ABSTRACT
Objective:To investigate the expression of circular RNA circOMA1 in children with acute myeloid leukemia (AML) and to investigate the effect and mechanism of circOMA1 on the cell proliferation and apoptosis of human acute monocytic leukemia cells (THP-1).Methods:Bone marrow samples of 14 children with AML at the initial diagnosis and after complete remission were collected as the initial diagnosis group and remission group, and bone marrow samples from 10 children without tumor or malignant blood disease in the same hospital and the same period were enrolled as the control group.Real-time quantitative polymerase chain reaction (qPCR) was used to detect the expression of circOMA1, miR-145 and myelocytomatosis viral oncogene homolog (MYC) mRNA in clinical AML samples and THP-1 cell line.The cells transfected with THP-1 were divided into groups, the cells transfected with circOMA1 alone (circOMA1 high expression group) were transfected with pcDNA3.1 empty vector cells as control (pcDNA3.1 control group); the cells co transfected with circOMA1 and miR-145 (circOMA1+ miR-145 group) were treated with pcDNA3.1 and miR-NC co transfected cells were used as control (pcDNA3.1+ miR-NC group, circOMA1+ miR-NC group). Cell counting kit (CCK8) was adopted to detect the effects of circOMA1 and miR-145 on the cell proliferation of THP-1.The effects of circOMA1 and miR-145 on cell apoptosis of THP-1 were detected using flow cytometry, and the effects of circOMA1 and miR-145 on MYC protein expression was detected via Western blot.The comparison between groups was analyzed by independent sample t-test or paired sample t-test, and the correlation was analyzed by Pearson correlation. Results:The expression of circOMA1 in the initial diagnosis group(4.408±3.607) was significantly increased compared with that in the control group (0.998±0.560) ( t=2.946, P<0.01); the expression of circOMA1 in remission group(1.582±0.950) was significantly decreased compared with that in the initial diagnosis group( t= 3.628, P<0.01). The THP-1 cell experiments showed that compared to the pcDNA3.1 control group, the expression of miR-145 in the circOMA1 high expression group decreased ( t= 4.21, P<0.05), cell proliferation was enhanced at 72 h and 96 h ( t=5.46, 7.40, all P<0.05), apoptosis was inhibited( t=6.44, P<0.01). The expression of MYC protein in circOMA1+ miR-NC group was higher than that of pcDNA3.1+ miR-NC group( t=5.72, P<0.01), the expression of MYC protein in circOMA1+ miR-145 group was lower than that in circOMA1+ miR-NC group ( t=4.56, P<0.05); at 72 h and 96 h, the cell proliferation level of circOMA1+ miR-NC group was higher than that of pcDNA3.1+ miR-NC group ( t=5.77, 7.30, all P<0.05), the level of cell proliferation in circOMA1+ miR-145 group was lower than that in circOMA1+ miR-NC group ( t=4.66, 6.17, all P<0.05); the apoptosis rate of circOMA1+ miR-145 group was higher than that of circOMA1+ miR-NC group ( t=4.25, P<0.05). circOMA1 expression was negatively correlated with miR-145 expression ( r=-0.62, P=0.016) and positively correlated with MYC gene expression ( r=0.64, P=0.013) in clinical samples. Conclusions:circOMA1 is highly expressed in children with AML, and can promote AML cell proliferation and inhibit apoptosis through miR-145/MYC pathway, which provides a basis for AML therapy and diagnosis.
ABSTRACT
Objective To establish and optimize a loop-mediated isothermal amplification (LAMP) method for the rapid detection of Escherichia coli and its microbial toxin.Methods The LAMP reaction system and reaction conditions were determined by optimizing LAMP reaction,and the optimized LAMP system was used for the detection.Results Primers targeting shiga toxin (stx) gene and O157 antigen gene rfbe were designed.The established and optimized LAMP amplification system contained 1.2 mmol/L dNTPs,10 mmol/L MgSO4,0.4 mol/L betaine,1 μl 10 × Bst DNA polymerase Buffer,8 U Bst DNA polymerase fragment,2 μl DNA template,and the ratio of inner-primer (FIP and BIP) and outerprimer (F3 and B3) were 8∶ 1.Time and temperature for LAMP was 60 min,60 ℃.The sensitivity was 103 times higher than polymerase chain reaction (PCR),reached 5 × 101 CFU/ml.When LAMP was applied to 19 reference strains,102 EHEC strains,the specification was 100％ while identification rate of rfbe,stx1 and stx2 gene reached 100％,95.2％,92.9％.Conclusions The LAMP method showed a promising prospect for the rapid detection of common nosocomial pathogens microbial toxin.
ABSTRACT
Objective To investigate the effect of miRNA -1 00 on the proliferation of human leukemia cells HL -60,and to explore the mechanism of this action.Methods The bioinformatics software and database were applied to predict and analyze target genes of miRNA -1 00.The vector contained the target gene 3′UTR portion cloned into a luciferase reporter construct.A luciferase reporter assay was performed following co -transfection of small molecular miRNA -1 00 mimics and target gene wild -type or mutant plasmid into HEK -293T cells.HL -60 cells were trans-fected with miRNA -1 00 mimics or anti -miRNA -1 00.After transfection,Western blot was applied to validate the expression of carboxy -terminal domain small phosphatase -like protein (CTDSPL),and the viability of HL -60 was measured by using cell counting kit (CCK -8)assay at 24 h,48 h,72 h,96 h.Results Online software predicted that CTDSPL was likely to be the target gene of miRNA -1 00.Dual luciferase reporter gene assay system showed that miRNA -1 00 could significantly suppress the activity of reporter gene containing CTDSPL 3′-UTR which decreased by about 57.1 %(P =0.000 7).Western blot showed that the expression of CTDSPL was increased after being trans-fected with miRNA -1 00 antisense oligonucleotides and decreased after being transfected with miRNA -1 00 mimics.At the same time,the growth rate of cells treated with miRNA -1 00 mimics or CTDSPL miRNA -1 00 was increased com-pared with that in control by CCK -8 test (P <0.05 ).Conclusions CTDSPL is a downstream target gene of miRNA -1 00.miRNA -1 00 can promote leukemia cell proliferation by inhibiting the expression of CTDSPL.