RESUMO
Objective@#To investigate the effects of miR-23a-3p on proliferation, migration and apoptosis on human acute myeloid leukemia (AML) cells by targeting SMC1A.@*Methods@#Microarray analysis was used to screen differentially expressed microRNAs and mRNAs in human AML cells. Real-time fluorescence quantitative PCR (RT-qRCR) was used to detect the expressions of miR-23a-3p and SMCA in human AML cell line U937. TargetScan database was used to analyze the correlation between miR-23a-3p and SMC1A. Double luciferase reporter gene was used to detect the interaction between miR-23a-3p and SMC1A. The effect of miR-23a-3p expression on the proliferation of U937 cells was detected by clonal assay. The migration, apoptosis, cell cycle and caspase-3 activity of U937 cells regulated by miR-23a-3p were detected by cell scratch assay and flow cytometry, respectively. Western blot was used to detect the expressions of Bax and Bcl-2 in U937 cells.@*Results@#Compared with human normal monocyte SC group (1.00), the expression of miR-23a-3p in U937 cells was up-regulated (2.56±0.78) (P<0.01), while the expression of SMC1A was down-regulated (0.48±0.56, P<0.01). miR-23a-3p specifically bond to SMC1A 3′UTR and regulated the expression activity of SMC1A. Overexpression of miR-23a-3p promoted the proliferation and migration of U937 cells and inhibited the apoptosis of U937 cells, while up-regulation of SMC1A inhibited the proliferation and migration of U937 cells and promoted the apoptosis of U937 cells. The percentages of G0/G1 phase, G2/M phase and S phase cells in the negative control group were (37.48±0.21)%, (16.78±0.18)% and (45.74±0.15)% respectively, and those in the miR-23a-3p mimics group were (19.96±0.11)%, (41.69±0.24)% and (38.24±0.34)%, respectively. The difference was statistically significant (all P<0.05). The proportions of G0/G1 phase, G2/M phase and S phase cells in the group of miR-23a-3p mimics+ pcDNA3.1-SMC1A were (36.88±0.21)%, (30.44±0.33)% and (32.88±0.16)%, respectively, without significant difference when compared with those of the miR-23a-3p mimics group (P>0.05). The relative expression levels of Bax and Bcl-2 protein in the negative control group were 0.55±0.45 and 0.31±0.54, respectively. Overexpression of miR-23a-3p inhibited the expression of Bax protein in U937 cells (0.23±0.13, P<0.001), promoted the expression of Bcl-2 protein (0.50±0.23, P<0.01), while SMC1A increased the expression of Bax protein in U937 cells (0.40±0.11, P<0.01), and inhibited the expression of Bcl-2 protein (0.37±0.15). In the negative control group, caspase-3 activity was (25.82±0.89)%. Overexpression of miR-23a-3p inhibited caspase-3 activity in U937 cells (3.64±0.56)%, P<0.01, while up-regulation of SMC1A promoted caspase-3 activity in U937 cells (15.29±0.85)%, P<0.01.@*Conclusion@#miR-23a-3p can inhibit the proliferation and migration and promote apoptosis of human AML cells by targeting SMC1A.
RESUMO
Objective To explore the effect of bosutinib on cell differentiation and apoptosis of human acute myeloid leukemia (AML) cell lines including HL-60, THP-1, and U937, and to clarify the related mechanisms. Methods MTT Assay was used to assess the proliferation of HL-60, THP-1, and U937 cells treated with various concentrations of bosutinib (0-20 μmol/L) for 48 h. Cell surface marker CD11b expression was detected by flow cytometry in HL-60, THP-1, and U937 cells treated with bosutinib (0-5 μmol/L) for 72 h. Apoptosis was evaluated by Annexin V-FITC/PI double stainning in the following two sets of experiments: 1 HL- 60, THP-1 and U937 cells were treated with various concentrations of bosutinib (0-10 μmol/L). 2 U937 cells were pretreated with caspases inhibitor benzyloxy-carbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) for 1 h and then exposed to bosutinib for 48 h. Western blot was used to examine the protein expression of differentiation related transcription factors C/EBPβ, P21, and c-Myc, and apoptosis related protein Mcl- 1, Bax, and Caspase 3 in HL- 60 and U937 cells treated with bosutinib for 24 h. Results Bosutinib dose- dependently inhibited AML cell growth. Treatment with various concentrations of bosutinib for 72 h significantly increased CD11b expression and apoptotic rate in AML cells as compared to blank control (P< 0.05 or P < 0.01). Bosutinib effectively upregulated protein expression levels of C/EBPβ and P21 in HL-60 cells, and induced down-regulation of Mcl-1 with up-regulation of Bax protein expression, and activated Caspase 3 in U937 ells. Pretreatment of U937 cells with Z-VAD-FMK significantly inhibited apoptosis induced by bosutinib. Conclusion Bosutinib effectively inhibits cell proliferation and induces cell differentiation with apoptosis in AML cells. It could be a potent therapeutic agent against AML.