Bmi-1-siRNA通过PTEN/pAKT通路调控K562细胞体内外的增殖能力
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摘要
目的:观察Bmi-1基因沉默对白血病K562细胞体内外增殖能力的影响并初步探究二者之间的分子机制是否与PTEN/pAKT通路有关。方法:将Bmi-1小干扰RNA(siRNA)序列转染到K562细胞中降低其Bmi-1的表达;应用MTT比色法和软琼脂集落形成实验检测Bmi-1-siRNA对K562细胞体外增殖的影响;裸鼠皮下接种各组细胞,观察Bmi-1-siRNA对K562细胞在裸鼠体内的致瘤能力的影响;应用Western blot检测Bmi-1、PTEN、p-AKT等蛋白的表达。结果:Bmi-1-siRNA有效沉默了Bmi-1基因mRNA和蛋白的表达;沉默Bmi-1基因的表达能够抑制K562细胞的增殖活性、集落形成及裸鼠成瘤能力;Bmi-1基因沉默后,干扰组PTEN表达明显升高,而p-AKT活性明显下降;p-AKT抑制剂LY294002处理K562细胞后,p-AKT表达降低,集落形成及裸鼠成瘤能力相比未处理K562细胞降低;PTEN抑制剂Bpv处理K562-S1细胞后,PTEN的表达降低,而p-AKT、集落形成及裸鼠成瘤能力得以重塑。结论:Bmi-1基因有可能参与了对白血病细胞K562的体内外增殖能力的调控,PTEN/pAKT信号通路可能是介导这一调控过程的分子机制之一。
Objective: To investigate the effect of Bmi-1 gene silence on the proliferation ability of K562 cells in vitro and in vivo, and to explore the relation of molecular mechanism between proliferation ability of K562 cells in vitro and in vivo with PTEN/pAKT signaling pathway. Methods: The Bmi-1 small interference RNA(siRNA) sequences were transfected into K562 cells for decreasing Bmi-1 expression. The effect of Bmi-1 siRNA on the proliferation of K562 cells in vitro and in vivo was detected by MTT method and colony-forming test, the effect of Bmi-1 siRNA on the tumorogenicity of K562 cells was observed by subcutaneous inoculation of K562 cells, LY294002 and Bpv treated K562 cells in nude mice, the expression of Bmi-1, PTEN and pAKT proteins were detected by Western blot. Results: The Bmi-1 siRNA could inhibit the proliferation activity, colony-forming and tumor-forming abilities of K562 cells. After the silence of Bmi-1 gene, the PTEN expression in Bmi-1 gene-silenced group was significantly enhanced. While the pAKT expression in Bmi-1 gene-silenced group was significantly reduced; after the K562 cells were treated with LY294002(an inhibitor of pAKT), the pAKT expression colony-forming and tumor forming abilities were reduced in comparison with untreated K562 cells; after the K562-S1 cells were treated with Bpv(an inhibitor of PTEN), the PTEN expression decreased, while the pAKT expression, colony forming and tumor-forming abilities were restored. Conclusion: The Bmi-1 gene possibly involves in regulation of K562 proliferation in vivo and in vitro, the effect of PTEN/pAKT signaling pathway maybe one of molecular mechanisms mediating this regulation.
Objective: To investigate the effect of Bmi-1 gene silence on the proliferation ability of K562 cells in vitro and in vivo, and to explore the relation of molecular mechanism between proliferation ability of K562 cells in vitro and in vivo with PTEN/pAKT signaling pathway. Methods: The Bmi-1 small interference RNA(siRNA) sequences were transfected into K562 cells for decreasing Bmi-1 expression. The effect of Bmi-1 siRNA on the proliferation of K562 cells in vitro and in vivo was detected by MTT method and colony-forming test, the effect of Bmi-1 siRNA on the tumorogenicity of K562 cells was observed by subcutaneous inoculation of K562 cells, LY294002 and Bpv treated K562 cells in nude mice, the expression of Bmi-1, PTEN and pAKT proteins were detected by Western blot. Results: The Bmi-1 siRNA could inhibit the proliferation activity, colony-forming and tumor-forming abilities of K562 cells. After the silence of Bmi-1 gene, the PTEN expression in Bmi-1 gene-silenced group was significantly enhanced. While the pAKT expression in Bmi-1 gene-silenced group was significantly reduced; after the K562 cells were treated with LY294002(an inhibitor of pAKT), the pAKT expression colony-forming and tumor forming abilities were reduced in comparison with untreated K562 cells; after the K562-S1 cells were treated with Bpv(an inhibitor of PTEN), the PTEN expression decreased, while the pAKT expression, colony forming and tumor-forming abilities were restored. Conclusion: The Bmi-1 gene possibly involves in regulation of K562 proliferation in vivo and in vitro, the effect of PTEN/pAKT signaling pathway maybe one of molecular mechanisms mediating this regulation.
