维生素K_2诱导肝癌细胞株Hep-G2凋亡机制研究

Apoptosis of hepatocellular carcinoma cell line Hep-G2 induced by vitamin K_2

目的将维生素K_2用于肝癌Hep-G2细胞株,观察它对BTG2和Cyclin D1表达的影响,以探讨其抑制细胞增殖和诱导细胞凋亡的机制。方法 CCK-8检测维生素K_2对Hep-G2细胞增殖的影响;流式细胞术检测维生素K_2对细胞周期及凋亡的影响;蛋白质印迹法检测BTG2和Cyclin D1蛋白的表达;RT-PCR检测BTG2和Cyclin D1的mRNA转录水平。结果随着浓度增加,维生素K_2(40、80、160μmol/L)可以抑制肝癌Hep-G2细胞的增殖,且这种抑制作用具有浓度和时间依赖性,药物干预细胞48h后IC_(50)=65.17μmol/L。流式细胞术结果显示,实验组G_1期细胞比例(55±1.0)%较对照组的(44±1.0)%明显升高,差异有统计学意义(P=0.000 3),说明维生素K_2可将细胞周期阻滞在G_1期。流式细胞术测细胞凋亡结果显示,实验组中晚期细胞凋亡率与对照组相比,差异有统计学意义(F=275.6,P<0.05),且该浓度的维生素K_2诱导细胞凋亡的作用具有时间依赖性,F=693.3,P<0.01。蛋白质印迹法结果显示,维生素K_2作用24、48和72h,BTG2蛋白表达分别为0.138±0.001、0.258±0.013和0.330±0.007,其表达呈上升趋势,与对照组(0.021±0.001)相比,差异有统计学意义,F=993.7,P<0.01;Cyclin D1蛋白表达分别为0.411±0.002、0.372±0.005和0.232±0.006,其表达呈下降趋势,与对照组(0.733±0.032)相比,差异有统计学意义,F=489.7,P<0.01。RT-PCR结果显示,维生素K_2作用24、48、72h,BTG2mRNA表达分别为1.13±0.46、1.15±0.03和1.22±0.37,其表达并没有随着维生素K_2作用时间的延长而出现明显上升趋势,且与对照组(1.00±0.00)相比,差异无统计学意义,F=0.30,P=0.83;Cyclin D1mRNA表达分别为0.63±0.03、0.44±0.05和0.14±0.03,其表达随着维生素K_2作用时间的延长而逐渐下调(P<0.01),与对照组(1.00±0.00)比较,差异有统计学意义,F=415.6,P<0.01。结论维生素K_2可抑制肝癌细胞Hep-G2的增殖,并诱导细胞凋亡,其机制可能与其上调BTG2表达和下调Cyclin D1表达有关,为维生素K_2成为治疗肝癌的天然药物提供了新的依据。

OBJECTIVE To observe the effect of vitamin K2（VK2）on expression of BTG2 and Cyclin D1 in Hepatocellular carcinoma Hep-G2 cell line,and to investigate the mechanism of inhibiting the proliferation and inducing apoptosis of Hep-G2 cells by VK2.METHODS The growth inhibition of Hep-G2 cells was detected by CCK-8 method.Effect of VK2 on cell apoptosis and proliferation was checked by flow cytometry.The expressions of BTG2 and Cyclin D1 protein were tested by Western blot,the transcription levels of BTG2 and Cyclin D1 mRNAs were checked by RT-PCR.RESULTS With the increase of concentration（40,80,160μmol/L）,the proliferations of Hep-G2 cells were inhibited in a dose and time dependent manner,and IC50 value was 65.17μmol/L after drug intervention for 48 hours.The results of flow cytometry showed that the cells proportion of G1 phase in the experimental group（55±1.0）% was significantly higher than that of the control group（44±1.0）%,and the difference was statistically significant（P=0.000 3）,which illustrated that VK2 could block the cell cycle to G1 phase.In addition,the results of apoptosis measured by flow cytometry showed that the apoptosis rate in the middle and late stage of the experiment group was significantly different from that in the control group（F=275.6,P〈0.05）.In addition,the rate of apoptosis was correlated positively with time（F=693.3,P〈0.01）.Western blot results showed that with the treating time protracting,the expression of BTG2 was0.138±0.001,0.258±0.013,0.330±0.007 respectively at 24,48 and 72 h,which showed an upward trend,and the difference was significant（F=993.7,P〈0.01）when compared with the control group（0.021±0.001）,while the expression of Cyclin D1 protein was 0.411±0.002,0.372±0.005,0.232±0.006 at the same time,which decreased gradually,in addation,compared to the control group（0.733±0.032）the difference was statistically significant（F=489.7,P〈0.01）.RT-PCR indicated that the expressions of BTG2 mRNA was 1.13±0.46,1.15±0.03,1.22±0.37 at 24,48 and72 h,which indicated that the variation trend of upregulation was not obvious with the extension of time（P〈0.05）.Compared with the control group（1.00±0.00）,the difference was not clear in the light of statistics（F=0.30,P=0.83）.However,the expressions of Cyclin D1 mRNA in the three period of time was 0.63±0.03,0.44±0.05,0.14±0.03,which showed that VK2 could downgradulate the expression of Cyclin D1 mRNA,and the difference was statistically significant（F=415.6,P〈0.01）comparing to the control group（1.00±0.00）.CONCLUSIONS VK2 could inhibit the proliferation of Hep-G2 cells and induce apoptosis,and the mechanisim may be related to the upregulation of BTG2 and downregulation of Cyclin D1 by VK2.This provides a new basis for VK2 as a natural medicine for the treatment of liver cancer.