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BackgroundMicroRNAs (miRNAs) play a critical role in responses to biotic and abiotic stress and have been characterized in a large number of plant species. Although flax (Linum usitatissimum L.) is one of the most important fiber and oil crops worldwide, no reports have been published describing flax miRNAs (Lus-miRNAs) induced in response to saline, alkaline, and saline-alkaline stresses.ResultsIn this work, combined small RNA and degradome deep sequencing was used to analyze flax libraries constructed after alkaline-salt stress (AS2), neutral salt stress (NSS), alkaline stress (AS), and the non-stressed control (CK). From the CK, AS, AS2, and NSS libraries, a total of 118, 119, 122, and 120 known Lus-miRNAs and 233, 213, 211, and 212 novel Lus-miRNAs were isolated, respectively. After assessment of differential expression profiles, 17 known Lus-miRNAs and 36 novel Lus-miRNAs were selected and used to predict putative target genes. Gene ontology term enrichment analysis revealed target genes that were involved in responses to stimuli, including signaling and catalytic activity. Eight Lus-miRNAs were selected for analysis using qRT-PCR to confirm the accuracy and reliability of the miRNA-seq results. The qRT-PCR results showed that changes in stress-induced expression profiles of these miRNAs mirrored expression trends observed using miRNA-seq. Degradome sequencing and transcriptome profiling showed that expression of 29 miRNA-target pairs displayed inverse expression patterns under saline, alkaline, and saline-alkaline stresses. From the target prediction analysis, the miR398a-targeted gene codes for a copper/zinc superoxide dismutase, and the miR530 has been shown to explicitly target WRKY family transcription factors, which suggesting that these two micRNAs and their targets may significant involve in the saline, alkaline, and saline-alkaline stress response in flax.ConclusionsIdentification and characterization of flax miRNAs, their target genes, functional annotations, and gene expression patterns are reported in this work. These findings will enhance our understanding of flax miRNA regulatory mechanisms under saline, alkaline, and saline-alkaline stresses and provide a foundation for future elucidation of the specific functions of these miRNAs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0808-2) contains supplementary material, which is available to authorized users.

Cancer cells are characterized by an uncontrolled increase in cell proliferation. G1 to S transition is one of the two main checkpoints used by cells to control the cell cycle progress and cell proliferation. G1/S progression is highly regulated by multiple intracellular signaling transduction cascades including Akt and p53 pathways, which therefore becomes a promising target for the development of novel anticancer therapy. Scutellaria barbata D. Don (SB) is a major component in many Chinese medicine formulas that have long been used in China to clinically treat various cancers including colorectal cancer (CRC). Recently, we reported that the ethanol extract of SB (EESB) is able to induce cancer cell apoptosis via activation of the mitochondrion-dependent pathway and inhibit tumor angiogenesis through suppression of Hedgehog signaling. To further elucidate the precise mechanisms of its antitumor activity, in the present study we evaluated the effect of EESB on the proliferation of human colon carcinoma HT-29 cells and investigated the underlying molecular mechanism. We found that EESB could inhibit the proliferation of HT-29 cells through blocking the G1/S cell cycle progression. In addition, EESB treatment profoundly promoted antiproliferative p21 expression, but inhibited the expression of pro-proliferative PCNA, cyclin D1 and CDK4 in HT-29 cells. Moreover, the phosphorylation/activation of Akt was significantly suppressed by EESB treatment, whereas that of p53 was enhanced. These results suggest that EESB could effectively induce G1/S arrest in human colon carcinoma cells via modulation of multiple cell cycle-related signaling pathways.

