Decreased miR-30b-5p expression by DNMT1 methylation regulation involved in gastric cancer metastasis

miRNAs have emerged as crucial regulators in the regulation of development as well as human diseases, especially tumorigenesis. The aims of this study are to evaluate miR-30b-5p expression pattern and mechanism in gastric carcinogenesis due to which remains to be determined. Expression of miR-30b-5p was analyzed in 51 gastric cancer cases and 4 cell lines by qRT-PCR. The effect of DNA methylation on miR-30b-5p expression was assessed by MSP and BGS. In order to know whether DNMT1 increased miR-30b-5p promoter methylation, DNMT1 was depleted in cell lines AGS and BGC-823. The role of miR-30b-5p on cell migration was evaluated by wound healing assays. Decreased expression of miR-30b-5p was found in gastric cancer samples. In tumor, the expression level of miR-30b-5p was profound correlated with lymph node metastasis (P = 0.019). The level of miR-30b-5p may be restored by DNA demethylation and DNMT1 induced miR-30b-5p promoter methylation. In vitro functional assays implied that enforced miR-30b-5p expression affected cell migration, consistent with tissues analysis. Our findings uncovered that miR-30b-5p is significantly diminished in gastric cancer tissues, providing the first insight into the epigenetic mechanism of miR-30b-5p down-regulation, induced by DNMT1, and the role of miR-30b-5p in gastric cancer carcinogenesis. Overexpression of miR-30b-5p inhibited cell migration. Thus, miR-30b-5p may represent a potential therapeutic target for gastric cancer therapy.     

Molecular cloning and expression of a C-type lectin gene from Venerupis philippinarum

C-type lectins have been demonstrated to play important roles in invertebrate innate immunity by mediating the recognition of pathogens and clearing the micro-invaders. In the present study, a C-type lectin gene (denoted as VpCTL) was identified from Venerupis philippinarum by expressed sequence tag and rapid amplification of cDNA ends approaches. The full-length cDNA of VpCTL consists of 904 nucleotides with an open-reading frame of 456 bp encoding a peptide of 151 amino acids. The deduced amino acid sequence of VpCTL shared high similarity with C-type lectins from other species. The C-type lectin domain and the characteristic EPN and WND motifs were found in VpCTL. The VpCTL mRNA was dominantly expressed in the haemocytes of the V. philippinarum. After Listonella anguillarum challenge, the temporal expression of VpCTL mRNA in haemocytes was increased by 97- and 84-fold at 48 and 96 h, respectively. With high expression level in haemocytes and hepatopancreas, and the up-regulated expression in haemocytes indicted that VpCTL was perhaps involved in the immune responses to L. anguillarum challenge.     

Cerebral ischemic preconditioning reduces glutamate excitotoxicity by up-regulating the uptake activity of GLT-1 in rats

Our previous study has shown that cerebral ischemic preconditioning (CIP) can up-regulate the expression of glial glutamate transporter-1 (GLT-1) during the induction of brain ischemic tolerance in rats. The present study was undertaken to further explore the uptake activity of GLT-1 in the process by observing the changes in the concentration of extracellular glutamate with cerebral microdialysis and high-performance liquid chromatography. The results showed that a significant pulse of glutamate concentration reached the peak value of sevenfold of the basal level after lethal ischemic insult, which was associated with delayed neuronal death in the CA1 hippocampus. When the rats were pretreated 2 days before the lethal ischemic insult with CIP which protected the pyramidal neurons against delayed neuronal death, the peak value of glutamate concentration decreased to 3.9 fold of the basal level. Furthermore, pre-administration of dihydrokainate, an inhibitor of GLT-1, prevented the protective effect of CIP on ischemia-induced CA1 cell death. At the same time, compared with the CIP + Ischemia group, the peak value of glutamate concentration significantly increased and reached sixfold of the basal level. These results indicate that CIP induced brain ischemic tolerance via up-regulating GLT-1 uptake activity for glutamate and then decreasing the excitotoxicity of glutamate.     

Virus-induced gene silencing for comparative functional studies in Gladiolus hybridus

Key message

Virus-induced gene silencing (VIGS) system could be performed successfully in Gladiolus hybridus with vacuum infiltration of cormels and young plants.

Abstract

Functional analysis of genes in gladiolus has previously been impractical due to the lack of an efficient stable genetic transformation method. However, virus-induced gene silencing (VIGS) is effective in some plants which are difficult to transform through other methods. Although the Tobacco rattle virus (TRV)-based VIGS system has been developed and used for verifying gene functions in diverse plants, an appropriate TRV-VIGS approach for gladiolus has not been established yet. In this report we describe the first use of the TRV-VIGS system for gene silencing in gladiolus. Vacuum infiltration of cormels and young plants with the GhPDS-VIGS vector effectively down-regulated the PHYTOENE DESATURASE ortholog GhPDS gene and also resulted in various degrees of photobleaching in Gladiolus hybridus. The reduction in GhPDS expression was tested after TRV-based vector infection using real-time RT-PCR. In addition, the progress of TRV infection was detected by fluorescence visualization using a pTRV2: CP-GFP vector. In conclusion, the TRV-mediated VIGS described here will be an effective gene function analysis mechanism in gladiolus.