摘要
目的:探讨三叶青总黄酮对乳腺癌细胞增殖、转移的影响,并分析其作用机制。方法:通过Cell Counting Kit-8/CCK8法检测不同质量浓度三叶青总黄酮(10 g·L^-1、20 g·L^-1及40 g·L^-1)作用24 h、48 h和72 h对MCF-7细胞增殖的影响。细胞集落实验测定不同质量浓度三叶青总黄酮作用24 h对MCF-7细胞增殖的影响。Transwell技术和流式细胞术检测三叶青总黄酮作用24 h后对细胞迁移、侵袭和细胞周期的影响。采用蛋白质印迹技术检测CyclinD1、C-Myc、E-cadherin、基质金属蛋白酶(matrix metalloproteinase,MMP)-9、β-catenin和p-β-catenin蛋白表达水平。实时荧光定量核酸扩增检测系统法(real-time quantitative PCR detecting system,q-PCR)检测CyclinD1、C-Myc、E-cadherin和MMP-9 mRNA的表达水平。结果:各组MCF-7细胞抑制率比较,差异有统计学意义(F组别=77.265,P=0.000),随着时间的延长,细胞抑制率逐渐增加(F时间=14.581,P=0.000)。药物处理24 h后,三叶青总黄酮10 g·L^-1组、20 g·L^-1组及40 g·L^-1组的细胞集落数分别为(90.43±9.17)个、(45.64±4.67)个和(10.21±1.26)个,均显著低于对照组(F=919.002,P<0.05)。三叶青总黄酮10 g·L^-1组、20 g·L^-1组及40 g·L^-1组的G0/G1期细胞比例分别为(70.59±7.13)%、(73.64±7.28)%和(78.83±7.89)%显著高于对照组(F=18.812,P=0.000)。三叶青总黄酮10 g·L^-1组、20 g·L^-1组及40 g·L^-1组的迁移、侵袭细胞数分别为(120.17±13.36)个、(96.75±9.81)个和(75.44±7.68)个,(84.08±8.62)个、(66.39±6.81)个和(42.45±4.41)个,均显著低于对照组(F=141.487、286.712,P=0.000)。三叶青总黄酮10 g·L^-1组、20 g·L^-1组及40 g·L^-1 CyclinD1、C-myc、E-cadherin和MMP-9蛋白及mRNA表达水平均低于对照组,p-β-catenin蛋白表达水平低于对照组,E-cadherin蛋白和mRNA水平高于对照组(P<0.05)。结论:三叶青总黄酮具有抑制乳腺癌细胞MCF-7增殖和侵袭的作用,其可能的机制是将细胞周期阻滞于G0/G1期,调节Wnt/β-catenin信号通路相关蛋白的表达。
Objective:To explore the effect of total flavonoids of Clover on breast cancer cell proliferation and metastasis,and analyze its mechanism of action.Methods:Cell Counting Kit-8/CCK8 method was used to detect the total flavonoids of clover(10 g·L^-1 group,20 g·L^-1 group and 40 g·L^-1 group) for 24 h,48 h and 72 h on the proliferation of MCF-7 cells.Cell colony experiment was used to determine the effect of different concentrations of total flavonoids on the proliferation of MCF-7 cells.Transwell technique and flow cytometry were used to detect the effect of total leaf flavonoids on cell migration,invasion and cell cycle after 24 h.The protein expression levels of CyclinD1,C-Myc,E-cadherin,MMP-9,β-catenin and p-β-catenin were detected by Western blotting.Real-time quantitative nucleic acid amplification detection system(Real-time Quantitative PCR Detecting System,q-PCR) was used to detect the expression levels of CyclinD1,C-Myc,E-cadherin and matrix metalloproteinase(MMP)-9 mRNA.Results:Compared with the inhibition rate of MCF-7 cells in each group,the difference was statistically significant(Group F=77.265,P=0.000).With the extension of time,the inhibition rate of cells gradually increased(Ftime=14.581,P=0.000).After 24 hours of drug treatment,the number of cell colonies in the total flavonoids of Trifolium pratense 10 g·L^-1 group,20 g·L^-1 group and 40 g·L^-1 group were(90.43±9.17) and(45.64± 4.67) and(10.21±1.26) were significantly lower than the control group(F=919.002,P<0.05).The ratios of G0/G1 phase cells in the 10 g·L^-1 group,20 g·L^-1 group and 40 g·L^-1 group were(70.59±7.13) %,(73.64±7.28) % and(78.83±7.89) % is significantly higher than the control group(F=18.812,P=0.000).The total flavonoids of C.adenophorum in the 10 g·L^-1 group,20 g·L^-1 group and 40 g·L^-1 group were(120.17±13.36),(96.75±9.81) and migrating cells respectively.(75.44±7.68),(84.08±8.62),(66.39±6.81) and(42.45±4.41) were significantly lower than the control group(F=141.487,286.712,P=0.000).The total flavonoids in cloverleaf 10 g·L^-1 group,20 g·L^-1 group and 40 g·L^-1 CyclinD1,C-myc,E-cadherin and MMP-9 protein and mRNA expression levels were lower than those in the control group The expression level of p-β-catenin protein was lower than that of the control group,and the level of E-cadherin protein and mRNA was higher than that of the control group(P<0.05).Conclusion:The total flavonoids of clover can inhibit the proliferation and invasion of breast cancer cells MCF-7.The possible mechanism is to block the cell cycle in G0/G1 phase and regulate the expression of Wnt/β-catenin signaling pathway-related proteins.
作者
杜闯
王燕
张莹莹
张健华
张临风
李靖若
DU Chuang;WANG Yan;ZHANG Yingying;ZHANG Jianhua;ZHANG Linfeng;LI Jingruo(The First Affiliated Hospital to Zhengzhou University,Zhengzhou Henan China 450000)
出处
《中医学报》
CAS
2020年第8期1717-1722,共6页
Acta Chinese Medicine
基金
河南省高等学校重点科研项目(19A320074)。
