Identification and characterization of microRNAs and their target genes from Nile tilapia (Oreochromis niloticus).

MicroRNAs (miRNAs) are a class of small single-stranded, endogenous 21-22 nt non-coding RNAs that regulate their target mRNA levels by causing either inactivation or degradation of the mRNAs. In recent years, miRNA genes have been identified from mammals, insects, worms, plants, and viruses. In this research, bioinformatics approaches were used to predict potential miRNAs and their targets in Nile tilapia from the expressed sequence tag (EST) and genomic survey sequence (GSS) database, respectively, based on the conservation of miRNAs in many animal species. A total of 19 potential miRNAs were detected following a range of strict filtering criteria. To test the validity of the bioinformatics method, seven predicted Nile tilapia miRNA genes were selected for further biological validation, and their mature miRNA transcripts were successfully detected by stem-loop RT-PCR experiments. Using these potential miRNAs, we found 56 potential targets in this species. Most of the target mRNAs appear to be involved in development, metabolism, signal transduction, transcription regulation and stress responses. Overall, our findings will provide an important foundation for further research on miRNAs function in the Nile tilapia.

Targeted suppression of µ-calpain and caspase 9 expression and its effect on caspase 3 and caspase 7 in satellite cells of Korean Hanwoo cattle.

The calpains play an important role in cell death and cell signalling. Caspases catalyse wholesale destruction of cellular proteins which is a major cause of cellular death. The current study looks at the function of µ-calpain and caspase 9, using RNAi (RNA interference)-mediated silencing, and to observe the mRNA expression level of caspase genes during satellite cell growth. The satellite cells were treated with siRNA (small interfering RNA) of µ-calpain and caspase 9 separately. There was reduction of 16 and 24% in CAPN1 (calpain1)-siRNA2 and CAPN1-siRNA3 transfected cells respectively, whereas it was 60 and 56% in CAPN1-siRNA1 and CAPN1-siRNA4 transfected cells respectively. CAPN1-siRNA4 and CAPN1-siRNA1 treated cells showed more reduction in caspase 3 and 7 gene expression. CARD9 (caspase recruitment domain 9)-siRNA1 and CARD9-siRNA2-treated cells showed reduction of 40 and 49% respectively. CARD9-siRNA1 and CARD9-siRNA2 showed an increase in caspase 3 gene expression, whereas CARD9-siRNA2 showed reduction in caspase 7 gene expression. These results suggest a strong cross-talk between µ-calpain and the caspase enzyme systems. Suppression of target genes, such as µ-calpain and caspase 9, might have genuine potential in the treatment of skeletal muscle atrophy.

Fabrication and growth mechanism of ZnO nanostructures and their cytotoxic effect on human brain tumor U87, cervical cancer HeLa, and normal HEK cells.

ZnO nanostructures of diverse shape were grown via a solution process with different precursors and conditions. Morphological investigation of the nanostructures was carried out using field emission scanning electron microscopy and transmission microscopy observations and revealed that the nanostructures exhibit a wurtzite phase with an ideal lattice fringe distance of approximately 0.52 nm. The powder crystallinity was examined via X-ray diffraction spectroscopy. Screening results from anticancer studies of the effects on human brain tumor U87, cervical cancer HeLa, and normal HEK cells of ZnO nanostructures of diverse shape were obtained and indicate promising activity that varies with changes in the structure and the size of the particles. Treatment-induced cell death [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and survival assay], growth inhibition, cytogenetic damage (formation of micronuclei), and apoptosis were studied as parameters for the cellular response. Treatment with nanostructures enhanced growth inhibition and cell death in a concentration-dependent manner in both U87 and HeLa cell lines. At higher concentrations (above 15.6 µg/ml) the cytotoxic effects of the nanoparticles were highly synergistic and mainly mediated through apoptosis, implying the possible interactions of lesions caused by the agents. The enhanced cell death due to nanoparticles was accompanied by a significant increase (2-3 fold at 31.25 µg/ml) in the formation of micronuclei in U87 cells. The increase in the formation of micronuclei observed after treatment indicates that these structures may interfere with the rejoining of DNA strand breaks. Among all the nanostructures, nanoparticles and sheets exhibited potent activity against both HeLa and U87 cells. However, despite potent in vitro activity, all nanostructures exhibited diminished cytotoxicity against normal human HEK cells at all effective concentrations.

