缺氧诱导因子抑制剂YC-1对缺氧肝癌细胞生物钟基因和蛋白表达及增殖的影响  

Effects of hypoxia-inducible factor inhibitor YC-1 on expression and proliferation of circadian clock genes and proteins in hypoxic hepatoma cells

作　　者：许静 孙诚谊 喻超 何晓晓 杨盛力[3] XU Jing1 , SUN Chengyi, YU Chao, HE Xiaoxiao, YANG Shengli(1 The Affiliated Hospital of Guizhou Medical University, Guiyang 550000, Chin)

机构地区：[1]贵州医科大学附属医院,贵阳550000 [2]华中科技大学同济医学院附属梨园医院 [3]华中科技大学同济医学院附属协和医院

出　　处：《山东医药》2018年第7期5-8,共4页Shandong Medical Journal

基　　金：国家自然科学基金青年基金项目(81702885);湖北省自然科学基金项目(2017CFB178);中央高校基本科研业务费专项资金项目:华中科技大学自主创新基金项目(0118530328);中国肝炎防治基金会天晴肝病研究基金项目(TQGB20170005)

摘　　要：目的观察缺氧诱导因子(HIF)抑制剂YC-1对缺氧肝癌细胞生物钟基因、生物钟蛋白表达及细胞增殖的影响。方法将肝癌细胞Huh7置于乏氧培养箱中培养,分为实验组和对照组,实验组加入50μmol/L的YC-1,对照组加入DMSO,培养48 h后分别提取mRNA和蛋白。采用real-time PCR法检测两组细胞中的Per1、Per2、Per3、Cry1、Cry2、Clock、Bmal1 mRNA,采用Western blotting法检测Per1、Per2、Per3、Cry1、Cry2、Clock、Bmal1蛋白。取对数生长期的Huh7细胞,分为实验组和对照组,实验组加入50μmol/L的YC-1,对照组加入等量生理盐水,在乏氧条件下培养细胞,采用克隆形成实验观察两组克隆形成情况,计算克隆形成率。结果实验组细胞中Clock、Per1、Per3、Cry1 mRNA及蛋白相对表达量低于对照组,Bmal1、Per2、Cry2 mRNA及蛋白相对表达量高于对照组(P均<0.05)。实验组克隆形成个数为83个、克隆形成率为27.67%,对照组分别为191个、63.67%,实验组克隆形成个数及克隆形成率均低于对照组(P均<0.05)。结论 YC-1作用后,缺氧环境下的肝癌细胞中Clock、Per1、Per3、Cry1基因及蛋白表达下调,Bmal1、Per2、Cry2基因及蛋白表达上调,细胞增殖和克隆形成能力减弱。Objective To observe the effects of hypoxia inducible factor（ HIF） inhibitor YC-1 on the circadian clock genes,circadian clock proteins and cell proliferation of hepatocellular carcinoma（ HCC） cells. Methods The HCC Huh7 cells were cultured in the hypoxia incubator and then were divided into the experimental group and control group.Cells in the the experimental group were added with 50 μmol/L YC-1,and cells in the control group were added with DMSO. At 48 h after culture,the mRNA and protein of two groups were exacted,respectively. The real-time PCR was used to detect the Per1,Per2,Per3,Cry1,Cry2,Clock,and Bmal1 mRNA,while the Per1,Per2,Per3,Cry1,Cry2,Clock,and Bmal1 proteins were detected by Western blotting in cells of the two groups. The Huh7 cells in the logarithmic growth phase were divided into the experimental group and control group. YC-1（ 50 μmol/L） was added into the experimental group,while the same amount of 0. 9% Na Cl solution was added into the control group. Cells were cultured under hypoxic conditions. Clone formation experiment was used to observe the proliferation,and then the clone formation rate was calculated. Results The relative mRNA and protein expression levels of Clock,Per1,Per3,and Cry1 in the experimental group were lower than those in the control group,but the relative mRNA and protein expression levels of Bmal1,Per2,and Cry2 were higher than those in the control group（ all P 〈 0. 05）. The number of clones was 83 and the colony formation rate was 27. 67% in the experimental group,versus 191 and 63. 67% in the control group,respectively. The number of colony formation and colony formation rate in the experimental group were all lower than those in the control group（ all P 〈0. 05）. Conclusions After YC-1 treatment,the expression of Clock,Per1,Per3,and Cry1 genes and proteins is downregulated in the HCC cells,while the expression of Bmal1,Per2,and Cry2 genes is up-regulated,and the cell proliferation and colony forming abilities decrease.

关 键 词：缺氧诱导因子抑制剂YC-1 肝细胞癌 生物钟 生物钟基因 细胞增殖 

分 类 号：R735.7[医药卫生—肿瘤]