Neurogenesis is regulated by a number of signaling pathways, including the retinoic acid (RA) pathway, a key regulator of neurogenesis in the subventricular zone (SVZ) and hippocampus. Acupuncture has been used to treat neurological conditions and is known to potentially enhance cell proliferation in the neurogenic area (hippocampal dentate gyrus and the SVZ of the lateral ventricle walls) in pathological conditions, which is associated with improved brain function. However, whether or not the neuroprotective effects of electroacupuncture (EA) are mediated by the regulation of the RA signaling pathway remains to be determined. Using a transient middle cerebral artery occlusion model, in the present study we evaluated the effect of EA on the neurological functional recovery, infarction volume and investigated the underlying molecular mechanisms. Two hundred and sixteen SD rats were randomly divided into 3 groups: sham, model group (ischemic rats without EA stimulation) and EA group (ischemic rats with EA stimulation on ST36 and LI11). Behavioral deficits were detected with high-resolution digital analysis of 24-h home-cage video recordings. Infarct volume was determined by triphenyltetrazolium hydrochloride staining and the expression of RA mRNA and protein was measured using RT-PCR and western blotting, respectively. We found that EA decreased the infarct volume, promoted neurological functional recovery and increased the RA mRNA and protein expression, compared with the model group. Findings of this study suggest that promoting neurological functional recovery by modulating RA expression in the post-ischemic brain is one of the mechanisms by which EA can be effective in the treatment of ischemic stroke.

Background Members of the WRKY protein family, one of the largest transcription factor families in plants, are involved in plant growth and development, signal transduction, senescence, and stress resistance. However, little information is available about WRKY transcription factors in flax (Linum usitatissimum L.). Results In this study, comprehensive genome-wide characterization of the flax WRKY gene family was conducted that led to prediction of 102 LuWRKY genes. Based on bioinformatics-based predictions of structural and phylogenetic features of encoded LuWRKY proteins, 95 LuWRKYs were classified into three main groups (Group I, II, and III); Group II LuWRKYs were further assigned to five subgroups (IIa-e), while seven unique LuWRKYs (LuWRKYs 96–102) could not be assigned to any group. Most LuWRKY proteins within a given subgroup shared similar motif compositions, while a high degree of motif composition variability was apparent between subgroups. Using RNA-seq data, expression patterns of the 102 predicted LuWRKY genes were also investigated. Expression profiling data demonstrated that most genes associated with cellulose, hemicellulose, or lignin content were predominantly expressed in stems, roots, and less in leaves. However, most genes associated with stress responses were predominantly expressed in leaves and exhibited distinctly higher expression levels in developmental stages 1 and 8 than during other stages. Conclusions Ultimately, the present study provides a comprehensive analysis of predicted flax WRKY family genes to guide future investigations to reveal functions of LuWRKY proteins during plant growth, development, and stress responses.

Abstract. In the present study, we investigated the effects of millimeter wave treatment on the activation of the p38MAPK signaling pathway in the process of NO-induced apoptosis in chondrocytes. Cartilage was isolated from the knee joint of SD rats and used to establish cultured primary chondrocytes. After identification using in situ staining of type II collagen, the passage 2 chondrocytes were incubated with or without sodium nitroprussiate (SNP) to induce apoptosis and treated with a millimeter wave for various times. The apoptosis of chondrocytes was detected using immunofluorescence, an MTT assay, and Annexin V-FITC labeling followed by fluorescence-activated cell sorting (FACS). The activity of caspase-3 was measured using colorimeters, and the levels of p38 and p53 were also detected using RT-PCR and Western blotting. After treatment with SNP, the OD values of the experimental groups were significantly lower than the control group (P<0.01). The 24-h interference of a millimeter wave significantly prevented apoptosis (P<0.01) and showed a dose dependency, and an identical trend of apoptosis was noted with normal cell number counting (P<0.01) and FACS (P<0.01). Consistently, the caspase 3 activity showed a reverse trend, with the highest activity in the experimental group receiving no millimeter wave treatment (P<0.01). The mRNA expression of p38 and p53 and the protein levels of phosphorylated p38 and p53 showed a similar trend (P<0.01) to that of caspase 3 activity. In conclusion, millimeter wave treatment inhibits the SNP-induced apoptosis of chondrocytes through the p38MAPK pathway.

