丁酸钠调控HepG2细胞的增殖、凋亡和侵袭
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摘要
目的研究丁酸钠对人肝癌细胞HepG2增殖和凋亡的影响,并探讨可能的作用机制。方法用不同浓度丁酸钠处理HepG2细胞后,MTT方法检测细胞的增殖能力,流式细胞技术检测细胞周期的分布和细胞凋亡,Transwell小室检测丁酸钠对HepG2细胞侵袭能力的影响。免疫荧光法检测HDAC4蛋白在HepG2细胞中的表达及定位。蛋白质印迹法(Western Blot)检测HDAC4蛋白的表达水平。结果随着丁酸钠处理浓度的增加和处理时间的延长,HepG2细胞增殖能力明显受抑制,细胞周期也发生阻滞,G1期细胞比例明显增加,而S期细胞比例明显减少。不同浓度(0,1,5,10 mmol/L)丁酸钠处理HepG2细胞24 h后,早期凋亡率分别为2.7%,4.5%,6.5%,6.7%,差异有统计学意义(F=15.1,P=0.001)。丁酸钠显著抑制细胞侵袭能力,侵袭细胞百分比分别降至72.7%(1 mmol/L)、41.7%(5 mmol/L)、21.3%(10 mmol/L),差异有统计学意义(F=202.1,P<0.001)。HDAC4蛋白在HepG2细胞中呈阳性表达,主要位于细胞质中。丁酸钠明显抑制HDAC4蛋白的表达,并呈浓度依赖性。结论丁酸钠抑制肝癌细胞系HepG2的增殖,调控细胞周期、促进凋亡,抑制细胞的侵袭能力。
Objective To determine the effect of sodium butyrate on HepG2 cells growth,apoptosis,invasion and to explore the potential mechanism.Methods HepG2 cell line was treated with different concentrations of Na Bu,and MTT assay was conducted to detect cell viability.Cell cycle distribution and apoptosis was evaluated with flow cytometry.Cell invasion was analyzed by transwell assays.Immunofluorescence staining was used to detect HDAC4 protein expression and localization in HepG2 cells.Western Blot test was performed to determine the expression of HDAC4 protein.Results With the increase of Na Bu concentration and the treatment time,the cell proliferation was significantly inhibited and the cell cycle was significantly blocked with the proportion of G1 cells increased significantly and the proportion of cells in the phase S significantly decreased.The early apoptotic rates of HepG2 cells treated with Na Bu at different concentrations(0,1,5,10 mmol/L) for 24 h were 2.7%,4.5%,6.5% and 6.7%,respectively,and the difference was statistically significant(F=15.1,P=0.001).Na Bu significantly inhibited cell invasion,and the percentage of invasive cells decreased to72.7%(1 mmol/L),41.7%(5 mmol/L) and 21.3%(10 mmol/L),respectively.The difference was statistically significant(F=202.1,P<0.001).HDAC4 protein was positively expressed in HepG2 cells and mainly located in cytoplasm.Na Bu significantly inhibited the expression of HDAC4 protein in a concentration dependent manner.Conclusion Na Bu inhibited the proliferation of HepG2 cells by regulating cell cycle,promoting apoptosis and inhibiting invasion ability.
Objective To determine the effect of sodium butyrate on HepG2 cells growth,apoptosis,invasion and to explore the potential mechanism.Methods HepG2 cell line was treated with different concentrations of Na Bu,and MTT assay was conducted to detect cell viability.Cell cycle distribution and apoptosis was evaluated with flow cytometry.Cell invasion was analyzed by transwell assays.Immunofluorescence staining was used to detect HDAC4 protein expression and localization in HepG2 cells.Western Blot test was performed to determine the expression of HDAC4 protein.Results With the increase of Na Bu concentration and the treatment time,the cell proliferation was significantly inhibited and the cell cycle was significantly blocked with the proportion of G1 cells increased significantly and the proportion of cells in the phase S significantly decreased.The early apoptotic rates of HepG2 cells treated with Na Bu at different concentrations(0,1,5,10 mmol/L) for 24 h were 2.7%,4.5%,6.5% and 6.7%,respectively,and the difference was statistically significant(F=15.1,P=0.001).Na Bu significantly inhibited cell invasion,and the percentage of invasive cells decreased to72.7%(1 mmol/L),41.7%(5 mmol/L) and 21.3%(10 mmol/L),respectively.The difference was statistically significant(F=202.1,P<0.001).HDAC4 protein was positively expressed in HepG2 cells and mainly located in cytoplasm.Na Bu significantly inhibited the expression of HDAC4 protein in a concentration dependent manner.Conclusion Na Bu inhibited the proliferation of HepG2 cells by regulating cell cycle,promoting apoptosis and inhibiting invasion ability.
