Abstract：
BACKGROUND: MicroRNAs (miRNAs)-a class of small endogenous non-coding RNAs-are widely involved in post-transcriptional gene regulation of numerous physiological processes. High-throughput sequencing revealed that the miR-192 expression level appeared to be significantly higher in the blood exosomes of sows at early gestation than that in non-pregnant sows. Furthermore, miR-192 was hypothesized to have a regulatory role in embryo implantation; however, the target genes involved in exerting the regulatory function of miR-192 required further elucidation.
METHODS: In the present study, potential target genes of miR-192 in porcine endometrial epithelial cells (PEECs) were identified through biotin-labeled miRNA pull-down; functional and pathway enrichment analysis was performed via gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment. Bioinformatic analyses were concurrently used to predict the potential target genes associated with sow embryo implantation. In addition, double luciferase reporter vectors, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), and Western blot were performed to verify the targeting and regulatory roles of the abovementioned target genes.
RESULTS: A total of 1688 differentially expressed mRNAs were identified via miRNA pull-down. Through RT-qPCR, the accuracy of the sequencing data was verified. In the bioinformatics analysis, potential target genes of miR-192 appeared to form a dense inter-regulatory network and regulated multiple signaling pathways, such as metabolic pathways and the PI3K-Akt, MAPKs, and mTOR signaling pathways, that are relevant to the mammalian embryo implantation process. In addition, CSK (C-terminal Src kinase) and YY1 (Yin-Yang-1) were predicted to be potential candidates, and we validated that miR-192 directly targets and suppresses the expression of the CSK and YY1 genes.
CONCLUSIONS: We screened 1688 potential target genes of miR-192 were screened, and CSK and YY1 were identified as miR-192 target genes. The outcomes of the present study provide novel insights into the regulatory mechanism of porcine embryo implantation and the identification of miRNA target genes.
METHODS: In the present study, potential target genes of miR-192 in porcine endometrial epithelial cells (PEECs) were identified through biotin-labeled miRNA pull-down; functional and pathway enrichment analysis was performed via gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment. Bioinformatic analyses were concurrently used to predict the potential target genes associated with sow embryo implantation. In addition, double luciferase reporter vectors, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), and Western blot were performed to verify the targeting and regulatory roles of the abovementioned target genes.
RESULTS: A total of 1688 differentially expressed mRNAs were identified via miRNA pull-down. Through RT-qPCR, the accuracy of the sequencing data was verified. In the bioinformatics analysis, potential target genes of miR-192 appeared to form a dense inter-regulatory network and regulated multiple signaling pathways, such as metabolic pathways and the PI3K-Akt, MAPKs, and mTOR signaling pathways, that are relevant to the mammalian embryo implantation process. In addition, CSK (C-terminal Src kinase) and YY1 (Yin-Yang-1) were predicted to be potential candidates, and we validated that miR-192 directly targets and suppresses the expression of the CSK and YY1 genes.
CONCLUSIONS: We screened 1688 potential target genes of miR-192 were screened, and CSK and YY1 were identified as miR-192 target genes. The outcomes of the present study provide novel insights into the regulatory mechanism of porcine embryo implantation and the identification of miRNA target genes.
摘要：
背景：微RNA（miRNA）-一类小的内源性非编码RNA-广泛参与许多生理过程的转录后基因调控。高通量测序显示，孕早期母猪血液外泌体中miR-192的表达水平明显高于非妊娠母猪。此外，据推测，miR-192在胚胎植入中具有调节作用；然而，参与miR-192调控功能的靶基因需要进一步阐明.
方法：在本研究中，通过生物素标记的miRNA下拉法鉴定了猪子宫内膜上皮细胞（PEEC）中miR-192的潜在靶基因；通过基因本体论分析和京都基因百科全书和基因组途径富集进行了功能和途径富集分析。同时使用生物信息学分析来预测与母猪胚胎植入相关的潜在靶基因。此外,双荧光素酶报告载体，逆转录-定量聚合酶链反应(RT-qPCR),并进行蛋白质印迹以验证上述靶基因的靶向和调节作用。
结果:通过miRNA下拉鉴定了总共1688个差异表达的mRNA。通过RT-qPCR,测序数据的准确性得到验证.在生物信息学分析中，miR-192的潜在靶基因似乎形成了一个密集的相互调节网络，并调节了多个信号通路，如代谢途径和PI3K-Akt，MAPK,和mTOR信号通路，与哺乳动物胚胎植入过程有关。此外,CSK(C-末端Src激酶)和YY1(Yin-Yang-1)被预测为潜在候选者,我们验证了miR-192直接靶向并抑制CSK和YY1基因的表达。
结论：我们筛选了1688个潜在的miR-192靶基因，CSK和YY1被鉴定为miR-192靶基因。本研究的结果为猪胚胎植入的调控机制和miRNA靶基因的鉴定提供了新的见解。
方法：在本研究中，通过生物素标记的miRNA下拉法鉴定了猪子宫内膜上皮细胞（PEEC）中miR-192的潜在靶基因；通过基因本体论分析和京都基因百科全书和基因组途径富集进行了功能和途径富集分析。同时使用生物信息学分析来预测与母猪胚胎植入相关的潜在靶基因。此外,双荧光素酶报告载体，逆转录-定量聚合酶链反应(RT-qPCR),并进行蛋白质印迹以验证上述靶基因的靶向和调节作用。
结果:通过miRNA下拉鉴定了总共1688个差异表达的mRNA。通过RT-qPCR,测序数据的准确性得到验证.在生物信息学分析中，miR-192的潜在靶基因似乎形成了一个密集的相互调节网络，并调节了多个信号通路，如代谢途径和PI3K-Akt，MAPK,和mTOR信号通路，与哺乳动物胚胎植入过程有关。此外,CSK(C-末端Src激酶)和YY1(Yin-Yang-1)被预测为潜在候选者,我们验证了miR-192直接靶向并抑制CSK和YY1基因的表达。
结论：我们筛选了1688个潜在的miR-192靶基因，CSK和YY1被鉴定为miR-192靶基因。本研究的结果为猪胚胎植入的调控机制和miRNA靶基因的鉴定提供了新的见解。
