Expression Patterns And Functions Of Long Non-coding RNA RMST On Early Embryonic Development In Pigs

Pre-implantation embryonic development refers to the series of divisions and developments of fertilized eggs,resulting in the formation of blastocysts capable of implantation.Various factors are involved in the regulation and influence of pig pre-implantation embryonic development,and the study of its regulatory mechanism is currently weak.Investigating the mechanism of preimplantation embryonic development has profound significance for reproductive biology and regenerative medicine,making it a highly anticipated research topic.Long non-coding RNAs(lncRNAs)are a class of non-coding RNAs with a length of more than 200 nucleotides,which do not encode proteins but play important roles in gene expression and regulation.With the development of sequencing technology,a large number of lncRNAs have been discovered in early embryos and have been shown to participate in multiple regulatory mechanisms of early embryonic development.Therefore,lncRNAs have attracted widespread attention and discussion in the field of early embryonic development in recent years.Rhabdomyosarcoma 2 associated transcript(RMST)is a long non-coding RNA identified in rhabdomyosarcoma,which is involved in embryonic stem cell differentiation,neurogenesis,muscle cell differentiation and maturation,and is associated with tumor development,cardiovascular disease,and nervous system disorders.However,it is unclear whether RMST is also involved in the regulation of early embryonic development in mammals.Therefore,this study explores the function and regulatory mechanism of RMST in pig early embryonic development using pig early embryos as a model.Firstly,we obtained the complete transcript information of pig RMST using Rapid Amplification of cDNA Ends(RACE)technology.Next,we used SYBR Green-based real-time quantitative PCR to detect the expression of RMST in mature pig oocytes and pre-implantation embryos at different stages.The results showed that RMST expression was low in mature pig oocytes,1-cell,and 2-cell embryos,increased in 4-cell embryos and reached a peak,decreased in 8-cell embryos,and increased again in day 7 blastocysts.Subcellular localization analysis showed that RMST was distributed in both the cytoplasm and nucleus of early embryo cleavage cells.To investigate whether RMST plays a regulatory role in pig early embryonic development,we injected si RNA targeting RMST in the pronuclear stage and tracked early embryonic development.The results showed that the blastocyst rate after RMST knockdown was only 9.53 ± 4.49%,and 55.39 ±7.77% of embryos were arrested at the 8-cell stage,indicating that RMST is essential for pig early embryonic development.As many lncRNAs exert their functions by regulating the expression of upstream and downstream genes in a cis-regulatory manner,we also examined the effects of RMST knockdown and overexpression on the expression of neighboring genes.The results showed that neither RMST knockdown nor overexpression affected the expression levels of the upstream gene NEDD1 or the downstream gene LOC102160458.Additionally,we found that neither knockdown nor overexpression of NEDD1 or LOC102160458 affected the expression level of RMST.In mice,Rmst can act as a "sponge" for miR-221-3p,regulating the expression of target genes by "absorbing" miR-221-3p.Therefore,we hypothesized that pig RMST could also act as a "sponge" for miR-221-3p.To test this hypothesis,we used a luciferase reporter system to detect whether miR-221-3p could interfere with the expression of a luciferase reporter containing a target RMST fragment.The results showed that miR-221-3p interfered with the expression of the luciferase reporter containing the target RMST fragment,but not the one without the target fragment,and the control si RNA also did not interfere with the expression of the luciferase reporter containing the target RMST fragment.This indicates that pig RMST can act as a "sponge" for miR-221-3p.Furthermore,we used a gel shift assay to verify whether the digoxigenin(DIG)-labeled miR-221-3p,biotin-labeled RMST,and AGO2 protein complex could interact in vitro.The results showed that miR-221-3p and RMST could interact in the presence of the AGO2 protein complex,validating the mechanism of RMST as a "sponge" for miR-221-3p.Additionally,we transfected DIG-labeled miR-221-3p and biotin-labeled RMST synthesized in vitro into pig fibroblasts,and then performed an immunoprecipitation assay using anti-AGO2 antibody.The RT-PCR results showed that miR-221-3p and RMST were present in the complex precipitated by the anti-AGO2 antibody,further validating the mechanism of RMST as a "sponge" for miR-221-3p.In order to identify the target gene protected by RMST as a "sponge" for miR-221-3p,we used the starbase v3.0 database to predict the target genes of miR-221-3p.From the prediction results,we chose p53 binding protein 2(TP53BP2)as the target gene for our study.We used a luciferase reporter system to detect whether miR-221-3p could interfere with the expression of a luciferase reporter containing a target TP53BP2 fragment.The results showed that miR-221-3p interfered with the expression of the luciferase reporter containing the target TP53BP2 fragment,but not the one without the target fragment,and the control si RNA also did not interfere with the expression of the luciferase reporter containing the target TP53BP2 fragment,indicating that pig miR-221-3p can indeed target TP53BP2.Furthermore,we used a gel shift assay to verify whether the DIG-labeled miR-221-3p,biotin-labeled RMST,and AGO2 protein complex could interact in vitro.The results showed that miR-221-3p and TP53BP2 could interact in the presence of the AGO2 protein complex.Additionally,we transfected DIG-labeled miR-221-3p and biotin-labeled TP53BP2 synthesized in vitro into pig fibroblasts,and then performed an immunoprecipitation assay using anti-AGO2 antibody.The RT-PCR results showed that miR-221-3p and TP53BP2 were present in the complex precipitated by the anti-AGO2 antibody,further validating that miR-221-3p can indeed target TP53BP2.In summary,our study suggests that RMST may act as a competing endogenous RNAs(ceRNA),competitively binding to miR-221-3p to regulate the expression level of TP53BP2,forming the RMST-miR-221-3p-TP53BP2(lncRNA-miRNA-m RNA)regulatory pathway.