Functional Study Of A Novel Long Non-coding RNA Lnc-370 In Early Porcine Embryonic Development

Pre-implantation embryo development in mammals begins at fertilization with the migration and fusion of the maternal and paternal pro-nuclei,followed by the degradation of inherited factors involved in germ cell specification and the zygotic gene activation?ZGA?required for subsequent cell divisions,compaction,and blastulation.Most studies about early embryogenesisthat carried on mouses or non-mammalian species,often need extrapolation of the findings to human development.pigs,as animal model of large mammals,become an important biomedical study model.Because the organ size of pigs are similar to human's,the pig can be used as an important organ model for human organ transplantation,which is essential for the regulation of reproduction in pigs.Long non-coding RNAs?lnc RNAs?were formerly suggested to be lacking of biological functions and considered as “dark matters”.However,it is now obvious that many lnc RNAs played an important role in various biological processes.In the cytoplasm,lnc RNAs couldincrease or decrease the stability of mRNAs,and promote or inhibit mRNAs translation,whichwould finally influence the expression of proteins-coding genes.Furthermore,some lnc RNAs acted as precursors or interacting partners of short noncoding RNAs.Because porcine ZGA generally occurs in the 4-cell stage,we analyzed the function of several highly expressed lnc RNA in the stage.In previous work,we identifed 7,618 novel linc RNAs from 4,776 loci based on published RNA-seq data.Constructed the linc RNA stage specific co-expression gene modules,Performed correlationanalysis,function enrichment analysis,and WGCNA analysis.In this study,We analyzed the structural characteristics and expression patterns of porcine lincRNA,As results,our newly identified linc RNAs in pig shared many characteristics with those in other mammalian species.Compared with protein codingtranscripts,they are lower in exon number,longer in exon,shorter in whole transcripts,lower in expression level and more specific in expression patterns.We choose 12 porcine linc RNA transcripts Whichwere verified by Q-PCR,to determine the expressionlevel pattern in porcine different embryonic development periods and different tissues.According to the results of Q-PCR,these novel lnc RNA transcripts show high expression in porcine testis and ovary tissues.According to the expression pattern,we choose TCONS₀0166370 transcripts as the research object,for convenience,this transcript was named Lnc-370,using locked nucleic acid?LNA?interference,the functional deficiency experiment indicate Thatmore than 90% of embryos arrested at 1-cell stage in porcine embryos,and these embryos can not develop beyond 1-cell stage.Then,we explored the mechanism of Lnc-370 interference after embryo arrest,tracking the whole process of the injection group and the control group of embryonic pronuclear formation and nuclear condensation.The results showed that Lnc-370 interference did not affcet nuclear expand and prokaryotic formation,but arise from the failure of male and female pronuclear fusion.At the same time,immunofluorescence staining of Ed U was used to determine the DNA replication of the blocked embryos.DNA damage and early apoptosis are detected through ?-H2 AX and Annexin-V.For the spindle of the experimental group and the control group embryos by using alpha-Tublin antibody staining,the results showed that the interference of Lnc-370 embryos could not form normal spindle,so we speculated that Lnc-370 would play an important role in spindle assembly.We also explored the stage of embryo arrest,and found that the embryo could not reach metaphase of mitosis.By co-injection of the Lnc370-LNA and Egfp mRNA in a single blastomere of 2-cell embryos,we observe that knockdown of Lnc-370 in one blastomere of the 2-cell embryo have no effect on the following cleavage,indicating Lnc-370 may be a specific regulator in the first cell cycle of zygote.In addition,our results showed that after Lnc-370 interference,the blastocyst rate was nearly to zero,so we inferred that the Lnc-370 takes part in dynamic control process during early embryonic development.But current research results showed that the detailed molecular mechanism of Lnc-370 regulation of early embryonic development in pigs remained obscure,it still takes a lot of time for us to explore.