PDF(Pubmed)
Abstract：
OBJECTIVE: The study was conducted to screen differentially expressed miRNAs in sows at early pregnancy by high-throughput sequencing and explore its mechanism of action on embryo implantation.
METHODS: The blood serum of pregnant and non-pregnant Landrace×Yorkshire sows were collected 14 days after artificial insemination, and exosomal miRNAs were purified for high throughput miRNA sequencing. The expression patterns of 10 differentially expressed (DE) miRNAs were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The qRT-PCR quantified the abundance of serum exosomal miR-192 in pregnant and control sows, and the diagnostic power was assessed by receiver operating characteristic (ROC) analysis. The target genes of DE miRNAs were predicted with bioinformatics software, and the functional and pathway enrichment analysis was performed on gene ontology and the Kyoto encyclopedia of genes and genomes terms. Furthermore, a luciferase reporter system was used to identify the target relation between miR-192 and integrin alpha 4 (ITGA4), a gene influencing embryo implantation in pigs. Finally, the expression levels of miRNAs and the target gene ITGA4 were analyzed by qRT-PCR, and western blot, with the proliferation of BeWo cells detected by cell counting kit-8 (CCK-8).
RESULTS: A total of 221 known miRNAs were detected in the libraries of the pregnant and non-pregnant sows, of which 55 were up-regulated and 67 were down-regulated in the pregnant individuals compared with the non-pregnant controls. From these, the expression patterns of 10 DE miRNAs were validated. The qRT-PCR analysis further confirmed a significantly higher expression of miR-192 in the serum exosomes extracted from pregnant sows, when compared to controls. The ROC analysis revealed that miR-192 provided excellent diagnostic accuracy for pregnancy (area under the ROC curve [AUC] = 0.843; p>0.001). The dual-luciferase reporter assay indicated that miR-192 directly targeted ITGA4. The protein expression of ITGA4 was reduced in cells that overexpressed miR-192. Overexpression of miR-192 resulted in the decreased proliferation of BeWo cells and regulated the expression of cell cycle-related genes.
CONCLUSIONS: Serum exosomal miR-192 could serve as a potential biomarker for early pregnancy in pigs. miR-192 targeted ITGA4 gene directly, and miR-192 can regulate cellular proliferation.
METHODS: The blood serum of pregnant and non-pregnant Landrace×Yorkshire sows were collected 14 days after artificial insemination, and exosomal miRNAs were purified for high throughput miRNA sequencing. The expression patterns of 10 differentially expressed (DE) miRNAs were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The qRT-PCR quantified the abundance of serum exosomal miR-192 in pregnant and control sows, and the diagnostic power was assessed by receiver operating characteristic (ROC) analysis. The target genes of DE miRNAs were predicted with bioinformatics software, and the functional and pathway enrichment analysis was performed on gene ontology and the Kyoto encyclopedia of genes and genomes terms. Furthermore, a luciferase reporter system was used to identify the target relation between miR-192 and integrin alpha 4 (ITGA4), a gene influencing embryo implantation in pigs. Finally, the expression levels of miRNAs and the target gene ITGA4 were analyzed by qRT-PCR, and western blot, with the proliferation of BeWo cells detected by cell counting kit-8 (CCK-8).
RESULTS: A total of 221 known miRNAs were detected in the libraries of the pregnant and non-pregnant sows, of which 55 were up-regulated and 67 were down-regulated in the pregnant individuals compared with the non-pregnant controls. From these, the expression patterns of 10 DE miRNAs were validated. The qRT-PCR analysis further confirmed a significantly higher expression of miR-192 in the serum exosomes extracted from pregnant sows, when compared to controls. The ROC analysis revealed that miR-192 provided excellent diagnostic accuracy for pregnancy (area under the ROC curve [AUC] = 0.843; p>0.001). The dual-luciferase reporter assay indicated that miR-192 directly targeted ITGA4. The protein expression of ITGA4 was reduced in cells that overexpressed miR-192. Overexpression of miR-192 resulted in the decreased proliferation of BeWo cells and regulated the expression of cell cycle-related genes.