引文
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12 Peng HX,Liu XD,Luo ZY.Upregulation of the proto-oncogene Bmi-1 predicts a poor prognosis in pediatric acute lymphoblastic leukemia.BMC Cancer,2017;17(1):76.
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15 Liu L,Li H,Zhang M,et al.Effects of targeted nano-delivery systems combined with hTERT-siRNA and Bmi-1-siRNA on MCF-7 cells.Int J Clin Exp Pathol,2015;8(6):6674-6682.
16 Li X,Zheng X,Xu B,et al.Lower Bmi-1 expression may predict longer survival of colon cancer patients.Cell Physiol Biochem,2016;39(6):2421-2426.
17 Zhang Z,Bu X,Chen H,et al.Bmi-1 promotes the invasion and migration of colon cancer stem cells through the downregulation of E-cadherin.Int J Mol Med,2016;38(4):1199-1207.
18 Lessard J,Sauvageau G.Bmi-1 determines the proliferative capacity of normal and leukemia stem cells.Nature,2003;423(6937):255-260.
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21 Dean M,Fojo T,Bates S.Tumour stem cells and drug resistance.Nat Rev Cancer,2005;5(4):275-284.
22 Yuan W,Yuan Y,Zhang T,et al.Role of Bmi-1 in regulation of ionizing irradiation-indu ced epithelial-mesenchymal transition and migration of breast cancer cells.PLoS One,2015;10(3):e0118799.
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24 Xu Z,Liu H,Lv X,et al.Knockdown of the Bmi-1 oncogene inhibits cell prolife ration and induces cell apoptosis and is involved in the decrease of Akt phosphorylation in the human breast carcinoma cell line MCF-7.Oncol Rep,2011;25(2):409-418.
25 Song LB,Li J,Liao WT,et al.The polycomb group protein Bmi-1 represses the tumor suppressor PTEN and induces epithelialmesenchymal transition in human nasopharyngeal epithelial cell.JClin Invest,2009;119(12):3626-3636.
26 Kearsley JH,Furlong KL,Cooke RA,et al.An immunohistochemical assessment of cellular proliferation markers in head and neck squamous cell cancers.Br J Cancer,1990;61(6):821-827.
27 Shirono K,Hattori T,Takatsuki K.A new classification of clinical stages of adult T-cell leuke mia based on prognosis of the disease.leukemia,1994;8(11):1834-1837.
28 Delahunt B,Bethwaite P,Thornton A,et al.Proliferation of renal cell carcinoma assessed by fixation-resistant polyclonal Ki-67antibody labeling.Cancer,1995;75(11):2714-2719.
2 Zhang X,Guo W,Wang X,et al.Antitumor activity and inhibitory effects on cancer stem cell-like properties of Adeno-associated virus(AAV)-mediated Bmi-1 interference driven by Bmi-1 promoter for gastric cancer.Oncotarget,2016;7(16):22733-22745.
3 Wang JF,Liu Y,Liu WJ,et al.Expression of Bmi-1 gene in esophageal carcinoma cell EC9706 and its effect on cell cycle,apoptosis and migration.Chin J Cancer,2010;29(7):689-696.
4 Zhang X,Wang CX,Zhu CB.Overexpression of Bmi-1 in uterine cervical cancer corre lation with clinicopathology and prognosis.Int J Gynecol Cancer,2010;20(9):1597-1603.
5 Wang MC,Jiao M,Wu T,et al.Polycomb complex protein BMI-1promotes invasion and metastasis of pancreatic cancer stem cells by activating PI3K/AKT signaling,an ex vivo,in vitro,and in vivo study.Oncotarget,2016;7(8):9586-9599.
6 Wang X,Wang C,Zhang X,et al.Bmi-1 regulates stem celllike properties of gastric canc er cells via modulating miRNAs.JHematol Oncol,2016;9(1):90.
7 Zheng X,Wang Y,Liu B,et al.Bmi-1-shRNA inhibits the proliferation of lung adenocarcinoma cells by blocking the G1/Sphase through decreasing cyclin D1 and increasing p21/p27 levels.Nucleic Acid Ther,2014;24(3):210-216.
