Identification And Characterization Of Regulatory Non-coding RNA(miRNA And LncRNA) In Development Of Porcine Placental Folds

The production of large litters with high quality piglets is one of the important indicators of porcine reproductive performance.Fetal growth depends on nutrients received through the placenta.The efficiency of nutrient transport is dependent on the surface area for maternal–fetal nutrient exchange.As a chorioallantoic placenta,a porcine trophectoderm is in contact with the uterine epithelium without invasion.The structure of porcine placenta is different from that of other species.The development of the microscopically folded structure of the porcine placenta is important as it expands the surface area for maternal-fetal exchange.Pig placental fold development includes two stages,namely placental fold formation and expansion.It is known that,during these two periods,the morphology and function of porcine placenta change significantly,but the relevant molecular mechanism remains to be analyzed in depth.A non-coding RNA(nc RNA)is an RNA molecule that is not translated into a protein.Two types of nc RNA,Micro RNA and lnc RNA,play an important function in regulating gene expression.Therefore,the study of non-coding RNA in pig placental fold development could provide the basis for elucidating the regulatory mechanism of placental fold development,while also helping to analyze the regulatory mechanism of porcine reproductive traits.The first part of the study is to analyze expression patterns and functions of mi RNAs in the development of porcine placental folds.Firstly,we identified mi RNAs expression patterns during placental folds formation(gestational day 26 and gestational day 50).We analyzed and validated the target gene and related regulatory networks to screen important mi RNA-target gene pairs.Secondly,the expression patterns of important mi RNA-target pairs were identified during placental folds extension(gestational day 50 and gestational day 95).mi RNAs involved in placental development were identified by an integrative analysis with mi RNAs expression patterns in the developing porcine placenta.In addition,we identified and validated SNPs in the mi RNA binding site by using bioinformatics analysis and experimental validation,respectively.The second part of the study is to analyze lnc RNA in porcine placental development.The transcriptional analysis of placental tissue during the placental fold expansion stage(gestational day 50 and gestational day 95 days)was performed using RNA-seq.We identified the function of lnc RNA in the porcine placenta.The main results are as follows:1.Identification and functional analysis of the mi RNA in porcine placental development(1)We used a micro RNA array to study the expression pattern of micro RNA in placental fold formation.42 mi RNAs were found to be differentially expressed during placental fold formation.28 mi RNAs were up-regulated and 14 mi RNAs were downregulated.14 mi RNAs(mi R-205,mi R-141,mi R-23 a,mi R-24,mi R-27 a,mi R-29 a,mi R-183,mi R-100,and mi R-30a)were chosen for validation by q RT-PCR.The bioinformatics functional analysis showed that the differentially expressed mi RNA were involved in ECM remodeling,cell proliferation,and tissue morphogenesis.(2)The expression patterns of 14 target genes,whose function were involved in ECM remodeling,cell proliferation,and tissue morphogenesis,were validated by q RTPCR in the stage of formation of porcine placental,and whose expression was inversely correlated with mi RNA.PPARG,CDH1,CLDN1,OCLN,TJP3,GATA2,TP53INP1,HBP1,and ULK1 were up-regulated target genes.ZEB2,COL1A2,COL3A1,LAMC1,and TGFBR1 were down-regulated target genes.The interaction between mi RNA and target(HBP1-mi R-17,HBP1-mi R-20 a,ULK1-mi R-20 a,PPARG-mi R-130 b,LAMC1-mi R-29 a,COL1A2-mi R-29 a,COL3A1-mi R-29 a,ZEB2-mi R-205,and ZEB2-mi R-200c)were validated by the luciferase.(3)We analyze mi RNA expression patterns during placental fold expansion.The result showed that only in placental fold formation could mi R-29 a and mi R-17 regulate target genes LAMC1,COL3A1,and HBP1,but mi R-130 b,mi R-205,and mi R-200 c could regulate the target genes PPARG,ZEB2,and CDH1 in both placental fold formation and extension.(4)SNPs inside mi RNA binding sites can disrupt the regulation of mi RNA on the target gene.We found 237 SNP-mi RNA-target pairs which are related to porcine placental fold development by using whole genome resequencing.11 SNPs were verified by sequencing.The binding efficiency of 4 SNP-mi RNA-target pairs were confirmed to be affected by SNPs.(5)We further analyzed the SNP rs55618224-mi R-18a-CDC42 in the above four settings.We found SNP rs55618224 was polymorphic in the porcine populations.mi R-18 a and CDC42 were negatively correlated during placental fold development.Immunohistochemical assays showed that CDC42 was expressed in trophoblast cells of porcine placenta.In vitro experiments showed that mi R-18 a could inhibit the expression of CDC42(SNP rs55618224 genotype is TT)m RNA and protein.Different genotypes of SNP rs55618224 could affect the CDC42 expression regulated by mi R-18 a.The expression levels of CDC42 m RNA were significantly lower in the TT genotype(mi R-18 a binding type)compared to the CC(mi R-18 a non-binding type)in porcine placenta.The above result showed that SNP rs55618224 could affect the binding efficiency of mi R-18 a and CDC42 in porcine placenta,which may regulate porcine placental folds development,while SNP rs55618224 may be a marker for porcine reproductive traits.2.Identification and functional analysis of lnc RNA in porcine placental development(1)299 lnc RNA were identified in the placental fold expansion stage.There were 62 differentially expressed lnc RNA(up-regulated 31 and down-regulated 31).Meanwhile,there were 1984 differentially expressed genes(up-regulated 1256 and down-regulated728).(2)11 differentially expressed gene were validated by q RT-PCR.Among them,HOXA5,CAV1,CAV2,IGF1,IGF1 R,VIM,ITGB2 and AKT3 were up-regulated in the placental folds expansion stage and BCR,CA4,and AGPAT5 were down-regulated.5differentially expressed lnc RNA were validated by q RT-PCR.The functional analysis showed that the up-regulated genes were involved in cell adhesion,focal adhesion,angiogenesis and immune response and the down-regulated genes were involved in metabolic process,SMAD signal transduction and Rho signal transduction.(3)20 target genes of the differentially expressed lnc RNA were predicted by bioinformatics.Among them,2 lnc RNA-target pairs were validated by using q RT-PCR(AGPAT5-XLOC?013767 and PLET1-XLOC?036871).The target genes were involved in cell proliferation,cell migration and angiogenesis.(4)The RNA-seq result showed that the anti-sense lnc RNA ZEB2-AS,an anti-sense lnc RNA located in the ZEB2 5'UTR region,was expressed in the porcine placenta.The luciferase result indicated that ZEB2 5'UTR u ORF could repress protein expression levels and ZEB2-AS could bind to ZEB2 5'UTR.