Effect Of Sperm-borne LncRNA Loc100847420 On The Early Development Of Cow Embryos

The effect of RNA brought by sperm on development has attracted more and more attraction from researchers.In the related research on humans and mice,researchers mainly focus on the intergenerational inheritance of the paternal line.Environment and disease can induce alterations in theRNA in sperm,which can further affect the offspring through the fertilization.Long non-codingRNA(lncRNA)is defined asRNA that is longer than 200 nt and does not encode protein.LncRNA is not only different from mRNA can encode protein,but also different from smallRNA with a clearer mechanism of action.Its functions are more complicated.In this study,we focus on the cow sperm-derived lncRNA.Using high-throughput sequencing technology and biological experiments,we screen the specific lncRNA carried in cow sperm and explore its regulatory role as a competitive endogenousRNA(ceRNA).The research content and results are as follows:(1)Through transcriptome sequencing of cow Metaphase II of meiosis(MII)oocytes and mature sperm,lncRNAs that are highly expressed in sperm but not expressed in oocytes were screened out.The expression of lncRNA in MII oocytes and in vitro fertilization(In vitro fertilization,IVF 2-cell embryos was detected by real-time fluorescent quantitative PCR,screening loc100847420,loc100848395,and loc104971118 in IVF 2-cell expression levels were significantly higher than MII oocytes.Using bioinformatics tools,we predict the bound miRNAs of loc100847420,loc100848395 and loc104971118,as well as the target genes of the bound miRNAs.We also preliminarily construct a ceRNA network.(2)The target genes of the miRNA bta-miR-154 b bound by lncRNA loc100847420 were subjected to KEGG and GO enrichment analysis.UBE2V1 and UBE2D3 were initially screened as research objects.Luciferase reporter gene experiment further showed that UBE2V1 is the target gene of bta-miR-154 b,and bta-miR-154 b can down-regulate UBE2V1 mRNA and protein levels.(3)By predicting the binding sites of loc100847420 and bta-miR-154 b,we construct the loc100847420 overexpression vector and the overexpression vector with the binding site mutated,and co-transfect them with bta-miR-154 b in fetal bovine fibroblasts.Set up pc DNA3.1(+)-empty / negative control mimic group(control group),pc DNA3.1(+)-empty /bta-miR-154 b mimic group,pc DNA3.1(+)-loc100847420 wild type/bta-miR-154 b mimic group and pc DNA3.1(+)-loc100847420 mutant/bta-miR-154 b mimic group respectively,pc DNA3.1(+)-empty / bta-miR-154 b mimic compared with the control group The mRNA level of UBE2V1 will be significantly reduced(p < 0.05),and when we co-transfected pc DNA3.1(+)-loc100847420 wild-type/bta-miR-154 b mimic,the mRNA level of UBE2V1 is not significantly different from the control group(p > 0.05)but significantly higher than the pc DNA3.1(+)-empty/bta-miR-154 b mimic group(p < 0.05),while co-transfected with pc DNA3.1(+)-loc100847420 mutant/bta-miR-154 b mimic was also significantly lower than the control group(p < 0.05).These results demonstrate that loc100847420 acts as a ceRNA to regulate the level of UBE2V1 mRNA through bta-miR-154 b.(4)In vitro transcribed loc100847420 and RNA with the binding site of bta-miR-154 b mutated,and injected the in vitro transcription product into MII oocytes.The results showed that the cleavage rate is remain unchanged after injection of wild-type loc100847420(p>0.05),the blastocyst rate was significantly increased(p < 0.05),and the quality of blastocysts was improved(p < 0.05);and the 2-cell and 4-cell of wild-type loc100847420 were injected,the mRNA level of UBE2V1 in embryos at different stages was significantly increased(p < 0.05),may affect early embryo development.These results show that the sperm-derived lncRNA loc100847420 can be function as a ceRNA to regulate the expression of UBE2V1,and can affect the development of early embryos.