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MicroRNAs (miRNAs) are widely expressed in different mammalian tissues and exert their biological effects through corresponding target genes. miRNA target genes can be rapidly and efficiently identified and screened by combining bioinformatics prediction and experimental validation. To investigate the possible molecular regulatory mechanisms involving miRNAs during uterine involution in postpartum ewes, we used Illumina HiSeq sequencing technology to screen for the number and characteristics of miRNAs in faster uterine involution and normal uterine involution group. A total of 118 differentially expressed miRNAs, including 33 known miRNAs and 85 new miRNAs, were identified in the hypothalamic library, whereas 54 miRNAs, including 5 known miRNAs and 49 new miRNAs, were identified in the uterine library. Screening with four types of gene prediction software revealed 73 target genes associated with uterine involution, and subsequently, GO annotation and KEGG pathway analysis were performed. The results showed that, in the hypothalamic–uterine axis, uterine involution in postpartum ewes might primarily involve two miRNA-target gene pairs, namely, miRNA-200a–PTEN and miRNA-133–FGFR1, which can participate in GnRH signal transduction in the upstream hypothalamus and in the remodeling process at the downstream uterus, through the PI3K–AKT signaling pathway to influence the recovery of the morphology and functions of the uterus during the postpartum period in sheep. Therefore, identification of differentially expressed miRNAs in this study fills a gap in the research related to miRNAs in uterine involution in postpartum ewes and provides an important reference point for a comprehensive understanding of the molecular mechanisms underlying the regulation of postpartum uterine involution in female livestock. [ABSTRACT FROM AUTHOR]

To examine the possible miRNA molecular regulatory mechanisms during maternal uterine involution after delivery, we selected ovary and uterus tissues that are structurally connected as experimental materials. We employed Illumina HiSeq sequencing to screen and analyze the quantity and characteristics of miRNA in postpartum ewes in the methylergometrine-treated group and physiological saline control group. Results showed that 16 miRNAs were identified in the ovary libraries, including 4 known miRNAs and 12 novel miRNAs. In the uterus libraries, 54 miRNAs were identified, which included 5 known miRNAs and 49 novel miRNAs. At the same time, target gene prediction, GO annotation, and KEGG signaling pathway enrichment analysis were employed. We found that maternal uterine involution after delivery may involve two miRNA-target gene pairs, i.e., miRNA-200a- ZEB1 and YAP1. The YAP1/Hippo signaling pathway is used to construct an ovary–uterine axial regulatory mechanism to regulate the restoration of postpartum maternal uterine morphology and function. In view of this, the identification of miRNAs with significant differences in this study fills a gap in research on miRNAs associated with regulation of postpartum uterine recovery in ewes and provided an important reference for comprehensive understanding and in-depth research on the regulatory molecular network mechanism for postpartum uterine involution in small ruminants. [ABSTRACT FROM AUTHOR]

Evidence has shown that microRNA-665 (miR-665) is highly expressed in the mid-luteal phase compared with the early and end-luteal phase of the corpus luteum (CL) life cycle. However, whether miR-665 is a positive regulator of the life span of the CL is still unknown. The objective of this study is to explore the effect of miR-665 on the structural luteolysis in the ovarian CL. In this study, the targeting relationship between miR-665 and hematopoietic prostaglandin synthase (HPGDS) was firstly verified by dual luciferase reporter assay. Then, quantitative real-time PCR (qRT-PCR) was used to detect the expression of miR-665 and HPGDS in luteal cells. Following miR-665 overexpression, the apoptosis rate of the luteal cells was determined using flow cytometry; B-cell lymphoma-2 (BCL-2) and caspase-3 mRNA and protein were measured using qRT-PCR and Western blot (WB) analysis. Finally, the DP1 and CRTH2 receptors of PGD 2 , a synthetic product of HPGDS, were localized using immunofluorescence. Results confirmed that HPGDS was a direct target gene of miR-665, and miR-665 expression was negatively correlated with HPGDS mRNA expression in luteal cells. Meanwhile, after miR-665 was overexpressed, the apoptotic rate of the luteal cells showed a significant decrease (P < 0.05) and this was accompanied by elevated expression levels of anti-apoptotic factor BCL-2 mRNA and protein and decreased expression levels of apoptotic factor caspase-3 mRNA and protein (P < 0.01). Moreover, the immune fluorescence staining results showed that the DP1 receptor was also significantly decreased (P < 0.05), but the CRTH2 receptor was significantly increased (P < 0.05) in luteal cells. Overall, these results indicate that miR-665 reduces the apoptosis of luteal cells via inhibiting caspase-3 expression and promoting BCL-2 expression, and the biological function of miR-665 may be attributed to its target gene HPGDS which regulates the balance of DP1 and CRTH2 receptors expression in luteal cells. As a consequence, this study suggests that miR-665 might be a positive regulator of the life span of the CL rather than destroy the integrity of CL in small ruminants. • miR-665 is a newly identified miRNA in reproduction, we firstly confirmed its regulatory mechanism on the corpus luteum in vitro. • We found that miR-665 is a positive regulator of the life span of the corpus luteum. • Potential mechanisms of miR-665 on luteum cells survival by which down-regulated DP1/CRTH2 receptor ratio reduces caspase-3 expression and increases BCL-2 expression to inhibit the apoptosis of the luteum cells. [ABSTRACT FROM AUTHOR]

Oomycete pathogens secrete numerous effectors to manipulate host immunity. While some effectors share a conserved structural fold, it remains unclear if any have conserved host targets. Avr3a‐like family effectors, which are related to Phytophthora infestans effector PiAvr3a and are widely distributed across diverse clades of Phytophthora species, were used to study this question. By using yeast‐two‐hybrid, bimolecular fluorescence complementation and co‐immunoprecipitation assays, we identified members of the plant cinnamyl alcohol dehydrogenase 7 (CAD7) subfamily as targets of multiple Avr3a‐like effectors from Phytophthora pathogens. The CAD7 subfamily has expanded in plant genomes but lost the lignin biosynthetic activity of canonical CAD subfamilies. In turn, we identified CAD7s as negative regulators of plant immunity that are induced by Phytophthora infection. Moreover, AtCAD7 was stabilized by Avr3a‐like effectors and involved in suppression of pathogen‐associated molecular pattern‐triggered immunity, including callose deposition, reactive oxygen species burst and WRKY33 expression. Our results reveal CAD7 subfamily proteins as negative regulators of plant immunity that are exploited by multiple Avr3a‐like effectors to promote infection in different host plants. [ABSTRACT FROM AUTHOR]