Microbial synthesis of gold nanoparticles using the fungus Penicillium brevicompactum and their cytotoxic effects against mouse mayo blast cancer C 2 C 12 cells.

Microorganisms, their cell filtrates, and live biomass have been utilized for synthesizing various gold nanoparticles. The shape, size, stability as well as the purity of the bio synthesized nanoparticles become very essential for application purpose. In the present study, gold nanoparticles have been synthesized from the supernatant, live cell filtrate, and biomass of the fungus Penicillium brevicompactum. The fungus has been grown in potato dextrose broth which is also found to synthesize gold nanoparticles. The size of the particles has been investigated by Bio-TEM before purification, following purification and after storing the particles for 3 months under refrigerated condition. Different characterization techniques like X-ray diffraction, Fourier transform infrared spectroscopy, and UV-visible spectroscopy have been used for analysis of the particles. The effect of reaction parameters such as pH and concentration of gold salt have also been monitored to optimize the morphology and dispersity of the synthesized gold nanoparticles. A pH range of 5 to 8 has favored the synthesis process whereas increasing concentration of gold salt (beyond 2 mM) has resulted in the formation of bigger sized and aggregated nanoparticles. Additionally, the cytotoxic nature of prepared nanoparticles has been analyzed using mouse mayo blast cancer C(2)C(12) cells at different time intervals (24, 48, and 72 h) of incubation period. The cells are cultivated in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum with antibiotics (streptopenicillin) at 37°C in a 5% humidified environment of CO(2). The medium has been replenished every other day, and the cells are subcultured after reaching the confluence. The viability of the cells is analyzed with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide method.

Identification and characterization of the Chinese giant salamander (Andrias davidianus) miRNAs by deep sequencing and predication of their targets.

MicroRNAs (miRNAs) are non-coding small RNA of approximately 22 nt in length. They are considered as key regulators for gene expression at the post-transcriptional level and play an important role in the regulation of many fundamental biological processes. Andrias davidianus, as one of the largest amphibian on earth, may represent the transitional type of animal from aquatic to terrestrial life, but so far, no miRNA has been identified in the species. In this study, Illumina deep sequencing was used for high-throughput analysis of miRNAs in a pooled small RNA library isolated from eight tissues sample of A. davidianus including the spleen, liver, muscle, kidney, skin, testis, gut, and heart. In total, 6,213,146 reads were obtained, 5,962,175 of which were related to 143 unique miRNAs, including 140 conserved and three novel A. davidianus-specific miRNAs. Among them, seven conserved miRNAs and one novel miRNA were selected to validate their expression pattern by stem-loop qRT-PCR. Moreover, 4700 potential target genes were predicted for 143 A. davidianus miRNAs; GO enrichment analysis and KEGG pathway showed that most of the targets are involved in diverse biological processes including ubiquitin-mediated proteolysis, FOXO signaling pathway, tight junction, and spliceosome. This study provides the first large-scale identification and characterization of A. davidianus miRNAs, and predicted their potential target genes; it will lay a valuable foundation for future understanding the role of these miRNAs on regulating diverse biological processes.

The effect of troglitazone on lipid accumulation and related gene expression in Hanwoo muscle satellite cell.

The current study was undertaken to determine the effect of the troglitazone (TGZ) on the expression of peroxisome proliferator-activating receptor (PPARγ), CCAAT/enhancer-binding protein, fatty acid binding protein 4, calpain 1 (CAPN1), and lipid accumulation in the myotube of Hanwoo muscle satellite cells. The satellite cells were treated with 5, 10, and 50 µM of TGZ for indicated time intervals. TGZ promoted the trans-differentiation with significant increase in glycerol accumulation. Polymerase chain reaction (PCR) and microarray results indicated that the TGZ treatment significantly increased the expression of adipogenic transcription factors. TGZ (10 and 50 µM) increased the CAPN1 gene expression 2.2- and 2.6-fold in real-time polymerase chain reaction analysis and 0.52- and 0.25-fold in microarray analysis, respectively, when compared with their respective controls. This result suggests that CAPN1 gene might be involved in the adipogenic differentiation programs. In addition, 13 genes were upregulated and 12 genes were downregulated in microarray analysis. Most of the up/downregulated genes were directly linked with adipogenesis.

