靶向沉默Notch1基因对骨髓瘤细胞增殖的影响
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摘要
目的:明确沉默Notch1基因对骨髓瘤细胞增殖和凋亡的影响,寻找骨髓瘤治疗的新靶点。方法:在多发性骨髓瘤RPMI8226细胞中转染Notch1-shRNA靶向沉默Notch1基因,用CCK-8及流式细胞术评价Notch1基因沉默后骨髓瘤细胞增殖和凋亡的变化,应用荧光定量PCR方法分析Notch1 mRNA表达变化,用Western blot方法检测Notch信号通路相关蛋白Hes-1、Jagged-1、Jagged-2、BCL-2、PTEN、AKT、p-AKT的表达变化。结果:骨髓瘤细胞转染Notch1-shRNA后Notch1基因和蛋白表达均受到显著抑制,Notch1 mRNA相对表达量下调(66±0.1)%,Notch1蛋白相对表达量下调(88.0±3.4)%,与对照组比较差异有统计学意义(P<0.05)。转染后48 h实验组细胞增殖的速度明显减低;流式细胞术结果显示实验组瘤细胞凋亡明显增加;Notch1基因沉默后下游蛋白Hes-1、p-AKT和BCL-2表达水平明显下调,PTEN表达水平明显升高。结论:靶向沉默Notch1基因可以抑制骨髓瘤细胞增殖,促进细胞凋亡进程,其作用机制与p-AKT信号活化和PTEN基因功能复活有关,Notch1信号可作为潜在的骨髓瘤治疗靶点。
Objective: To investigate the effects of Notch1 gene silencing on the proliferation and apoptosis of multiple myeloma cells,and to find the new targets for the treatment of multiple myeloma. Methods: Notch1-shRNA targeted silencing Notch1 gene was transfected into multiple myeloma RPMI8226 cells,the CCK-8 and flow cytometry were used to detect the proliferation and apoptosis of myeloma cells after Notch1-shRNA transfection,the real-time fluorescence quantitative PCR was used to analyze expression level of Notch1 mRNA,and the Western blot were used to detect the expression level of Notch1 signaling pathway-related protein,such as Hes-1,Jagged-1,Jagged-2,BCL-2,PTEN,AKT and P-AKT. Results: The mRNA and protein expression levels of Notch1-shRNA transfected cells were significantly inhibited in the experimental group assayed by real time fluorescence quantitative PCR and Western blot,the mRNA and protein expression level were down-regulated to 66% + 0. 1%,88% + 3. 4% respectively,as compared with the control group( P < 0. 05). CCK-8 results confirmed that the cell proliferation rate was significantly decreased in the experimental group 48 hours after transfection. Flow cytometry results showed that the cell apoptosis rate was significantly higher in the experimental group than that in the control group. The expression levels of downstream protein Hes1,p-AKT and BCL-2 were decreased,the level of PTEN increased significantly after Notch1 gene silencing. Conclusion: Notch1 gene silencing by transfection of Notch1-shRNA can inhibit the proliferation of myeloma cells and induce their apoptosis,and its mechanism is related to the activation of PTEN gene and p-AKT signaling. Notch1 signal can be used as a potential target for multiple myeloma therapy.
Objective: To investigate the effects of Notch1 gene silencing on the proliferation and apoptosis of multiple myeloma cells,and to find the new targets for the treatment of multiple myeloma. Methods: Notch1-shRNA targeted silencing Notch1 gene was transfected into multiple myeloma RPMI8226 cells,the CCK-8 and flow cytometry were used to detect the proliferation and apoptosis of myeloma cells after Notch1-shRNA transfection,the real-time fluorescence quantitative PCR was used to analyze expression level of Notch1 mRNA,and the Western blot were used to detect the expression level of Notch1 signaling pathway-related protein,such as Hes-1,Jagged-1,Jagged-2,BCL-2,PTEN,AKT and P-AKT. Results: The mRNA and protein expression levels of Notch1-shRNA transfected cells were significantly inhibited in the experimental group assayed by real time fluorescence quantitative PCR and Western blot,the mRNA and protein expression level were down-regulated to 66% + 0. 1%,88% + 3. 4% respectively,as compared with the control group( P < 0. 05). CCK-8 results confirmed that the cell proliferation rate was significantly decreased in the experimental group 48 hours after transfection. Flow cytometry results showed that the cell apoptosis rate was significantly higher in the experimental group than that in the control group. The expression levels of downstream protein Hes1,p-AKT and BCL-2 were decreased,the level of PTEN increased significantly after Notch1 gene silencing. Conclusion: Notch1 gene silencing by transfection of Notch1-shRNA can inhibit the proliferation of myeloma cells and induce their apoptosis,and its mechanism is related to the activation of PTEN gene and p-AKT signaling. Notch1 signal can be used as a potential target for multiple myeloma therapy.
