Based On Ovarian Transcriptome And Metabolome,screen Mrna And Non-coding RNA Influencing Litter Size Of Sow And Verify The Functions Of MiR-183-circTCP1 Pathway

Litter size,an important reproduction index,reflects the production level and eco nomic benefits of a pig farm.The ovary is an important reproductive organ of sow,which goes through a series of biological processes in each estrus cycle.The comple x transcription network of coding genes and non-coding genes of the ovaries regulate s the reproduction process of sows.However,the pathways influencing on the litter s ize of sows is still unclear.In this study,eight Large white and Landrace hybrid so ws were selected as the research object,and they were all sera and PCR negative f or PRRSV and PRV.The differential expression coding gens and non-coding gens,in cluding micro RNA,circ RNA and lnc RNA,as well as the metabolites in ovaries of th e pigs with a character of larger litter size or small litter size were analyzed based o n the data of RNA Sequencing and ovarian metabolomics.And according to GO and KEGG pathways analysis,a possible related pathway,mi R-183-circ TCP1,was picked out and verified in vitro by RT-q PCR,Edu staining,CCK-8,Western blot,Flow cyto metry and Double luciferase detection.According to our study,the following results were obtained.1.Screen candidate genes that affect litter size of sow based on whole ovarian t ranscriptomeThe transcriptome in ovaries from large white and Landrace hybrid sows were o btained using high-throughput sequencing.About 100 million clean reads were obtain ed from each sample,and the matching rate with pig reference genome was 96.3% a t the highest level and 94.1% at the lowest.Through differential expression analysis,a total of 2708 differentially expressed genes were identified in ovaries of LLS grou p compared with SLS group,including 1110 up-regulated and 1598 down-regulated g enes.Through GO and KEGG analysis,14 candidate genes,including STAR,HSD17B2,HSD17B12,BMP6,BMP4,INHBA,BMP2,GDF7,TGFBR1,TGFB3,BMP7,AK R1C1,CYP21A2 and HSD17B8 were obtained,which were located in 4 pathways of Hippo signaling pathway,TGF-beta signaling pathway,steroid hormone synthesis pat hway and ovarian steroid hormone synthesis pathway.Among them STAR,HSD17B2,HSD17B12,INHBA,TGFBR1 and AKR1C1 were up regulated and BMP6,BMP4,BMP2,BMP7,GDF7,TGFB3,CYP21A2 and HSD17B8 were down regulated in the LLS group compared with the SLS group.2.Study on non-coding RNAs that affect litter size of sow based on ovarian wh ole transcriptomeA total of 33,302 and 548 differentially expressed mi RNAs,lnc RNAs and circ R NAs in group LLS and SLS were selected,respectively.The expression levels of so me non-coding RNAs that possible involving regulation ovary development or ovary steroid hormone synthesis were verified using RT-PCR.Moreover,the ce RNA network of lnc RNA-mi RNA-m RNA and circ RNA-mi RNA-m RNA were constructed.And 2 ke y candidate mir RNAs(mir-183,mir-1343),4 key candidate lnc RNAs(LOC110259814,LOCLOC106504714,LOC106505099 and LOC110257180),and 2 candidate key circ RNAs that possibly influence litter size of sows were picked out.3.Screen metabolic materials and related genes that affect litter size of sow bas ed on ovary metabolism studyBased on the metabolism study,a total of 55 different metabolites were obtained in the ovaries of pigs from group LLS and SLS.And they were clustered in 18 dif ferent pathways.The found key metabolites are aniline,histamine and 1-piperidine,a nd six genes,including ANXA8,LOC100524773,LOC110258110,DDO,SMIM1 and AZGP1 may play a role in regulating the metabolism of them.These genes are pote ntial factors that affect the reproductive performance of sow.4.Identification the function of mi R-183 and Verification mir-183-circtcp1 pathw ay(1)mi R-183 promotes the proliferation of porcine ovarian granulosa cells(GCs)and has no significant effect on the apoptosis of GCs.(2)mi R-183 inhibits the synthesis of estradiol(E2)and promotes the synthesis o f progesterone(P4)in the S phase of granulosa cells.Three beta-hsd is a key functi onal gene in the synthesis of P4,and Cyp19a1 plays an important role in the synthe sis of E2.(3)The expression level of mi R-183 was significantly up-regulated in LLS grou p,so as to produce more follicular granulosa cells and larger litter size.(4)circ-TCP1 competitively adsorbs mir-183 and possibly plays a key regulatory role in the biological processes related to sow fertility.