CONCLUSIONS: Serum exosomal miR-192 could serve as a potential biomarker for early pregnancy in pigs. miR-192 targeted ITGA4 gene directly, and miR-192 can regulate cellular proliferation.
摘要：
■进行该研究，以通过高通量测序在妊娠早期母猪中筛选差异表达的miRNA，并探讨其对胚胎着床的作用机制。
■在人工授精后14天收集妊娠和非妊娠长白猪×约克郡母猪的血清，和外泌体miRNA被纯化用于高通量miRNA测序。通过qRT-PCR验证了10个差异表达(DE)miRNA的表达模式。定量逆转录-聚合酶链反应(qRT-PCR)定量检测妊娠母猪和对照母猪血清外泌体miR-192的丰度,并通过接收器工作特性（ROC）分析评估诊断能力。利用生物信息学软件对DEmiRNAs的靶基因进行预测,并对基因本体论（GO）和京都基因和基因组百科全书（KEGG）术语进行了功能和途径富集分析。此外，荧光素酶报告系统用于鉴定miR-192和整合素α4（ITGA4）之间的靶关系，影响猪胚胎植入的基因。最后,通过qRT-PCR分析miRNAs和靶基因ITGA4的表达水平,和westernblot,CCK-8检测到BeWo细胞的增殖。
■在妊娠和非妊娠母猪的文库中总共检测到221种已知的miRNA，与非妊娠对照相比，妊娠个体中55例上调，67例下调。从这些,验证了10个DEmiRNAs的表达模式。qRT-PCR分析进一步证实了miR-192在妊娠母猪血清外泌体中的表达显著增高,与对照组相比。ROC分析显示miR-192为妊娠提供了极好的诊断准确性(ROC曲线下面积[AUC]=0.843;P>0.001)。双荧光素酶报告基因测定表明miR-192直接靶向ITGA4。在过表达miR-192的细胞中,ITGA4的蛋白表达降低。miR-192的过表达导致BeWo细胞的增殖减少，并调节细胞周期相关基因的表达。
■血清外泌体miR-192可以作为猪早期妊娠的潜在生物标志物。miR-192直接靶向ITGA4基因，miR-192可以调节细胞增殖。
■在人工授精后14天收集妊娠和非妊娠长白猪×约克郡母猪的血清，和外泌体miRNA被纯化用于高通量miRNA测序。通过qRT-PCR验证了10个差异表达(DE)miRNA的表达模式。定量逆转录-聚合酶链反应(qRT-PCR)定量检测妊娠母猪和对照母猪血清外泌体miR-192的丰度,并通过接收器工作特性（ROC）分析评估诊断能力。利用生物信息学软件对DEmiRNAs的靶基因进行预测,并对基因本体论（GO）和京都基因和基因组百科全书（KEGG）术语进行了功能和途径富集分析。此外，荧光素酶报告系统用于鉴定miR-192和整合素α4（ITGA4）之间的靶关系，影响猪胚胎植入的基因。最后,通过qRT-PCR分析miRNAs和靶基因ITGA4的表达水平,和westernblot,CCK-8检测到BeWo细胞的增殖。
■在妊娠和非妊娠母猪的文库中总共检测到221种已知的miRNA，与非妊娠对照相比，妊娠个体中55例上调，67例下调。从这些,验证了10个DEmiRNAs的表达模式。qRT-PCR分析进一步证实了miR-192在妊娠母猪血清外泌体中的表达显著增高,与对照组相比。ROC分析显示miR-192为妊娠提供了极好的诊断准确性(ROC曲线下面积[AUC]=0.843;P>0.001)。双荧光素酶报告基因测定表明miR-192直接靶向ITGA4。在过表达miR-192的细胞中,ITGA4的蛋白表达降低。miR-192的过表达导致BeWo细胞的增殖减少，并调节细胞周期相关基因的表达。
■血清外泌体miR-192可以作为猪早期妊娠的潜在生物标志物。miR-192直接靶向ITGA4基因，miR-192可以调节细胞增殖。