引文
1 Dispenzieri A,Rajkumar SV,Gertz MA,et al.Treatment of newly diagnosed multiple myelolma based on Mayo Stratification of Myeloma and Risk-adapted Therapy(m SMART):consensus statement.Mayo Clin Proc,2007;82(3):323-341.
2 Artavanis-Tsakonas S,Rand MD,Lake RJ.Notch signaling:cell fate control and signal integration indevelopment.Science,1999;284(5415):770-776.
3 Borggrefe T,Oswald F.The Notch signaling Pathway:transcriptional regulation at Notch target genes.Cell Mol Life Sci,2009;66(10):1631-1646.
4 Jundt F,Prebsting KS,Anagnostopoulos I,et al.Jagged1-induced Notch signaling drives proliferation of multip le myeloma cells.Blood,2004;103(9):3511-3535.
5 Kogoshi H,Sato T,Koyama T,et al.Gamma-secretase inhibitions suppress the growth of leukemia and lymphoma cells.Oncol Rep,2007;18(1):77-80.
6 Nefedova Y,Sullivan DM,Bolick SC,et al.Inhibition of Notch signaling induces apoptosis of myeloma cells and enhances sensitivity to chemotherapy.Blood,2008;111(4):2220-2229.
7 Leiba M,Jakubikova J,Klippel S,et al.Halofuginone inhibits multiple myeloma growth in vitro and in vivo and enhances cytotoxicity of conventional and novel agents.Br J Haematol,2012;157(6):718-731.
8 洪秀理,张泽川,赵江宁,等.异硫氰酸苯已酯抑制骨髓瘤细胞Notch信号表达的研究。中国实验血液学杂志,2011;19(4):940-943.
9 Peng KF,Wu XF,Zhao HW,et al.Advanced oxidation protein products induce monocyte chemoattractant protein-1 expression via p38 mitogen-activated protein kinase activation in rat vascular smooth muscle cells.Chin Med J,2006;119(13):1088-1093.
10 Xiang J,Ouyang Y,Cui Y,et al.Silencing of Notch3 Using shRNA driven by survivin promoter inhibits growth and promotes apoptosis of human T-cell acute lymphoblastic leukemia cells.Clin Lymphoma Myeloma Leuk,2012;12(1):59-65.
11 Zhou L,Wang DS,Li QJ,et al.The down-regulation of Notch1 inhibits the invasion and migration of hepatocellular carcinoma cells by inactivating the cyclooxygenase-2/Snail/E-cadherin pathway in vitro.Dig Dis Sci,2013;58(4):1016-1025.
12 Wang Z,Banerjee S,Li Y,et al.Down-regulation of Notch-1 inhibits invasion by inactivation of nuclear factor-κB,vascular endothelial growth factor,and matrix metalloproteinase-9 in pancreatic cancer cells.Cancer Res,2006;66(5):2778-2784.
13 Bin Hafeez B,Adhami VM,Asim M,et al.Targeted knockdown of Notch1 inhibits invasion of human prostate cancer cells concomitant with inhibition of matrix metalloproteinase-9 and urokinase plasminogen activator.Clin Cancer Res,2009,15(2):452-459.
14 Yu H,Zhao X,Huang SH,et al.Blocking Notch1 signaling by RNA interference can induce growth inhibition in He La cells.Int J Gynecol Cancer,2007;17(2):511-516.
15 Ramakrishnan V,Ansell S,Haug J,et al.MRK003,a gammasecretase inhibitor exhibits promising in vitro pre-clinical activity in multiple myeloma and non-Hodgkin's lymphoma.Leukemia,2012;26(2):340-348.
16 Nefedova Y,Sullivan DM,Bolick SC,et al.Inhibition of Notch signaling induces apoptosis of myeloma cells and enhances sensitivity to chemotherapy.Blood,2008;111(4):2220-2229.
17 Chang F,Lee JT,Navolanic PM,et al.Involvement of PI3K/Akt pathway in cell cycle progression,apoptosis,and neoplastic transformation:a target for cancer chemotherapy.Leukemia,2003;17(3):590-603.
18 Song G,Ouyang G,Bao S.The activation of Akt/PKB signaling pathway and cell survival.J Cell Mol Med,2005;9(1):59-71.
19 Wang Z,Li Y,Banerjee S,et al.Down-regulation of Notch-1 and Jagged-1 inhibits prostate cancer cell growth,migration and invasion,and induces apoptosis via inactivation of Akt,m TOR,and NF-kappa B signaling pathways.J Cell Biochem,2010;109(4):726-736.
20 Palomero T,Sulis ML,Cortina M,et al.Mutational loss of PTEN induces resistance to NOTCH1 inhibition in T-cell leukemia.Nat Med,2007;13(10):1203-1210.
