[Identify Myeloid Differentiation-Related MiRNAs Response to ATRA Induction by RNA Sequencing and CRISPR/Cas9 Gene Editing].

OBJECTIVE: To identify differentiation related miRNA and evaluate roles of miRNA during ATRA induced myeloid differentiation. METHODS: The small RNA sequencing was used to analyze differential expressed miRNAs in ATRA induced NB4 cells. Then the several up or down-regulated miRNA were selected as the research candidates. SgRNAs targeting the genome of each miRNA were designed and NB4 cells with inducible expression of Cas9 protein were generated. After transduced sgRNA into NB4/Cas9 cells, the mutation level by PCR and surveyor assay were evaluated. The cell differentiation level was investigated by surface CD11b expression via flow cytometry. RESULTS: A total of 410 mature miRNAs which expressed in NB4 cells were detected out after treated by ATRA, 74 miRNAs were up-regulated and 55 were down-regulated miRNAs with DNA cleavage generated by CRISPR/Cas9 was assayed directly by PCR or surveyor assay, quantitative PCR showed that the expression of miRNA was downregulated, which evaluated that gene edition successfully inhibitied the expression of mature miRNA. MiR-223 knockout showed the myeloid differentation of NB4 significantly inhibitied, while miRNA-155 knockout showed the myeloid differentation of NB4 cells significantly increased. CONCLUSION: CRISPR/Cas9 is a powerful tool for gene editing and can lead to miRNA knockout. Knockouts of miR-223 and miR-155 have shown a differentiation-related phenotype, and the potential mechanism is the integrative regulation of target genes.

[Knockout of Micro-RNA-21 Gene in DLBCL OCI-Ly3 cells by TALEN Technique].

OBJECTIVE: To explore an efficient way to knockout microRNA genes in hemapoietic cell lines with a very low transfection efficiency, so as to facilitate the study of microRNA function in hematopoietic malignancies. METHODS: TALE-nucleases was utilized to knockout the microRNA-21 gene in human diffuse large B-cell lymphoma cells (OCI-Ly3). The OCI-Ly3 single cell clones without expression of miR-21 were established through eGFP(+) enrichment, PCR screening, and microRNA quantification. Finally, the miR-21 changes of mutant clones were identified by sequencing. RESULTS: Four miR-21-knockouted OCI-Ly3 single-cell-derived clones were established after 2 round transfection and screening. The miR-21 knockout efficiency was around 10/10(6) original cells. Sequencing the mutant clones indicated that miR-21 expression could be drastically reduced by simply altering sequences immediately adjacent to the microRNA duplex. CONCLUSION: This strategy may be applied to knockout any microRNA of interest even in hemapoietic cell lines with very low transfection efficiency.

[Biological responses of maize seedlings to single and combined stress of cadmium and phenanthrene].

Maize (Zea mays L.) pot-culture experiments were conducted in this study for exploring the biological responses of maize seedlings to single and joint stress of Cadmium (Cd) and phenanthrene. The results showed that single-Cd treatment with Cd concentration ranging from 0 to 50 mg/kg had no significant influences on the above-ground biomass, root biomass, and the soluble protein of maize seedlings; with the increase of Cd concentrations, the maize leaf SOD activities appeared as an increasing-decreasing trend, while the POD, CAT, APX activities increased persistently. The APX is very sensitive to Cd stress, which increased 38% than control treatment at lower Cd treatment (1 mg/kg). The accumulation of O2-* is one of the main cause of single-Cd toxicity. Under the Cd-phenanthren combined stress, the SOD activity increased at lower Cd concentration (1 mg/kg) , which can reached to 1.66 times of control treatment, and decreased when Cd concentration becomes high. The SOD activity is a sensitive index in response to the Cd-phenanthren combined pollution. Unlike SOD activity, the activities of POD, CAT and APX were obviously induced by combined pollution of Cd and phenanthren; but these enzyme activities decreased when compared with the single-Cd stress, which suggested that the combined toxicity of Cd and phenanthrene is stronger than the single-Cd stress. And the accumulation of O2-* and *OH is one of the main cause of Cd-phenanthrene combined toxicity.

2-chlorophenol induced ROS generation in fish Carassius auratus based on the EPR method.

In the present study, a secondary spin trapping technique was used followed by electron paramagnetic resonance (EPR) analysis, to study the potential of reactive oxygen species (ROS) production after fish (Carassius auratus) were injected i.p. with different doses (50, 100, 200, 250, 500mgkg(-1)) of 2-chlorophenol (2-CP). The ROS signal intensity of the EPR spectrum showed a significant increase (p<0.05, compared with the control) when the 2-CP dose was as low as 50mgkg(-1). There is a good relationship between the 2-CP administered doses and ROS generation. Based on the hyperfine splitting constants and shape of the EPR spectrum, the ROS which was generated in fish liver after intraperitoneal (i.p.) injection of 2-CP was identified as ()OH. SOD and CAT activities were found to be induced at lower doses of 2-CP. GSH levels fell below the control level following all treatments with 2-CP, and GSSG levels changed along with those of GSH. These observations indicated that the fish experienced oxidative stress. The strong positive correlation (r=0.966, p<0.005) between ()OH radical and lipid peroxidation suggested that lipid peroxidation was possibly induced by ()OH. The phase II detoxification enzyme glutathione-S-transferase (GST) may play an important role in 2-CP metabolism or excretion and, consequently, reduce ROS production. This study provides strong evidence that level of ROS is significantly increased in 2-CP stressed fish, and ROS may serve as a potential biomarker to indicate 2-CP contamination.