引文
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[13]赵璐,薛晓文,李志裕.大环类HDAC抑制剂研究进展[J].海峡药学,2016,28(1):1-5.
[14]DAVIE JR.Inhibition of histone deacetylase activity by butyrate[J].J Nutr,2003,133(7 Suppl):2485S-2493S.
[15]AHN MY,KANG DO,NA YJ,et al.Histone deacetylase inhibitor,apicidin,inhibits human ovarian cancer cell migration via class IIhistone deacetylase 4 silencing[J].Cancer Lett,2012,325(2):189-199.
[16]王显艳,高峰,赵春明,等.microRNA-140通过下调HDAC4抑制胃癌细胞迁移和侵袭能力[J].癌症进展,2016,14(3):223-227.
[17]张艳春,李家明,王杰,等.具有去甲斑蝥素结构的HDAC抑制剂的设计与合成[J].安徽医药,2015,19(6):1065-1067.
[2]FERLAY J,SHIN HR,BRAY F,et al.Estimates of worldwide burden of cancer in 2008:GLOBOCAN 2008[J].Int J Cancer,2010,127(12):2893-2917.
[3]陈恳,丁益宏,朱郁飞,等.肝细胞癌患者血清中循环无细胞DNA、甲胎蛋白及a-L-岩藻糖苷酶联合检测的意义[J].安徽医药,2016,20(9):1715-1719.
[4]CANDIDO EP,REEVES R,DAVIE JR.Sodium butyrate inhibits histone deacetylation in cultured cells[J].Cell,1978,14(1):105-113.
[5]PANT K,YADAV AK,GUPTA P,et al.Butyrate induces ROS-mediated apoptosis by modulating miR-22/SIRT-1 pathway in hepatic cancer cells[J].Redox Biol,2017,12:340-349.
[6]SALIMI V,SHAHSAVARI Z,SAFIZADEH B,et al.Sodium butyrate promotes apoptosis in breast cancer cells through reactive oxygen species(ROS)formation and mitochondrial impairment[J].Lipids Health Dis,2017,16(1):208.
[7]SEIFRTOVM,HAVELEK R,CAHLKOVL,et al.Haemanthamine alters sodium butyrate-induced histone acetylation,p21WAF1/Cip1 expression,Chk1 and Chk2 activation and leads to increased growth inhibition and death in A2780 ovarian cancer cells[J].Phytomedicine,2017,35:1-10.
[8]LI J,CHEN J,RICUPERO CL,et al.Nuclear accumulation of HDAC4 in ATM deficiency promotes neurodegeneration in ataxia telangiectasia[J].Nat Med,2012,18(5):783-790.
[9]OZCAN L,GHORPADE DS,ZHENG Z,et al.Hepatocyte DACH1is increased in obesity via nuclear exclusion of HDAC4 and promotes hepatic insulin resistance[J].Cell Rep,2016,15(10):2214-2225.
[10]GAUR V,CONNOR T,SANIGORSKI A,et al.Disruption of the class IIa HDAC corepressor complex increases energy expenditure and lipid oxidation[J].Cell Rep,2016,16(11):2802-2810.
[11]DAWSON MA,KOUZARIDES T.Cancer epigenetics:from mechanism to therapy[J].Cell,2012,150(1):12-27.
[12]PEREGO P,ZUCO V,GATTI L,et al.Sensitization of tumor cells by targeting histone deacetylases[J].Biochem Pharmacol,2012,83(8):987-994.
[13]赵璐,薛晓文,李志裕.大环类HDAC抑制剂研究进展[J].海峡药学,2016,28(1):1-5.
[14]DAVIE JR.Inhibition of histone deacetylase activity by butyrate[J].J Nutr,2003,133(7 Suppl):2485S-2493S.
[15]AHN MY,KANG DO,NA YJ,et al.Histone deacetylase inhibitor,apicidin,inhibits human ovarian cancer cell migration via class IIhistone deacetylase 4 silencing[J].Cancer Lett,2012,325(2):189-199.
[16]王显艳,高峰,赵春明,等.microRNA-140通过下调HDAC4抑制胃癌细胞迁移和侵袭能力[J].癌症进展,2016,14(3):223-227.
[17]张艳春,李家明,王杰,等.具有去甲斑蝥素结构的HDAC抑制剂的设计与合成[J].安徽医药,2015,19(6):1065-1067.