8 Meng XX,Wang YF,Zheng XY,et al.ShRNA-mediated knockdown of Bmi-1 inhibit lung adenocarcinoma cell migration and metastasis.Lung Cancer,2012;77(1):24-30.
9 Jiang Y,Su BL,Meng XX,et al.Effect of siRNA-mediated silencing of Bmi-1 gene expression on HeLa cells.Cancer,2010;101(2):379-386.
10 Nishida Y,Maeda A,Kim MJ,et al.The novel BMI-1 inhibitor PTC-596 downregulates MCL-1 and induces p53-independent mitochondrial apoptosis in acute myeloid leukemia progenitor cells.Blood Cancer J,2017;7(2):e527.
11 Wang Y,Sun HH,Sui MH,et al.MiR-218 inhibits acute promyelocytic leukemia cell growth by tar geting BMI-1.Oncol Lett,2017;14(6):8078-8083.
12 Peng HX,Liu XD,Luo ZY.Upregulation of the proto-oncogene Bmi-1 predicts a poor prognosis in pediatric acute lymphoblastic leukemia.BMC Cancer,2017;17(1):76.
13 Meng XX,Liu WH,Liu DD,et al.Construction of antisense Bmi-1 expression plasmid and its inhibitory effect on K562 cells proliferation.Chin Med J,2005;118(16):1346-1350.
14李建勇.血液疾病诊断流程与治疗策略.北京:科学出版社,2007;447.
15 Liu L,Li H,Zhang M,et al.Effects of targeted nano-delivery systems combined with hTERT-siRNA and Bmi-1-siRNA on MCF-7 cells.Int J Clin Exp Pathol,2015;8(6):6674-6682.
16 Li X,Zheng X,Xu B,et al.Lower Bmi-1 expression may predict longer survival of colon cancer patients.Cell Physiol Biochem,2016;39(6):2421-2426.
17 Zhang Z,Bu X,Chen H,et al.Bmi-1 promotes the invasion and migration of colon cancer stem cells through the downregulation of E-cadherin.Int J Mol Med,2016;38(4):1199-1207.
18 Lessard J,Sauvageau G.Bmi-1 determines the proliferative capacity of normal and leukemia stem cells.Nature,2003;423(6937):255-260.
19 Wei XD,He J,Wang JY,et al.Bmi-1 is essential for the oncogenic potential in CD133(+)human laryngeal cancer cells.Tumour Biol,2015;36(11):8931-8942.
20 Bracken AP,Kleine-Kohlbrecher D,Dietrich N,et al.The Polycomb group proteins bind throughout the INK4A-ARF locus and are disassociated in senescent cells.Genes Dev,2007;21(5):525-530.
21 Dean M,Fojo T,Bates S.Tumour stem cells and drug resistance.Nat Rev Cancer,2005;5(4):275-284.
22 Yuan W,Yuan Y,Zhang T,et al.Role of Bmi-1 in regulation of ionizing irradiation-indu ced epithelial-mesenchymal transition and migration of breast cancer cells.PLoS One,2015;10(3):e0118799.
23 Li X,Yang Z,Song W,et al.Overexpression of Bmi-1 contributes to the invasion and metastasis of hepatocellular carcinoma by increasing the expression of matrix metalloproteinase(MMP)2,MMP-9 and vascular endothelial growth factor via the PTEN/PI3K/Akt pathway.Int J Oncol,2013;43(3):793-802.
24 Xu Z,Liu H,Lv X,et al.Knockdown of the Bmi-1 oncogene inhibits cell prolife ration and induces cell apoptosis and is involved in the decrease of Akt phosphorylation in the human breast carcinoma cell line MCF-7.Oncol Rep,2011;25(2):409-418.
25 Song LB,Li J,Liao WT,et al.The polycomb group protein Bmi-1 represses the tumor suppressor PTEN and induces epithelialmesenchymal transition in human nasopharyngeal epithelial cell.JClin Invest,2009;119(12):3626-3636.
26 Kearsley JH,Furlong KL,Cooke RA,et al.An immunohistochemical assessment of cellular proliferation markers in head and neck squamous cell cancers.Br J Cancer,1990;61(6):821-827.
27 Shirono K,Hattori T,Takatsuki K.A new classification of clinical stages of adult T-cell leuke mia based on prognosis of the disease.leukemia,1994;8(11):1834-1837.
28 Delahunt B,Bethwaite P,Thornton A,et al.Proliferation of renal cell carcinoma assessed by fixation-resistant polyclonal Ki-67antibody labeling.Cancer,1995;75(11):2714-2719.