自噬基因Beclin1抑制后增强人髓性白血病耐药细胞K562/IMA对于伊马替尼的药物敏感性
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摘要
目的研究自噬基因Beclin1沉默后增强伊马替尼耐药的人髓性白血病细胞K562/IMA的药物敏感性的作用。方法采用首剂大剂量冲击和逐步增加剂量相结合的方法构建伊马替尼耐药的人髓性白血病耐药细胞K562/IMA,伊马替尼干预K562和K562/IMA细胞株后,采用CCK-8检测细胞活力,流式细胞术检测细胞凋亡率确定药物耐药水平,Western-blot检测耐药株中Beclin1的表达。采用小干扰RNA技术(siRNA)转染Beclin1的siRNA-Beclin1沉默K562/IMA的Beclin1基因。设置对照组(control)、siRNA阴性对照组(siRNA-NC)和Beclin1 siRNA组(siRNA-Beclin1)。采用RT-QPCR和Western-blot法鉴定沉默后各组细胞Beclin1蛋白和mRNA的表达水平。CCK-8法检测各组细胞对于伊马替尼的敏感性,并计算半数抑制浓度IC_(50)。流式细胞术检测各组细胞的凋亡率。Western-blot法检测Beclin1、Bcl-2、Bax、cleaved-caspase 3、caspase-3、细胞色素C(cyto-C)的表达水平,Transwell小室检测细胞转移侵袭能力。结果 K562/IMA细胞株对10μmol·L~(-1)的伊马替尼耐药,且细胞中Beclin1的表达水平高于K562细胞株,在梯度浓度范围中K562/IMA的细胞活力显著高于K562,而细胞凋亡率低于K562细胞。siRNA沉默Beclin1后,siRNA-Beclin1组细胞活力相比control组显著降低,细胞凋亡率显著增高,而半数抑制浓度IC_(50)值显著低于control组。细胞中凋亡相关蛋白Bax、cleaved-caspase 3、caspase-3、cyto-C的水平显著高于control组,Bcl-2水平低于control组,侵袭试验结果显示,Beclin1沉默后细胞侵袭能力显著减弱,相比control组具有统计学意义。结论 Beclin1在人髓性白血病对于伊马替尼耐药中具有重要作用,自噬可能参与了耐药过程。沉默Beclin1后可以降低细胞耐药水平,提高药物敏感性。
OBJECTIVE To investigate the effect of autophagy gene Beclin1 silencing on enhancing the drug sensitivity of imatinib-resistant human myeloid leukemia cell line K562/IMA. METHODS Imatinib-resistant human myeloid leukemia cell line K562/IMA was constructed by the combination of the first dose of high-dose shock therapy and gradual increase of dose. Imatinib interfered with K562 and K562/IMA cell lines. Cell viability was detected by CCK-8. Apoptosis rate was detected by flow cytometry. Westernblot assay was used to determine the drug resistance level. The expression of Beclin1 in drug resistant strains was detected. Small interfering RNA(siRNA) transfected Beclin1 siRNA-Beclin1 was used to silence the Beclin1 gene of K562/IMA. Control group(control),siRNA negative control group(siRNA-NC) and Beclin1 siRNA group(siRNA-Beclin1) were set up. RT-QPCR and Western-blot were used to identify the expression level of Beclin1 protein and mRNA in each group after silencing. CCK-8 method was used to detect the sensitivity of each group of cells to imatinib,and the half inhibitory concentration IC_(50)was calculated. The apoptosis rate of each group was detected by flow cytometry. The expression levels of Beclin 1,Bcl-2,Bax,cleaved-caspase 3,caspase-3 and cytochrome C(cytoC) were detected by Western-blot assay,and the metastatic and invasive ability of cells was detected by Transwell chamber.RESULTS K562/IMA cell lines were resistant to imatinib at 10 μmol·L~(-1),and the expression of Beclin1 was higher than that of K562 cell lines. The cell viability of K562/IMA was significantly higher than that of K562 cell lines in the gradient concentration range,while the apoptosis rate was lower than that of K562 cell lines. After siRNA silencing Beclin~(-1),the cell viability and apoptosis rate of siRNA-Beclin 1 group were significantly lower than those of control group,and the IC_(50)value of half inhibitory concentration was significantly lower than that of control group. The levels of Bax,cleaved-caspase 3,caspase-3 and cyto-C in the cells were significantly higher than those in the control group, while the levels of Bcl-2 were significantly lower than those in the control group.CONCLUSION Beclin1 plays an important role in imatinib resistance in human myeloid leukemia,and autophagy may be involved in the drug resistance process. After silencing Beclin1,cell resistance level can be reduced and drug sensitivity improved.
