ABSTRACT
Objective To investigate the effect of down-regulation of lysine specific demethylase 1 (LSD1) by shRNA on the apoptosis and cell cycle of human acute myelogenous leukemia cells.Methods The lentiviral vector-mediated LSD1-shRNA was transfected into human acute promyelocytic leukemia HL-60 cells and acute monocytic leukemia SHI-1 cells.The expressions of LSD1 mRNA and protein were examined by real time quantitative PCR and Western blot,respectively.The flow cytometry was applied to detect the apoptosis and cell cycle distribution after AnnexinV-PE/7-AAD and PI dying,respectively.Results The expressions of LSD1 mRNA and protein in HL-60 and SHI-1 LSD1-shRNA group were significantly decreased compared with the blank control group and the negative shRNA group (P ＜ 0.01,respectively).The apoptosis levels of HL-60 and SHI-1 cells were significantly increased after knockdown of LSD1 (P ＜ 0.01).Moreover,the cell cycle distribution in the G0/G1 phases was also significantly increased(P ＜ 0.01).Conclusion LSDI-shRNA promotes cell apoptosis and increases the percentage of cells in the G0/G1 phases of human acute myelogenous leukemia cells.
ABSTRACT
Objective To investigate the effects of shRNA interference (RNAi) targeting the histone lysine specific demethylase 1 (LSD1) on the proliferation and apoptosis in acute leukemia (AL) cells. Methods LSD1 shRNA vectors were constructed and transduced into HL-60 and SHI-1 AL cell lines. Inhibitory efficiency of LSD1 was detected by real-time quantitative PCR (RT-qPCR) and Western blot respectively. Cell proliferation was assessed by methyl thiazolyl tetrazolium (MTT), and cell apoptosis was measured by flow cytometry. Results After interference of LSD1, the expression levels of LSD1 mRNA and protein in HL-60 and SHI-1 cells (mRNA: 0.242 ±0.023, 0.207 ±0.006; Protein: 0.256 ±0.015, 0.486 ±0.042) were decreased compared with blank control group without transfection process (mRNA: 1.021 ±0.178, 1.039 ±0.395; Protein:0.552 ±0.026, 0.754 ±0.060) and empty vector negative control group (shNC group) (mRNA: 0.935 ±0.136, 1.016±0.203;Protein: 0.500±0.026, 0.750±0.049) (P<0.05). The levels of their cell proliferation (absorbance value: 0.712±0.010, 0.549±0.007) were inhibited compared with blank control group (absorbance value:0.823±0.010, 0.625±0.005) and shNC group(absorbance value: 0.818±0.019, 0.621±0.003) (P< 0.05). While cell apoptosis rates were increased [(32.80 ±1.35) %, (23.49 ±1.40) %] compared with blank control group [(8.08 ±0.62) %, (7.28 ±1.17) %] and shNC group [(8.08 ±0.62) %, (7.28 ±1.17) %] (P< 0.05). Conclusions Lentivirus-mediated shRNA interferencing LSD1 can inhibit cells ' proliferation and promote apoptosis of HL-60 and SHI-1 AL cell lines, indicating that LSD1 may be a potential biological molecular marker and a new treatment target for AL.
ABSTRACT
Objective To investigate the effect and mechanism of cell differentiation agent Ⅱ (CDA-Ⅱ) on the differentiation of human acute mycloid leukemia (APL) HL-60 cells.Methods The cell morphology and differentiation was detected by Wright-Giemsa staining,the expression of cell surface differentiation antigen CD11b of HL-60 was detected by flow cytometry,the differentially expressed genes were screened by gene expression profile chip (NimbleGen).Results The result of Wright-Giemsa staining showed that CDA-Ⅱ induced HL-60 differentiation towards mature stages in a time-dependent manner.After treated with CDA-Ⅱ,the percentage of CD11b-positive HL-60 cells significantly increased in a time-and dose-dependent manner.The result of gene expression profiling indicated differentially expressed genes including 113 up-regulation genes and 140 down-regulation genes.The up-regulation expression genes involved in six pathways including mineral absorption,complement and coagulation cascades,down-regulation expression genes involved in nine pathways including pyrimidine metabolism,RNA polymerase,purine metabolism and so on.Conclusion CDA-Ⅱ can induce HL-60 differentiation and make gene differentially expressed.The data provide the feasibility of CDA-Ⅱ in differentiation induction therapy for APL.
ABSTRACT
ObjectiveTo investigate the effect of artesunate on the expression of vascular endothlial growth factors(VEGF)and VEGFR in SHI-1 cell line.MethodsEnzyme-linked immunosorbent assay analysis was performed to detect the amount of VEGF in culture supernatants of SHI-1 cell in the condition of artesunate or not. The expression of VEGFR1 and VEGFR2 in SHI-1 cell in the condition of artesunate or not were detected by flow cytometry.ResultsWithout artesunate,the concentration of VEGF in the culture supernatant of SHI-1 cell were (980.3±2.2) pg/ml in 24 h and (982.4±2.3) pg/ml in 48 h. The expression of VEGFRI in SHI-1 cell were (6.40±3.11) ％ in 24 h and (6.45±2.85) ％ in 48 h. The expression of VEGFR2 in SHI-1 cell were (13.90±2.26) ％ in 24 h and (13.95±1.96) ％ in 48 h. With artesunate at 5, 10, 20 ng/ml, the concentration of VEGF in culture supematant of SHI-1 cell were (234.6±1.8)pg/ml, (114.9±1.6)pg/ml, (108.8±1.5) pg/ml in 24 h and (62.3±1.7) pg/ml, (60.9±1.6) pg/ml, (32.7±1.7) pg/ml in 48 h, respectively. The levels of VEGF in SHI-1 cells treated with artesunate at different concentrations decreased significantly (P ＜0.05).There was significant difference between 24 hours group and 48 hours group(P ＜0.05).The expression of VEGFR1 in SHI-1 cell were (4.30±2.21) ％, (4.20±1.37) ％, (3.90±1.86) ％ in 24 h and (3.80±2.87) ％, (3.60±1.73) ％, (3.00±1.82) ％ in 48 h, respectively. The expression of VEGFR1 in SHI-1 cell treated with artesunate at different concentrations were not significantly different (P ＞0.05). No significant difference between 24 hours group and 48 hours group was observed (P ＞0.05). VEGFR2 expression of SHI-1 cell were(4.40±1.15) ％, (3.10±0.68) ％, (1.10±0.72) ％ in 24 h and (3.00±1.68) ％, (2.20±0.93) ％, (0.60±0.92) ％ in 48 h, respectively. The results indicated that the expression of VEGFR2 in SHI-1 cells treated with artesunate at different concentrations reduced significantly (P ＜0.05),but there was no significant difference between 24 h group and 48 h group (P ＞0.05). ConclusionThe concentration of VEGF in SHI-1 cell was high, and artesunate can down-regulate the expression of VEGF and VEGFR2,but the effect of artesunate on the VEGFR1 was not significant.