Screening Of Key Placental MiRNAs Affecting Intrauterine Growth Retardation And Its Molecular Mechanisms In Pigs

In modern swine industry,the litter size of sows has improved significantly with the rapid development of genetic improvement and reproduction technology,but the increase in the number of weak piglets and neonates with low birth weight caused by intrauterine growth retardation(IUGR)seriously affect economic benefits of pig production.The placenta and umbilical cord are important organs for transporting nutrients,oxygen and other active factors between mother and fetus during pregnancy,so placental insufficiency has been considered to be one of the main causes of intrauterine fetal growth retardation.micro RNA(miRNA)has been found to be an important epigenetic modification participated in regulating placental function and pregnancy outcomes in mammals.However,whether placental miRNAs are involved in the occurrence and development of IUGR in pigs was rarely reported.Therefore,the purpose of present study was to screen out important miRNAs and their target genes associated with intrauterine growth retardation by comparing placental miRNA and mRNA expression profiles and umbilical cord serum metabolic profiles of IUGR piglets and normal birth weight(NBW)piglets,and in vitro experiments were used to explore the effects of key miRNAs on the biological functions of porcine trophoblast cells to preliminarily elucidating their molecular regulatory mechanisms.The main results of research were as follows:(1)In the present study,25 stillbirths and 259 live-born piglets were produced by20 sows,including 31 IUGR piglets and 183 NBW piglets,accounting for 11.97% and70.66% of the live-born piglets,respectively.By comparing the metabolic profiles of cord serum between IUGR and NBW piglets,49 differential metabolites were identified,among which 42(including cysteine,phenylalanine,linoleic acid,fumaric acid)and 7 metabolites were significantly down-regulated and up-regulated in cord serum of IUGR piglets,respectively.These differential metabolites were mainly involved in linoleic acid metabolism,tricarboxylic acid cycle,niacin and niacinamide metabolism and pyruvate metabolism.(2)The piglet birth weight,placental weight,placental efficiency,villus number and capillary number per unit area in placenta of IUGR group were significantly lower than those of NBW group(P<0.05),implying IUGR piglets suffered from placental dysplasia.A total of 81 differential expressed miRNAs(DEmiRNAs)in the placentas between IUGR and NBW groups were identified through miRNA sequencing,among which 63 and 18 miRNAs were significantly up-regulated and down-regulated in the placentas of IUGR piglets,respectively.Predicted target genes of identified DEmiRNAs were mainly related to metabolism,signal transduction,immune system,catalytic activity,receptor activity,etc.There were 726 genes were identified as differential expressed genes(DEGs)in the placentas between IUGR and NBW groups by mRNA sequencing,of which 567 were significantly up-regulated and159 were significantly down-regulated in the IUGR group.GO and KEGG enrichment analysis showed that these DEGs were mainly related to immune system,endocrine system,lipid metabolism,amino acid metabolism,cell growth and death,and cytokine-cytokine receptor interaction.The intersection of target genes predicted by DEmiRNAs and DEGs was used to construct miRNA-mRNA interaction network diagram to screening out 11 DEmiRNAs,such as ssc-miR-339-5p,ssc-miR-7136-5p,ssc-miR-7138-5p,ssc-miR-125 a,ssc-miR-7134-5p,ssc-miR-7139-5p,ssc-miR-493-3p,ssc-miR-34,ssc-miR-30a-3p,ssc-miR-128,ssc-miR-125 b.ssc-miR-339-5p was found to have the most DEGs,among which GRIK3,SLC7A2,BFSP1,MORN5,IL17 REL,PRR35,PALM3,LOC100514786 were negatively correlated with the expression of ssc-miR-339-5p.(3)The ssc-miR-339-5p mimics and ssc-miR-339-5p inhibitor vectors were constructed to transfect porcine trophoblast cells(PTr2).The results showed that the overexpression of ssc-miR-339-5p inhibited the proliferation and migration of PTr2 cells.Double luciferase reporter experiment revealed that there was a targeting relationship between ssc-miR-339-5p and GRIK3,and ssc-miR-339-5p negatively regulated the expression of GRIK3.The expression of ssc-miR-339-5p also affected the expression levels of genes related to the cytokine-cytokine receptor interaction pathway.In this study,candidate miRNAs and genes that may be associated with porcine IUGR as well as cord serum metabolites were screened out by high-throughput sequencing and metabolomics,and it was further found that the overexpression of sscmiR-339-5p in placenta may inhibit the proliferation and migration of trophoblast cells by negatively regulating the expression of GRIK3.Abnormal trophoblastic development and inflammatory responses in the placenta,coupled with the dysplasia of the placental villi and blood vessels may affect the transport and metabolism of amino acids and energy substances,resulting in intrauterine growth retardation of pig fetuses.This study can provide theoretical basis and important evidence for the genetic improvement of reproductive traits of pigs.