Saline-alkaline stress is a universal abiotic stress that adversely affects plant growth and productivity. Saline-alkaline conditions results in plant abnormal transcriptome expression finally manifesting as defective phenotypes. Considerable research has revealed the active role of microRNA in various stress conditions. This study was aimed to identify novel miRNAs and the miRNA expression patterns in the leguminous model plant R108 (Medicago truncatula). The miRNA contained in the total RNA extracted from Medicago truncatula seedlings (72 h) that had been treated with solutions mimicking saline and alkaline soils was subjected to miRNA deep sequencing. The Illumina HiSeq sequencing platform was used to analyze nine small RNA libraries of three treatment groups: distilled water, 20 mM NaCl + Na2SO4 and 5 mM Na2CO3 + NaHCO3. Sequencing revealed that 876 miRNAs including 664 known miRNAs and 212 potential novel miRNAs were present in all the libraries. The miR159 family, miR156 family, miR2086-3p, miR396, miR166, miR319, miR167, miR5213-5p, miR1510 and miR2643 were among the most expressed miRNAs in all libraries. The results of miRNAs expression under treatments were validated by reverse-transcription quantitative PCR (RT-qPCR). Target gene prediction through computational analysis and pathway annotation analysis revealed that the primary pathways affected by stress were related to plant development, including metabolic processes, single-organism processes and response to the stimulus. Our results provide valuable information towards elucidating the molecular mechanisms of salt/alkali tolerance in Medicago truncatula and provide insight into the putative role of miRNAs in plant stress resistance.

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The mammalian gonadotropin-inhibitory hormone (GnIH) ortholog, RFamide-related peptide (RFRP), is considered to act on gonadotropin-releasing hormone (GnRH) neurons and the pituitary to inhibit gonadotropin synthesis and release. However, there is little evidence documenting whether RFamide-related peptide 3 (RFRP-3) plays a primary role in inhibition of the hypothalamo-pituitary-gonadal (HPG) axis prior to the onset of puberty. The present study aimed to understand the functional significance of the neuropeptide on pubertal development. The developmental changes in reproductive-related gene expression at the mRNA level were investigated in the hypothalamus of female mice. The results indicated that RFRP-3 may be an endogenous inhibitory factor for the activation of the HPG axis prior to the onset of puberty. In addition, centrally administered RFRP-3 significantly suppressed plasma luteinizing hormone (LH) levels in prepubertal female mice. Surprisingly, centrally administered RFRP-3 had no effects on plasma LH levels in ovariectomized (OVX) prepubescent female mice. In contrast, RFRP-3 also inhibited plasma LH levels in OVX prepubescent female mice that were treated with 17beta-estradiol replacement. Our study also examined the effects of RFRP-3 on plasma LH release in adult female mice that were ovariectomized at dioestrus, with or without estradiol (E2). Our results showed that the inhibitory effects of RFRP-3 were independent of E2 status. Quantitative real-time PCR and immunohistochemistry analyses showed that RFRP-3 inhibited GnRH expression at both the mRNA and protein levels in the hypothalamus. These data demonstrated that RFRP-3 could effectively suppress pituitary LH release, via the inhibition of GnRH transcription and translation in prepubescent female mice, which is associated with estrogen signaling pathway and developmental stages.

