUGR at a conservative Bonferroni significance level, which inferred the high heterogeneity of our IUGR samples.(2) MMC identified two modules, one representing IUGR placentas (Module3) and one representing preeclamptic placentas (Module5). Module3is composed with only IUGR placentas, while module5is composed with both preeclampsia and IUGR placentas, but in the7IUGR placentas,4were diagnostic as preeclampsia at the same time. (3) Based on the new classification generated by MMC,326differentially expressed genes in the module representing IUGR and889differentially expressed genes in a module representing preeclampsia were identified at Bonferroni corrected significant level.2100differentially expressed genes and5184differentially expressed genes were identified in module representing IUGR and preeclampsia respectively, at FDR<0.05significant level.(4) Functional analysis of molecular signatures associated with IUGR identified P13K/AKT, mTOR, p70S6K, apoptosis and IGF-1signaling as being affected. Functional analysis of molecular signatures associated with preeclampsia identified EIF2signaling, RhoGDI signaling, and mTOR signaling and immune responses such as leukocyte extravasation signaling, Fcg receptor-mediated phagocytosis in macrophages and monocytes, and CXCR4signaling as being affected.(5) Six top candidate microRNAs (miR-520A-5p, miR-194, miR-412, miR-149, miR-483, and miR-503) were selected from the predicted list generated by Gene Setes Enrichment Analysis (GSEA). Result of qRT-PCR indicated that miR-194was significantly down-regulated both in preeclampsia (p=0.0001) and IUGR (p=0.0304), and miR-149was significantly down-regulated in preeclampsia (p=0.0168).(6) Decision trees were constructed for the prediction of preeclampsia and IUGR respectively. By partrition test, they are statistically significant at p<0.05.(7) Thre universal expressed microRNAs (miR-16, miR-15, and miR-24) were stable in plasma with no affection by room temperature and repeat frozen-thraw cycling. The same was abserved on the differentiall expressed microRNAs (miR-149and miR-194), suggesting the microRNAs secreted from organs exist in circulating system in a very stable form.On the other hand, since genomic imprinting is important for both development of mammalian embryo and postnatal growth, and it is found that many imprinted genes were affected in abnormal placental transcriptom. And also because some genes affecting quantitative traits of live stocks were imprinted. The other aim of this study is to identify the imprinting status of MAGEL2in two embryo stages in the model of porcine, and analyze the association between polymorphism sites of porcine MAGEL2and carcass traits. The main results are listed below:(1) The same as its human and mouse homologue, porcine MAGEL2is also a no-intron gene, which was observed to be predominantly expressed in fetal brain and placenta, followed by heart. And the expression level in liver, spleen, lung, kidney, stomach, small intestine and skeletal muscle are relatively low.(2) Porcine MAGEL2was paternally expressed in day65fetal tissues, including heart, liver, spleen, lung, kidney, stomach, small intestine, skeletal muscle, brain, and placenta. Interestingly, we observed an imprinting variance of MAGEL2in day30fetuses. In the16heterozygous embryos, two embryos lost imprint and biallelically expressed, which suggests that imprinting was not estabilished well in the early embryo development.(3) Association analysis in a Yorkshire×Meishan F2resource population showed that the mutation of g.2592A>C was significantly associated with dressed carcass percentage (p<0.05) and buttock fat thickness (p<0.05), and mutation of g.3277T>C was significantly associated with bone percentage (p<0.05). |
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