MicroRNA-21 gene and cancer.

MicroRNAs (miRNAs) are a class of 18-27-nucleotides single-stranded RNA molecules that regulate gene expression at the post-transcriptional level. It has been demonstrated that miRNAs regulate a variety of physiological functions, including development, cell differentiation, proliferation, and apoptosis. There are growing evidence showed that miRNAs can affect the genesis and development of tumor and play a kind of tumor suppressor or oncogenic function by regulating its targetted gene-related signal pathway. miRNA-21 is one of the early discovered miRNAs in human cells, and the expression of miRNA-21 is significantly upregulated in different kinds of solid tumors. Its abnormal expression levels are closely associated with pathogenesis of cancers. This review summarizes the recent study on the field of miRNA-21 and its association with cancer.

Changes in proteolytic enzymes mRNAs and proteins relevant for meat quality during myogenesis and hypoxia of primary bovine satellite cells.

The current study was conducted to evaluate the functions of µ-calpain (CAPN1), calpastatin, HSPs (heat shock proteins), and caspases during myogenesis and cell death induced by sodium azide (NaN(3)) hypoxia. The cell samples were divided into three groups: satellite cells formed at confluent monolayer (stage 1), stage 1 cells fusion into myotubes on d eight post-differentiation (stage 2), and stage 2 cells treated with 1 mM NaN(3) for 24 h (stage 3). Real-time RT-PCR showed that stage 2 cells had increased CAPN1, calpastatin, caspase 7, and CARD9 (Caspase activation and recruitment domain 9) mRNA expressions compared to stage 1 cells (*p < 0.05). By Western blotting caspase 3, caspase 7, caspase 8, and caspase 9 protein levels increased in cells at stage 2 compared to cells at stage 1 (*p < 0.05). Real-time RT-PCR showed that stage 3 cells had increased CAPN1, calpastatin, caspase 7, HSP70 (70 kDA heat shock proteins), and HSP90 (90 kDA heat shock proteins-alpha) and decreased CARD9 mRNA expression compared to stage 2 cells (*p < 0.05). Stage 3 samples had increase caspase 7 and caspase 12 activities compared to stage 2 samples, and by Western blotting protein levels of both HSP70 and HSP90 expressions, increased significantly under hypoxia condition (*p < 0.05). Here, we conclude that CAPN1, calpastatin, caspase 3, caspase 7, caspase 8, and CARD9 have important roles for satellite cell myogenesis; and that caspase 7, 12, HSP70, and HSP90 are involved in the process of apoptotic cell death under hypoxia conditions and we speculate that these proteins may be involved in early postmortem proteolysis and meat tenderization.

Platinum quantum dots and their cytotoxic effect towards myoblast cancer cells (C2C12).

The cytotoxic effect towards myoblast cancer cells (C3C12) of well-crystalline colloidal Pt quantum dots (QDs) was examined and presented in this paper. The Pt QDs were synthesized by facile colloidal process and characterized by transmission electron microscopy (TEM) and high-resolution TEM (HR-TEM), which confirmed that the QDs are prepared in high-density, possessing a well-crystalline structure. To examine the cytotoxicity, various doses of as-prepared QDs were treated with C2C12 myoblast cancer cells at different incubation intervals; 24, 48, 72, and 96 hours, and the viability of cells was analyzed with MTT assay. The detailed analyses revealed that in the presence of Pt-QDs, with increasing the incubation time, the number of cancer cells decreases. Moreover, with increasing concentration of Pt-QDs, the cancer cell death increases, confirming that the concentration of Pt-QDs has a significant role in controlling the number of cancer cells. The present research demonstrated that Pt-QDs can efficiently be used as anticancer agents.