Studies On Mechanisms Of MiR-17-92 Cluster In The Development Of NDFMS

Macrosomia was defined as those neonates whose birth weight (BW) was≥4000 g. As the rising of the level and quality of human life, a rapid increase in rate of macrosomia has been reported. Macrosomia is associated not only with higher risks of shoulder dystocia, traumatic birth injuries, asphyxia, and perinatal mortality, but also with long-term effects on health and disease in adult life. Children born with macrosomia are prone to neonatal complications and may be more likely to develop cardiovascular disease, obesity, diabetes, and other metabolic diseases in life.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. We are facing a worldwide problem of newborn health risk caused by the abnormal weight, and how to prevent adult diseases in advance is the focus in the field of health. Therefore, it is very important to reveal the pathogenesis of NDFMS and seek for more effective treatment and intervention, which is of great significance to improve the quality of the population.Placenta is involved in in the development of macrosomia. Studies have shown miRNAs expressed in the human placenta, and play an important role in the development and function of the placenta. Abnormal miRNAs expression was closely associated with fetal growth and development. The objective of this study is to attain miRNA expression profiles of placentas of NCFMS and controls, and verify the molecular regulation mechanism in vitro, then study the role and mechanism of miR-17-92 cluster involved in NDFMS, providing theoretical basis for pathogenesis and potential diagnosis and treatment of biological markers of NDFMS.Part I Expression profile of miRNA in placentas of NDFMS and normalObjectiveTo screen for differential expression of miRNA in placentas of NCFMS and attain miRNA expression profiles of placentas of NCFMS and controls.MethodsA total of 20 placentas including 10 NCFMS and 10 controls were collected, and total RNA were extracted. miRNA expression profiles were conducted by TaqMan(?) MicroRNA chip, then verified by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR).ResultsmiRNA microarray show that the expression level of 55 miRNAs was 8 times higher in NDFMS group than that in control, with 15 miRNAs upregulated and 32 downregulated. Six members of miR-17-92 cluster were differently expressed.Conclusion1. miRNA microarray can be used for analyzing miRNA expression profiles of placentas.2. TaqMan microRNA chip revealed that different expression of miRNAs in placentas of NDFMS.3. Six members of miR-17-92 cluster were differently expressed, which provided the bases for further functional studies in the development of NDFMS.Part Ⅱ The role of miR-17-92 cluster in development of NDFMSObjectiveAccording to the previous results revealed by microRNA chips, we investigated the role and mechanism of miR-17-92 cluster members in the development of NDFMS.MethodsThe expression of miRNA and mRNA in placentas were detected by qRT-PCR. Abnormal expression of miR-17-92 cluster in HTR-8/SVneo cells was conducted by transient transfection. The function of miR-17-92 cluster was determined using transwell, CCK-8, EdU, and flow cytometer. In order to find out potential target genes of miR-17-92 cluster, we collected the target genes of the involved miRNA members from miRTarBase database. Total proteins were quantified using Western blot analyses. To determine how miR-17-92 members regulate their target genes, we employed luciferase reporter assays.Results1. Cell cycle pathway was identified to be the most relevant pathways regulated by miR-17-92 cluster miRNAs.2. miR-17-92 cluster may not only regulate the expression of SMAD4, SMAD3, RBI and EP300 transcriptionally, but also SMAD4 and RB1 post-transcriptionally.3. miR-17-92 cluster increased proliferation, attenuated cell apoptosis and accelerated cells entering S phase in HTR8/SVneo cells.4. The expression levels of Drosha and Dicer, two enzymes during the maturation of miRNA, were significantly higher in NDFMS than those in normal control group.5. The expression of miR-17-92 cluster in serum had a high diagnostic sensitivity and specificity for macrosomia (AUC:80.53%; sensitivity:82.61%; specificity: 69.57%).Conclusion1. The high expression of miR-17-92 gene cluster may regulate the expression of SMAD4 and RB1 protein, and change the proliferation and apoptosis of placental trophoblast cells, so as to promote the occurrence of NDFMS.2. miR-17-92 cluster can be recognized as a noninvasive predictive biomarker for macrosomia.