A genetic map is an important and valuable tool for quantitative trait locus (QTL) mapping, marker-assisted selection (MAS)-based breeding, and reference-assisted chromosome assembly. In this study, 112 F2 plants from a cross between Linum usitatissimum L. “DIANE” and “NY17” and parent plants were subjected to high-throughput sequencing and specific-locus amplified fragment (SLAF) library construction. After preprocessing, 61.64 Gb of raw data containing 253.71 Mb paired-end reads, each 101 bp in length, were obtained. A total of 192,797 SLAFs were identified, of which 23,115 were polymorphic, with a polymorphism rate of 11.99%. Finally, 2,339 SLAFs were organized into a linkage map consisting of 15 linkage groups (LGs). The total length of the genetic map was 1483.25 centimorgans (cM) and the average distance between adjacent markers was 0.63 cM. Combined with flax chromosome-scale pseudomolecules, 12 QTLs associating with 6 flax fiber-related traits were mapped on the chromosomal scaffolds. This high-density genetic map of flax should serve as a foundation for flax fine QTL mapping, draft genome assembly, and MAS-guided breeding. Ultimately, the genomic regions identified in this research could potentially be valuable for improving flax fiber cultivars, as well as for identification of candidate genes involved in flax fiber formation processes.Significance statementA high-density genetic map of flax was constructed, and QTLs were identified on the sequence scaffolds to be interrelated with fiber-related traits. The results of this study will not only provide a platform for gene/QTL fine mapping, map-based gene isolation, and molecular breeding for flax, but also provide a reference to help position sequence scaffolds on the physical map and assist in the process of assembling the flax genome sequence.

The aim of this study was to evaluate the therapeutic efficacy of Rubus aleaefolius Poir total alkaloids (TARAP) against hepatocellular carcinoma growth in vivo and in vitro, and to investigate the possible molecular mechanisms mediating its biological activity. Nude mice were implanted with HepG2 human hepatocellular carcinoma cells and fed with vehicle (physiological saline) or 3 g/kg/d dose of TARAP, 5 days per week, for 21 days. The in vivo efficacy of TARAP against tumor growth was investigated by evaluating its effect on tumor volume and tumor weight in mice with HCC xenografts and its adverse effect was determined by measuring the body weight gain. The in vitro effect of TARAP on the viability of HepG2 cells was determined by MTT assay. HepG2 cell morphology was observed via phase-contrast microscopy. Apoptosis in tumor tissues or in HepG2 cells was analyzed by TUNEL assay or FACS analysis with Annexin V/PI, respectively. The loss of mitochondrial membrane potential in HepG2 cells was determined via JC-1 staining followed by FACS analysis. Activation of caspase-9 and -3 in HepG2 cells was examined by a colorimetric assay. The mRNA and protein expression of Bcl-2 and Bax in tumor tissues were measured by RT-PCR and immunohistochemistry. TARAP reduced tumor volume and tumor weight, but had no effect on the body weight gain in HCC mice. TARAP decreased the viability of HepG2 cells and induced cell morphological changes in vitro in a dose- and time-dependent manner. In addition, TARAP induced apoptosis both in tumor tissues and in HepG2 cells. Moreover, TARAP treatment resulted in the collapse of mitochondrial membrane potential in HepG2 cells, as well as the activation of caspase-9 and -3. Furthermore, administration of TARAP increased the pro-apoptotic Bax/Bcl-2 ratio in HCC mouse tumors, at both transcriptional and translational levels. TARAP inhibits hepatocellular carcinoma growth both in vivo and in vitro probably through the activation of mitochondrial-dependent apoptosis, which may, in part, explain its anticancer activity. These results suggest that total alkaloids in Rubus aleaefolius Poir may be a potential novel therapeutic agent for the treatment of hepatocellular carcinoma and other cancers.