| The early embryonic development of mammals is a highly precise process,involving a series of biological processes such as fertilization,cleavage,and the first lineage differentiation,laying the foundation for the future development of the individual.Long non-coding RNA(lncRNA)is a type of RNA that lacks coding potential and has a length greater than 200 nucleotides.lncRNA was previously considered a simple non-coding "junk" RNA;however,research has shown that lncRNA plays an important role in various physiological and pathological processes.Many lncRNAs are highly expressed in early embryos,and some of them are crucial for maintaining embryonic development.Long non-coding RNA growth arrest-specific transcript 5(GAS5)is a novel tumor suppressor that is downregulated in malignant tumors such as breast cancer,gastric cancer,lung cancer,and prostate cancer.Studies have shown that high levels of GAS5 expression inhibit cell cycle progression,while inhibiting GAS5 reduces cell apoptosis and promotes cell division.Currently,research on GAS5 mainly focuses on humans and mice,with no studies on pigs.Compared to mice,pigs are more similar to humans in terms of body size,physiological structure,and gene expression levels.Therefore,the function and mechanism of GAS5 in the early embryonic development of pigs are worth investigating.In this study,we first compared the GAS5 sequences of mice and humans to the pig genome and identified the most similar sequence,ultimately determining that the LOC110255511 gene located on chromosome 9 is the main GAS5 gene in pigs.We then analyzed the expression pattern of GAS5 in pig oocytes and early embryos and found that GAS5 is expressed from the4-cell stage,with expression gradually increasing from the 4-cell to the morula stage and gradually decreasing from the blastocyst to the day 7 embryo stage.This result indicates that GAS5 has a unique expression pattern during early embryonic development in pigs,suggesting that it may have a regulatory function in pig early embryonic development.Subsequently,we performed subcellular localization analysis of GAS5 in blastocysts and found that GAS5 is expressed in both the cytoplasm and nucleus,indicating that GAS5 may have functions in both the cytoplasm and nucleus.To investigate whether GAS5 is involved in the regulation of early embryonic development in pigs,we interfered with GAS5 during the prokaryotic period.The statistical results of embryonic development showed that embryonic development was arrested at the 8-cell stage and the morula stage,indicating that GAS5 is necessary for early embryonic development in pigs.To explore the mechanism of GAS5 in regulating early embryonic development in pigs,we interfered and overexpressed GAS5 during the prokaryotic period,and found that interfering or overexpressing GAS5 had no effect on the expression levels of its upstream and downstream genes DARS2 and ZBTB37,indicating that GAS5 does not use cis-regulatory mechanisms to affect the expression of surrounding genes.In mice,GAS5 can act as an endogenous competing RNA(ce RNA)to "sponge" miR103,thereby playing a protective role for target genes.Therefore,we verified this mechanism in pigs.Using a dual luciferase reporter system and gel shift assay,we found that pig GAS5 can "sponge" miR103.Through website prediction,we found a series of target genes of miR103-3P,including PTEN.PTEN is a tumor suppressor gene discovered in recent years,which regulates many cellular processes,including proliferation and survival.Therefore,we chose PTEN for further verification.Using a dual luciferase reporter system and gel shift assay,we found that pig PTEN is a target gene of miR103-3P.Additionally,we found that interfering with GAS5 led to a decrease in PTEN expression levels,while overexpressing GAS5 led to an increase in PTEN expression levels.In summary,pig GAS5 can act as a ceRNA to "sponge" miR103-3P and promote the expression of PTEN.The conclusion of this paper provides a reference for the study of the function and mechanism of pig GAS5 and promotes the research on the mechanism of early embryonic development in pigs. |
