Analysis Of LncRNA Expression And Regulatory Network In The Ovary Of Xiang Pig During Estru

The fecundity of sows is considered to an important trait in the pig industry to improve continuously pork production.The molecular regulation of estrus cycles can affect the fecundity of female animals.Lnc RNAs are thought to involve in regulation of multiple biological processes.It was found that lncRNAs are crucial for maintaining the normal estrus cycle of animals and may serve as the regulators of the reproductive process in pigs.In order to understand the roles of lncRNAs during estrus cycles,the lncRNAs expression profiles were investigated in ovaries from Xiang pig sows during diestrus and estrus stages by RNA-seq technology.We identified the hub lncRNAs that regulate the estrus cycle in Xiang pigs ovaries.The hub lncRNAs associated networks for regulating the estrus cycle were further constructed based on competing endogenous RNA(ceRNA)mechanism.It might help to reveal the mechanism involved in regulating the estrus cycle for lncRNAs in Xiang pigs.The main results are as follows:1.Identification and characterization of lncRNAs in Xiang pigs ovaries.6,740 unique lncRNAs were identified from groups of diestrus and estrus ovaries.of which,4,662 lncRNAs were expressed in ovaries from two groups.Great amounts of lncRNAs were derived from introns and intergenic regions and expressed less than 100 CPM in two stages.In our study,the genomic characterization of lncRNAs between estrus and diestrus samples did not present significant difference,but the identified lncRNAs in Xiang pig ovaries have many obvious characteristics.They have shorter transcript and ORF length,fewer exon numbers and GC content,and lower expression.It was found that there were significant differences in the genomic characteristics of lncRNAs between the ovaries of Xiang pigs and existing database.We also found that SINE,LINE and LTR were abundant repetitive elements in lncRNAs of Xiang pigs ovaries.2.Host gene function analysis of lncRNAs in Xiang pigs ovaries.66.86% and 65.48 % of lncRNAs in the ovary samples at diestrus and estrus stages were originated by the host genes,respectively.Lnc RNAs at diestrus ovaries were derived from 2150 host genes,while lncRNAs at estrus ovaries were derived from 2190 host genes.The functional analyses of host genes from 4,662 expressed lncRNAs showed that these host genes were involved in 14 KEGG pathways with oocyte development,oocyte maturation and ovary function.The 14 KEGG pathways were including Wnt signaling pathway,Hippo signaling pathway,thyroid hormone signaling pathway,Erb B signaling pathway,TGF-beta signaling pathway,GnRH signaling pathway,apelin signaling pathway,prolactin signaling pathway,relaxin signaling pathway,progesterone-mediated oocyte maturation,aldosterone synthesis and secretion,AMPK signaling pathway,FoxO signaling pathway and estrogen signaling pathway.we identified 17 differentially expressed host genes associated with ovarian function in Xiang pigs,including SMAD4、SOX17、APC2、CTNNA2、YWHAQ、ESR1、ATP1A1、FOXO1、COMMD6、NOS2、PRKCE、GABARAPL1、BCL6、NFATC3、ANAPC1、IRS1 and TEAD1.These results provide that the lncRNAs derived from these host genes may be related to ovarian function in Xiang pigs.3.Expression and function analyses of DE-lncRNAs in the diestrus and estrus period of Xiang pigs.Furthermore,we found that 775 transcripts were DE lncRNAs(395 upregulated while 380 downregulated in estrus ovaries).Among all 775 DE lncRNAs,274 DE-lncRNAs were transcribed close to 327 target genes(Cis-regulation)and 749 dysregulated lncRNAs could regulate the expression of 2,252 target genes via co-expression regulatory mechanisms(Trans-regulation).Simultaneously,functional analyses of target genes from 775 DE-lncRNAs indicated that eight signaling pathways involved in ovarian functions and reproduction were significantly enriched(such as cellular senescence,p53 signaling pathway,FoxO signaling pathway,Hippo signaling pathway,progesterone-mediated oocyte maturation,cell cycle,oocyte meiosis,and steroid biosynthesis).Compare of the lncRNAs-target genes association network between diestrus and estrus groups revealed that 79 differentially expressed target genes interacting with 579DE-lncRNAs associated with ovarian functions and reproduction.7 DE-lncRNAs and 13 target genes were selected randomly and verified by q RT-PCR.4.Screening of hub lncRNAs and recognizing their regulatory roles as ceRNA in Xiang pigs ovaries during estrus stage.In addition,basing on the lncRNA-target gene interactive networks,we used a connectivity degree ≥ 20 as the threshold to select 43 hub lncRNAs in the regulation of gene expression during estrus stage.Through target gene enrichment analysis,we predicted that these hub lncRNAs might not only participate in complex gene regulation processes in disease,but also mainly be involoved in gene regulation of of ovarian function and reproductive in Xiang pigs.Several studies have proposed a ceRNA hypothesis,which revealed that lncRNAs and circRNAs can act as endogenous “miRNA sponges” and modulate the derepression of miRNA targets.The hub lncRNAs asociated networks for regulating the estrus cycle were further constructed based on competing endogenous RNA(ceRNA)mechanism.In the present study,we established the interactive networks of hub lncRNA-miRNA-mRNA on the basis of signaling pathways associated with ovarian functions and reproduction.By Comparing ceRNA networks between the diestrus and estrus groups,we identified the networks containing 40 hub lncRNAs(such as lncRNA＿MSTRG.10458.2,lncRNA＿MSTRG.18773.1,lncRNA＿MSTRG.8729.10 and lncRNA＿MSTRG.6716.1),64 miRNAs(such as let-7a,let-7c,miR-23 and miR-320)and 20 target genes(CCNB3,CCNB1,SMAD4 and AR).These 20 target genes were related to ovarian function and reproductive pathways.This ceRNAs may affect oocyte maturation,ovarian hormone synthesis and regulation in Xiang pigs during estrus cycle.The dual luciferase assay was used to validate the targeted relationship of two networks(lncRNA＿MSTRG.18778.1/ssc-miR-214-3p/ANAPC,lncRNA＿MSTRG.6716.1/ssc-miR-455-3p/SMAD2).The interactive networks of hub lncRNA/circRNA-miRNA-mRNA involved in regulating the estrus of Xiang pigs were also established on the basis of ceRNA hypothesis.We also identified this interactive networks containing 38 hub lncRNAs,15 circRNAs,16 miRNAs and 13 target genes.In this ceRNA network,hub lncRNAs can influence the expression of related miRNAs(such as ssc-miR-149,ssc-miR-31,ssc-miR-202-3p and miR-330)by collaborating with circRNAs.These related miRNAs participate in the regulation of the estrus cycle of Xiang pigs by influencing genes(such as ANAPC1、CDK1、CCNB3 and AR)related to biological processes such as oocyte meiosis,follicular development,oocyte maturation,granulosa cell proliferation and differentiation,and steroid production.In summary,to obtain more knowledge on the roles of lncRNAs in Xiang pig ovary during estrus cycle,we performed a genome-wide analysis of transcripts of ovary tissues from Xiang pig sows at diestrus and estrus phases using RNA sequencing method.We investigated the expression profiles of lncRNAs in ovarian tissues and identified the hub lncRNAs during estrus cycle in Xiang pigs.At the same time,we constructed the hub lncRNAs associated networks for regulating the estrus cycle basing on ceRNA mechanism.and it is of great significance to further analyze the molecular regulation mechanism of porcine ovarian,and it is of great significance to further analyze the molecular regulatory mechanism of lncRNAs in porcine ovarian function.