Effect Of SiRNA Down-regulation Of BECN1 Gene Expression On Chemosensitivity Of Leukemia K₅₆₂/AO₂ Cells

ObjectiveTo investigate the association between autophagy-related gene BECN1 and Chemosensitivity of leukemia K₅₆₂/AO₂ cells,and to reveal the possible mechanism of multidrug resistance in K₅₆₂/AO₂ cells.MethodsK₅₆₂/AO₂ cells were cultured in vitro,and the cells were inoculated into a6-well plate when they were in logarithmic growth phase,2ml medium per each well,attention to ensure that cells can reach 70%-90%within 24 hours;K₅₆₂/AO₂ cells were treated with different concentrations of doxorubicin for 48 h,andthemorphologicalchangeswereobservedundernormal microscope;BECN1-SiRNA of different density were transfected to K₅₆₂/AO₂cells with Lipofectamine^TM2000,the transfection efficiency was observed under fluorescence microscope by training after 48 hours,then the highest transfection efficiency concentration were selected.CCK-8 kit was used to detect the proliferation activity and IC50 of adriamycin-treated BECN1-siRNA transfected K₅₆₂/AO₂ cells and untransfected K₅₆₂/AO₂ cells.The beclin-1?LC3and drug-resistant protein P-gp expression level of the untransfected group(K₅₆₂/AO₂ cells group),the negative control group(NC-SiRNA-K₅₆₂/AO₂ cells group,NC group),the positive control group(GA-SiRNA-K₅₆₂/AO₂ cells group,GA group),the positive control group(BECN1-SiRNA-K₅₆₂/AO₂ cells group)were detected by Western Blotting.Results1.Inverted microscope demonstrated that treatment of K₅₆₂/AO₂ cells with adriamycin for 48h showed the Cell swelling,disintegration and other morphological changes;2.The highest transfection efficiency of K₅₆₂/AO₂ cells was selected when the transfection concentration was 50nmol/L and the transfection time was 48hours by Fluorescence microscopy.3.Compared with the untransfected group,the sensitivity of the transfection group(BECN1-SiRNA-K₅₆₂/AO₂ cells group)to adriamycin was significantly increased,and IC50 was obviously decreased?P<0.05?.4.The expression standards of autophagy protein?Beclin-1,LC3?and P-glycoprotein?P-gp?in BECN1-siRNA transfected cells was significantly decreased compared with untransfected cells?P<0.05?,it was suggesting that BECN1-siRNA can down-regulaing BECN1 gene expression and reducing the autophagy level of K₅₆₂/AO₂ cells.Conclusion1.Down-regulation the expression of autophagy gene BECN1 by BECN1-siRNA,that can inhibiting the autophagy level of K₅₆₂/AO₂ cells.2.Decreasing the autophagy level of K₅₆₂/AO₂ cells may increase the sensitivity of K₅₆₂/AO₂ cells to adriamycin.The mechanism may be related to the decrease of P-glycoprotein?P-gp? expression.