21 Palomero T,Dominguez M,Ferrando AA.The role of the PTEN/AKT Pathway in NOTCH1-induced leukemia.Cell Cycle,2008;7(8):965-970.
2 Artavanis-Tsakonas S,Rand MD,Lake RJ.Notch signaling:cell fate control and signal integration indevelopment.Science,1999;284(5415):770-776.
3 Borggrefe T,Oswald F.The Notch signaling Pathway:transcriptional regulation at Notch target genes.Cell Mol Life Sci,2009;66(10):1631-1646.
4 Jundt F,Prebsting KS,Anagnostopoulos I,et al.Jagged1-induced Notch signaling drives proliferation of multip le myeloma cells.Blood,2004;103(9):3511-3535.
5 Kogoshi H,Sato T,Koyama T,et al.Gamma-secretase inhibitions suppress the growth of leukemia and lymphoma cells.Oncol Rep,2007;18(1):77-80.
6 Nefedova Y,Sullivan DM,Bolick SC,et al.Inhibition of Notch signaling induces apoptosis of myeloma cells and enhances sensitivity to chemotherapy.Blood,2008;111(4):2220-2229.
7 Leiba M,Jakubikova J,Klippel S,et al.Halofuginone inhibits multiple myeloma growth in vitro and in vivo and enhances cytotoxicity of conventional and novel agents.Br J Haematol,2012;157(6):718-731.
8 洪秀理,张泽川,赵江宁,等.异硫氰酸苯已酯抑制骨髓瘤细胞Notch信号表达的研究。中国实验血液学杂志,2011;19(4):940-943.
9 Peng KF,Wu XF,Zhao HW,et al.Advanced oxidation protein products induce monocyte chemoattractant protein-1 expression via p38 mitogen-activated protein kinase activation in rat vascular smooth muscle cells.Chin Med J,2006;119(13):1088-1093.
10 Xiang J,Ouyang Y,Cui Y,et al.Silencing of Notch3 Using shRNA driven by survivin promoter inhibits growth and promotes apoptosis of human T-cell acute lymphoblastic leukemia cells.Clin Lymphoma Myeloma Leuk,2012;12(1):59-65.
11 Zhou L,Wang DS,Li QJ,et al.The down-regulation of Notch1 inhibits the invasion and migration of hepatocellular carcinoma cells by inactivating the cyclooxygenase-2/Snail/E-cadherin pathway in vitro.Dig Dis Sci,2013;58(4):1016-1025.
12 Wang Z,Banerjee S,Li Y,et al.Down-regulation of Notch-1 inhibits invasion by inactivation of nuclear factor-κB,vascular endothelial growth factor,and matrix metalloproteinase-9 in pancreatic cancer cells.Cancer Res,2006;66(5):2778-2784.
13 Bin Hafeez B,Adhami VM,Asim M,et al.Targeted knockdown of Notch1 inhibits invasion of human prostate cancer cells concomitant with inhibition of matrix metalloproteinase-9 and urokinase plasminogen activator.Clin Cancer Res,2009,15(2):452-459.
14 Yu H,Zhao X,Huang SH,et al.Blocking Notch1 signaling by RNA interference can induce growth inhibition in He La cells.Int J Gynecol Cancer,2007;17(2):511-516.
15 Ramakrishnan V,Ansell S,Haug J,et al.MRK003,a gammasecretase inhibitor exhibits promising in vitro pre-clinical activity in multiple myeloma and non-Hodgkin's lymphoma.Leukemia,2012;26(2):340-348.
16 Nefedova Y,Sullivan DM,Bolick SC,et al.Inhibition of Notch signaling induces apoptosis of myeloma cells and enhances sensitivity to chemotherapy.Blood,2008;111(4):2220-2229.
17 Chang F,Lee JT,Navolanic PM,et al.Involvement of PI3K/Akt pathway in cell cycle progression,apoptosis,and neoplastic transformation:a target for cancer chemotherapy.Leukemia,2003;17(3):590-603.
18 Song G,Ouyang G,Bao S.The activation of Akt/PKB signaling pathway and cell survival.J Cell Mol Med,2005;9(1):59-71.
19 Wang Z,Li Y,Banerjee S,et al.Down-regulation of Notch-1 and Jagged-1 inhibits prostate cancer cell growth,migration and invasion,and induces apoptosis via inactivation of Akt,m TOR,and NF-kappa B signaling pathways.J Cell Biochem,2010;109(4):726-736.
20 Palomero T,Sulis ML,Cortina M,et al.Mutational loss of PTEN induces resistance to NOTCH1 inhibition in T-cell leukemia.Nat Med,2007;13(10):1203-1210.
21 Palomero T,Dominguez M,Ferrando AA.The role of the PTEN/AKT Pathway in NOTCH1-induced leukemia.Cell Cycle,2008;7(8):965-970.