OBJECTIVE To investigate the effect of autophagy gene Beclin1 silencing on enhancing the drug sensitivity of imatinib-resistant human myeloid leukemia cell line K562/IMA. METHODS Imatinib-resistant human myeloid leukemia cell line K562/IMA was constructed by the combination of the first dose of high-dose shock therapy and gradual increase of dose. Imatinib interfered with K562 and K562/IMA cell lines. Cell viability was detected by CCK-8. Apoptosis rate was detected by flow cytometry. Westernblot assay was used to determine the drug resistance level. The expression of Beclin1 in drug resistant strains was detected. Small interfering RNA(siRNA) transfected Beclin1 siRNA-Beclin1 was used to silence the Beclin1 gene of K562/IMA. Control group(control),siRNA negative control group(siRNA-NC) and Beclin1 siRNA group(siRNA-Beclin1) were set up. RT-QPCR and Western-blot were used to identify the expression level of Beclin1 protein and mRNA in each group after silencing. CCK-8 method was used to detect the sensitivity of each group of cells to imatinib,and the half inhibitory concentration IC_(50)was calculated. The apoptosis rate of each group was detected by flow cytometry. The expression levels of Beclin 1,Bcl-2,Bax,cleaved-caspase 3,caspase-3 and cytochrome C(cytoC) were detected by Western-blot assay,and the metastatic and invasive ability of cells was detected by Transwell chamber.RESULTS K562/IMA cell lines were resistant to imatinib at 10 μmol·L~(-1),and the expression of Beclin1 was higher than that of K562 cell lines. The cell viability of K562/IMA was significantly higher than that of K562 cell lines in the gradient concentration range,while the apoptosis rate was lower than that of K562 cell lines. After siRNA silencing Beclin~(-1),the cell viability and apoptosis rate of siRNA-Beclin 1 group were significantly lower than those of control group,and the IC_(50)value of half inhibitory concentration was significantly lower than that of control group. The levels of Bax,cleaved-caspase 3,caspase-3 and cyto-C in the cells were significantly higher than those in the control group, while the levels of Bcl-2 were significantly lower than those in the control group.CONCLUSION Beclin1 plays an important role in imatinib resistance in human myeloid leukemia,and autophagy may be involved in the drug resistance process. After silencing Beclin1,cell resistance level can be reduced and drug sensitivity improved.
引文
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[5]WANG S M,LI X H,XIU Z L.Over-expression of Beclin-1 facilitates acquired resistance to histone deacetylase inhibitor-induced apoptosis[J].Asian Pac J Cancer Prev,2014,15(18):7913-7917.
[6]SUN Y,LIU J H,JIN L,et al.Effect of autophagy-related beclin1 on sensitivity of cisplatin-resistant ovarian cancer cells to chemotherapeutic agents[J].Asinn Pacific J Cancer Prevent,2015,16(7):2785-2791.
[7]BAO L,JARAMILLO M C,ZHANG Z,et al.Induction of autophagy contributes to cisplatin resistance in human ovarian cancer cells[J].Mol Med Rep,2015,11(1):91-98.
[8]WADA N,KUROKAWA Y,TAKAHASHI T,et al.Detecting secondary C-KIT mutations in the peripheral blood of patients with imatinib-resistant gastrointestinal stromal tumor[J].Oncology,2016,90(2):112-117.
[9]GARCIAPRAT L,MARTINEZVICENTE M,PERDIGUERO E,et al.Autophagy maintains stemness by preventing senescence[J].Nature,2016,529(7584):37-42.
[10]KOCHERGIN I A,ZAKHAROVA M N.The role of autophagy in neurodegenerative diseases[J].Neurochem J,2016,10(1):7-18.
[11]ZHOU W,YUE C,DENG J,et al.Autophagic protein Beclin1 serves as an independent positive prognostic biomarker for non-small cell lung cancer[J].PLoS One,2015,8(11):e80338.
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[15]WATANABE Y,TSUJIMURA A,TAGUCHI K,et al.HSF1stress response pathway regulates autophagy receptor SQSTM1/p62-associated proteostasis[J].Autophagy,2017,13(1):133-148.
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[17]KIM S H,KIM K Y,PARK S G,et al.Mitochondrial ROS activates ERK/autophagy pathway as a protected mechanism against deoxypodophyllotoxin-induced apoptosis[J].Oncotarget,2017,8(67):111581-111596.
[18]SHAN M,QIN J,JIN F,et al.Autophagy suppresses isoprenaline-induced M2 macrophage polarization via the ROS/ERK and m TOR signaling pathway[J].Free Radic Biol Med,2017,110:432-443.
[19]MUZ B,KUSDONO H D,AZAB F,et al.Tariquidar sensitizes multiple myeloma cells to proteasome inhibitors via reduction of hypoxia-induced P-gp-mediated drug resistance[J].Leuk Lymphoma,2017,58(12):1-10.
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