BackgroundOvarian cancer is a leading cause of the death from gynecologic malignancies. Hypoxia is closely related to the malignant growth of cells. However, the molecular mechanism of hypoxia-regulated ovarian cancer cells remains unclear. Thus, this study was conducted to identify the key genes and pathways implicated in the regulation of hypoxia by bioinformatics analysis.MethodsUsing the datasets of GSE53012 downloaded from the Gene Expression Omnibus (GEO), the differentially expressed genes (DEGs) were screened by comparing the RNA expression from cycling hypoxia group, chronic hypoxia group, and control group. Subsequently, cluster analysis was performed followed by the construction of the protein-protein interaction (PPI) network of the overlapping DEGs between the cycling hypoxia and chronic hypoxia using ClusterONE. In addition, gene ontology (GO) functional and pathway enrichment analyses of the DEGs in the most remarkable module were performed using Database for Annotation, Visualization and Integrated Discovery (DAVID) software. Ultimately, the signaling pathways associated with hypoxia were verified by RT-PCR, WB, and MTT assays.ResultsA total of 931 overlapping DEGs were identified. Nine hub genes and seven node genes were screened by analyzing the PPI and pathway integration networks, including ESR1, MMP2, ErbB2, MYC, VIM, CYBB, EDN1, SERPINE1, and PDK. Additionally, 11 key pathways closely associated with hypoxia were identified, including focal adhesion, ErbB signaling, and proteoglycans in cancer, among which the ErbB signaling pathway was verified by RT-PCR, WB, and MTT assays. Furthermore, functional enrichment analysis revealed that these genes were mainly involved in the proliferation of ovarian cancer cells, such as regulation of cell proliferation, cell adhesion, positive regulation of cell migration, focal adhesion, and extracellular matrix binding.ConclusionThe results show that hypoxia can promote the proliferation of ovarian cancer cells by affecting the invasion and adhesion functions through the dysregulation of ErbB signaling, which may be governed by the HIF-1α-TGFA-EGFR-ErbB2-MYC axis. These findings will contribute to the identification of new targets for the diagnosis and treatment of ovarian cancer.Electronic supplementary materialThe online version of this article (10.1186/s13048-018-0388-x) contains supplementary material, which is available to authorized users.

A full-length cDNA, named MsNHX1, encoding a vacuolar Na+/H+ antiporter was cloned from alfalfa (Medicago sativa), using degenerate primers, followed by 3' and 5' RACE. The cDNA sequence was 2225 bp long and included an open reading frame encoding a deduced protein of 541-amino-acid polypeptide. The deduced amino acid sequence showed high similarity (more than 73%) to those of the previously cloned Na+/H+ antiporters form Arabidopsis thaliana, Qryza sativa, Atriplex gemlinin, Beta vulgaris and Hordeum vulgare. Southern hybridization showed that MsNHX1 has only one copy in alfalfa genome. Semiquantitative RT-PCR analysis indicated that the mRNA level of MsNHX1 was moderate without stress and markedly up-regulated after treatment by NaCl and ABA (abscisic acid). Those results suggest that the MsNHX1 product play an important role in salt tolerance of the alfalfa, and its transcript expression is possibly partially regulated through ABA-dependent signaling pathway.

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Ovaries, which provide a place for follicular development and oocyte maturation, are important organs in female mammals. Follicular development is complicated physiological progress mediated by various regulatory factors including microRNAs (miRNAs). To demonstrate the role of miRNAs in follicular development, this study analyzed the expression patterns of miRNAs in granulosa cells through investigating three previous datasets generated by Illumina miRNA deep sequencing. Furthermore, via bioinformatic analyses, we dissected the associated functional networks of the observed significant miRNAs, in terms of interacting with signal pathways and transcription factors. During the growth and selection of dominant follicles, 15 dysregulated miRNAs and 139 associated pathways were screened out. In comparison of different styles of follicles, 7 commonly abundant miRNAs and 195 pathways, as well as 10 differentially expressed miRNAs and 117 pathways in dominant follicles in comparison with subordinate follicles, were collected. Furthermore, SMAD2 was identified as a hub factor in regulating follicular development. The regulation of miR-26a/b on smad2 messenger RNA has been further testified by real time PCR. In conclusion, we established functional networks which play critical roles in follicular development including pivotal miRNAs, pathways, and transcription factors, which contributed to the further investigation about miRNAs associated with mammalian follicular development.

A suppression subtraction hybridization (SSH) cDNA library had been constructed to identify differentially expressed genes. Based on the sequence of an expressed sequence tag (EST) homologous to Pisum sativum zinc finger protein mRNA (Accession number: AF160911), the full-length cDNA of 1,676 nucleotides was cloned from alfalfa by rapid amplification of cDNA ends (RACE). It was designated as MsZFN, encoding a protein of 418 amino acids. The amino acid sequence compared by blast revealed high homology with zinc finger protein of other plants. Sequence comparison showed that there were five conserved typical zinc finger motifs, and one sugar transfer protein signature. The calculated molecular weight of the MsZFN protein was 45.8 k Da, and theoretical isoelectric point was 8.13. The MsZFN localized in nucleus. Under normal growth conditions, differential expression of MsZFN exhibited that the expression was the highest in leaf and the lowest in root. MsZFN was quickly and transiently induced by NaCl treatment and reached its maximum at 30 min.