摘要
目的探讨扶正抗癌方阻滞H460细胞周期的分子机制。方法CCK8检测细胞存活率,流式细胞术检测细胞周期,Real-time PCR检测miR221-3p及p27^(Kip1)mRNA表达,Western blot检测p27^(Kip1)、Cyclin D1、Cyclin E、CDK2和CDK4表达,荧光素酶报告基因检测miR221-3p是否与p27^(Kip1)3’UTR结合。结果与对照组比较,给药组细胞存活率降低(P<0.01),G0/G1期细胞增加、S期细胞减少(P<0.05),Cyclin D1、Cyclin E、CDK2和CDK4蛋白表达降低(P<0.05),p27^(Kip1)蛋白和mRNA表达增加(P<0.05),miR221-3p表达降低(P<0.01);与阴性对照组比较,miR221-3p mimics组p27^(Kip1)mRNA表达降低(P<0.01);与nc-inhibitor组比较,miR221-3p mimics组p27^(Kip1)mRNA表达增加(P<0.01);与阴性对照组比较,转染p27^(Kip1)野生型质粒的荧光素酶活性降低(P<0.01)。结论扶正抗癌方下调miR221-3p表达,上调p27^(Kip1)表达,调控Cyclin D1、Cyclin E、CDK2和CDK4表达,阻滞细胞周期在G0/G1期抑制增殖。
Objective To discuss the molecular mechanism of Fuzheng Kang-Ai Decoction inducing cell cycle arrest in H460 cells.Methods The cell viability and the cell cycle are detected by the CCK8 assay and the flow cytometry analysis,respectively.The expression of miR221-3p and p27^(Kip1) mRNA are detected by Real-time PCR assay.The expression of p27^(Kip1),Cyclin D1,Cyclin E,CDK2 and CDK4 are detected by western blot assay.Luciferase reporter gene assay is used to detect whether miR221-3p binds to the 3’UTR site of p27^(Kip1).Results Compared with the blank control group,the H460 cell viability in Fuzheng Kang-Ai Decoction groups are reduced(P<0.01),the number of cells in G0/G1 phase increased and in S phase reduced(P<0.05),the expression of Cyclin D1,Cyclin E,CDK2 and CDK4 are reduced(P<0.05),the protein and mRNA expression of p27^(Kip1) are increased(P<0.05),the expression of miR221-3p is reduced(P<0.01).Compared with the negative group,the mRNA expression of p27^(Kip1) in miR221-3p mimics group is reduced(P<0.01).Compared with the nc-inhibitor group,the mRNA expression of p27^(Kip1) in miR221-3p mimics group is increased(P<0.01).Compared with the negative group,the luciferase activity of cells transfected with p27^(Kip1) wild-type plasmid is reduced(P<0.01).Conclusion Fuzheng Kang-Ai Decoction can inhibit proliferation by inducing cell cycle arrested at G0/G1 phase in H460 Cells,through which up-regulates the expression of p27^(Kip1) by down-regulating the expression of miR221-3p,and regulates the expression of Cyclin D1,Cyclin E,CDK2 and CDK4.
作者
李龙妹
甘紫胭
杨小兵
河文峰
廖桂雅
李秋萍
吴万垠
LI Long-mei;GAN Zi-yan;YANG Xiao-bing;HE Wen-feng;LIAO Gui-ya;LI Qiu-ping;WU Wan-yin(The Second Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510370,China)
出处
《现代中药研究与实践》
CAS
2021年第3期16-20,24,共6页
Research and Practice on Chinese Medicines
基金
国家自然科学基金青年项目(81803919,81974543)
广东省自然科学基金博士启动项目(2018A030310601)。
