1,25二羟维生素D3对肝癌HepG2细胞增殖侵袭影响

娄方方; 孙建超; 黄健; 孔维莹; 黄音员祝

doi:10.11847/zgggws2016-32-01-24

1,25二羟维生素D3对肝癌HepG2细胞增殖侵袭影响

doi: 10.11847/zgggws2016-32-01-24

1.
贵州医科大学检验学院临床生化教研室, 贵州贵阳 550004;
2.
贵州省人民医院临床检验中心

基金项目:

贵州省科技厅基金(黔科合LH字[2014]7114)

详细信息

作者简介:
娄方方(1988-),女,贵州遵义人,硕士在读,研究方向:临床生物化学。

通讯作者:
黄音员祝E-mail:374185146@qq.com

中图分类号: R395.6
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出版历程
- 收稿日期: 2015-09-16
- 刊出日期: 2016-01-10

Effects of 1,25(OH)2D3 and its combination with LY294002 on proliferation and invasion of human hepatocellular carcinoma HepG2 cells

1.
Clinical Biochemistry Department, School of Medical Laboratory, Guizhou Medical University, Guiyang, Guizhou Province 550004, Chnia

摘要

摘要: 目的探讨1,25二羟维生素D3[1,25(OH)₂D₃]及PI3K/AKT信号通路抑制剂(LY294002)对人肝癌HepG2细胞的增殖、侵袭影响及作用机制。方法实验设10^-8、10^-7、10^-6 mol/L 1,25(OH)₂D₃组及2.5、5.0、10.0、20.0、40.0、80.0μmol/L LY294002组,10^-7 mol/L 1,25(OH)₂D₃+5μmol/L LY294002联合组,噻唑蓝法检测人肝癌HepG2细胞增殖抑制率;CompuSyn软件计算联合指数;Tanswell小室检测HepG2细胞侵袭数;Western blot检测HepG2细胞增殖细胞核抗原(PCNA),细胞基质金属蛋白酶9(MMP-9)、磷酸化丝氨酸苏氨酸蛋白激酶(p-AKT)、10号染色体缺失且与张力蛋白同源物磷酸脂酶基因(PTEN)蛋白。结果 1,25(OH)₂D₃及LY294002对人肝癌HepG2细胞增殖抑制率呈时间-剂量依赖效应(P<0.05);1,25(OH)₂D₃联合LY294002组细胞增殖抑制率明显高于二者单独处理组(P<0.05),联合指数=0.728,2者具有协同效应;10^-7 mol/L 1,25(OH)₂D₃组、5μmol/L LY294002组以及联合组HepG2细胞侵袭数[分别为(45.9±6.4)、(49.9±6.0)、(27.8±4.0)个]明显低于对照组[(64.6±8.0)个](P<0.05)。与对照组比较,10^-7 mol/L 1,25(OH)₂D₃组及联合组HepG2细胞PTEN蛋白表达量增加,差异有统计学意义(P<0.05),10^-7 mol/L 1,25(OH)₂D₃组、5μmol/L LY294002组及联合组HepG2细胞PCNA、MMP-9、p-AKT蛋白表达均降低,差异有统计学意义(P<0.05),且联合组蛋白表达量低于二者单独处理组(P<0.05)。结论 1,25(OH)₂D₃可抑制人肝癌HepG2细胞增殖、侵袭,其机制可能与上调PTEN表达,抑制PI3K/AKT信号通路活性,下调PCNA、MMP-9蛋白有关;与LY294002合用具有协同效应。
- 1,25(OH)2D3 /
- HepG2细胞 /
- 增殖 /
- 侵袭 /
- PI3K/AKT
Abstract: Objective To explore effects and mechanism of 1,25-dihydroxyvitamin D3(1,25[OH]₂D₃) and LY294002(an inhibitor of phosphatidylinositol 3-kinase/serine/threonine protein kinase[PI3K/AKT]signal pathway) on proliferation,migration potentiality and metastasis of human hepatocellular carcinoma HepG2 cells.Methods HepG2 cells were treated with 1,25(OH)₂D₃(10^-8,10^-7,10^-6 mol/L),LY294002(2.5,5,10,20,40,80 umol/L) and the combination of 1,25(OH)₂D₃(10^-7 mol/L) and LY294002(5 umol/L),repectively.Cell proliferation was assayed by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) method;invasion ability of HepG2 cells was detected by transwell assay;expressions of proliferating cell nuclear antigen(PCNA),matrix metalloprotein-9(MMP-9),phosphorylase protein serine threonine kinase(p-AKT),and phosphatase and tensin homologue deleted on chromosome 10(PTEN)in HepG2 cells were assessed by Western blot;the combination indexes were calculated with CompuSyn software.Results The inhibitive effect of 1,25(OH)₂D₃ and LY294002 on HepG2 cells' proliferation showed a time-dose dependent manner(P<0.05).The proliferation inhibitive rate of HepG2 cells with combined treatment of 1,25(OH)₂D₃ and LY294002 was significantly higher than that of the cells treated by 1,25(OH)₂D₃ or LY294002 separately(both P<0.05),with a combination index of 0.728 and suggesting a synergistic effect of the two agents.The numbers of invasion cells were 45.9±6.4,49.9±6.0,and 27.8±4.0 for the HepG2 cells treated with 1,25(OH)₂D₃(10^-7 mol/L),LY294002(5 umol/L),and the two agents combined at the same doses,which were significantly lower than that (64.6±8.0) of the control HepG2 cells(P<0.05 for all).The expression level of PTEN in HepG2 cells treated with 1,25(OH)₂D₃(10^-7 mol/L) and 1,25(OH)₂D₃(10^-7 mol/L) combined with LY294002(5 μmol/L) was up-regulated significantly(both P<0.05),while the expressions of PCNA,MMP-9,and p-AKT in HepG2 cells treated with 1,25(OH)₂D₃(10^-7 mol/L),LY294002(5 μmol/L),and the two agents combined at the same doses were significantly down-regulated(P<0.05 for all) and the expressions of the proteins in HepG2 cells with the combined treatment of the two agents were significantly higher that those in the cells with separate treatment of 1,25(OH)₂D₃ or LY294002(both P<0.05).Conclusion The results of the study suggest that 1,25(OH)₂D₃ can inhibit the proliferation and invasion ability of human hepatocellular carcinoma HepG2 cells and the mechanism may be correlated to the up-regulation of PTEN,the inhibition of PI3K/AKT signal,and down-regulation of PCNA and MMP-9 protein;the results also demonstrate that combined treatment of 1,25(OH)₂D₃ and LY294002 has a synergistic effect on proliferation and invasion of HepG2 cells.
- 1,25(OH)2D3 /
- HepG2 cell /
- proliferation /
- invasion /
- phosphatidinositol 3-kinase/serine/threonine protein kinase

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