柴胡皂甙d上调CDKN1B抑制脑胶质瘤细胞增殖的机制研究
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摘要
目的:探讨柴胡皂甙d(Saikosaponins-d,SSd)对脑胶质瘤细胞增殖的作用及调控机制。方法:体外培养脑胶质瘤Μ251细胞株,加入终浓度分别为0、5、10、20μg/m L的SSd,采用CCK-8法检测Μ251细胞的增殖率,通过流式细胞仪分析细胞周期及细胞凋亡率的改变,RT-PCR法检测细胞CDKN1B mRNA的表达水平。通过CDKN1B siRNA复原转染实验,以10μg/m L的SSd为对照,检测CDKN1B在SSd影响胶质瘤增殖、细胞周期及凋亡中的作用。结果:SSd可抑制脑胶质瘤Μ251细胞的增殖水平,且随SSd浓度越高,抑制作用越明显。同时SSd阻滞细胞周期在G0/G1期,促进胶质瘤细胞凋亡。CDKN1B siRNA能明显促进胶质瘤的增殖,CDKN1B表达下调能明显恢复SSd抑制的胶质瘤细胞增殖能力,同时能恢复SSd阻滞的胶质瘤细胞周期,抑制胶质瘤细胞的凋亡水平。结论:柴胡皂甙d能够通过抑制增殖并促进凋亡对人脑胶质瘤Μ251细胞生长起到抑制作用,其机制可能与上调CDKN1B的表达有关。
Objective: To investigate the effect and mechanism of Saikosaponins-d( SSd) on the cell proliferation glioma cell. Methods: Gliomas U251 cell line was cultured in vitro and the proliferation of U251 cell was detected by CKK-8 method after adding different final concentrations of SSd( 0 ug/m L,5 ug/m L,10 ug/m L,and 20 ug/m L). Flow cytometry was used to monitor changes in U251 cell cycle and apoptosis. The mRNA expression of CDKN1 B was detected by RT-PCR. The effect of CDKN1 B on SSd( influencing the proliferation,cell cycle,and apoptosis rates) in U251 cells was observed after transfection with CDKN1 B siRNA.Results: With the increasing concentrations of SSd,the proliferation rates decreased remarkedly. The activity of U251 cells was inhibited,the cell cycle was arrested in G0/G1 phase,and the cell apoptosis was increased after using SSd. CDKN1 B siRNA could promote the proliferation of UC251,down-expression of CDKN1 B restored the proliferation inhibition and G0-G1 phase transition,also inhibited the apoptosis level of UC251 regulated by SSd. Conclusion: SSd may inhibit the cell activity of glioma cell line U251 and promote cell apoptosis in vitro via increasing the CDKN1 B expression.
Objective: To investigate the effect and mechanism of Saikosaponins-d( SSd) on the cell proliferation glioma cell. Methods: Gliomas U251 cell line was cultured in vitro and the proliferation of U251 cell was detected by CKK-8 method after adding different final concentrations of SSd( 0 ug/m L,5 ug/m L,10 ug/m L,and 20 ug/m L). Flow cytometry was used to monitor changes in U251 cell cycle and apoptosis. The mRNA expression of CDKN1 B was detected by RT-PCR. The effect of CDKN1 B on SSd( influencing the proliferation,cell cycle,and apoptosis rates) in U251 cells was observed after transfection with CDKN1 B siRNA.Results: With the increasing concentrations of SSd,the proliferation rates decreased remarkedly. The activity of U251 cells was inhibited,the cell cycle was arrested in G0/G1 phase,and the cell apoptosis was increased after using SSd. CDKN1 B siRNA could promote the proliferation of UC251,down-expression of CDKN1 B restored the proliferation inhibition and G0-G1 phase transition,also inhibited the apoptosis level of UC251 regulated by SSd. Conclusion: SSd may inhibit the cell activity of glioma cell line U251 and promote cell apoptosis in vitro via increasing the CDKN1 B expression.
引文
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[16] BOCHIS O V,IRIMIE A,PICHLER M,et al. The role of Skp2 and its substrate CDKN1B(p27)in colorectal cancer[J]. J Gastrointestin Liver Dis,2015,24(2):225-234.
[2]程海波,沈政洁,孙东东,等.抗肿瘤中药对肿瘤微环境的干预作用评述[J].中医杂志,2014,55(15):1343-1346.
[3]彭顺利,张巍,李妍.柴胡皂甙d的作用及其机制的研究进展[J].吉林医药学院学报,2014,35(1):66-68.
[4] ZHONG D,ZHANG H J,JIANG Y D,et al. Saikosaponin-d:A potential chemotherapeutics in castration resistant prostate cancer by suppressing cancer metastases and cancer stem cell phenotypes[J].Biochem Biophys Res Commun,2016,474(4):722-729.
[5] MILLER J J,SHIH H A,ANDRONESI O C,et al. Isocitrate dehydrogenase-mutant glioma:Evolving clinical and therapeutic implications[J].Cancer:2017.
[6] CHIANG L C,NG L T,LIΜL T,et al. Cytotoxicity and anti-hepatitis B virus activities of saikosaponins from Bupleurum species[J].Planta Med,2003,69(8):705-709.
[7] ZHONG D,ZHANG H J,JIANG Y D,et al. Saikosaponin-d:A potential chemotherapeutics in castration resistant prostate cancer by suppressing cancer metastases and cancer stem cell phenotypes[J].Biochem Biophys Res Commun,2016,474(4):722-729.
[8] CAI C,ZHANG H,OΜY,et al. Saikosaponin-d suppresses cell growth in renal cell carcinoma through EGFR/p38 signaling pathway[J]. Neoplasma,2017,64(4):518-525.
[9]吴文安,赵金,雎岩,等.柴胡皂甙D对肺癌A549细胞放射敏感性的影响及机制[J].现代肿瘤医学,2014,22(2):290-291.
[10] ZHΜB H,PΜR,ZHANG G P,et al. Effect of Saikosaponins-d on reversing malignant phenotype of Hep G2 cells in vitro[J]. Zhonghua Gan Zang Bing Za Zhi,2011,19(10):764-767.
[11]徐明明.柴胡皂甙D诱导人宫颈癌Hela细胞系凋亡的研究[J].医药论坛杂志,2011,32(8):89-91.
[12] YAO M,YANG J,CAO L,et al. Saikosaponind inhibits proliferation of DΜ145 human prostate cancer cells by inducing apoptosis and arresting the cell cycle at G0/G1 phase[J]. Mol Med Rep,2014,10(1):365-372.
[13] WANG B F,DAIi Z J,WANG X J,et al. Saikosaponin-d increases the radiosensitivity of smmc-7721 hepatocellular carcinoma cells by adjusting the g0/g1 and g2/m checkpoints of the cell cycle[J]. BMC Complement Altern Med,2013,13:263.
[14] OΜL,FERREIRA A M,OTIENO S,et al. Incomplete folding upon binding mediates Cdk4/cyclin D complex activation by tyrosine phosphorylation of inhibitor p27 protein[J]. J Biol Chem,2011,286(34):30142-30151.
[15] VERVOORTS J,LΜSCHER B. Post-translational regulation of the tumor suppressor p27(KIP1)[J]. Cell Mol Life Sci,2008,65(20):3255-3264.
[16] BOCHIS O V,IRIMIE A,PICHLER M,et al. The role of Skp2 and its substrate CDKN1B(p27)in colorectal cancer[J]. J Gastrointestin Liver Dis,2015,24(2):225-234.