摘要
目的:观察mi R-150对药物代谢酶CYP3A4的调控作用。方法:用不同浓度游离长链脂肪酸(FFA)刺激张氏肝细胞,使之成为脂肪变性肝细胞。通过荧光定量PCR和Western blot测定CYP3A4 m RNA和蛋白表达以及mi R-150表达。生物信息学分析并构建野生型CYP3A4 3'-UTR双荧光素酶报告载体,与mi R-150模拟物共转染。另外,在张氏肝细胞和脂肪变性肝细胞中分别转染mi R-150模拟物和抑制剂,检测CYP3A4 m RNA和蛋白表达。结果:张氏肝细胞经1 mmol/L FFA诱导24 h后,与对照组相比,脂肪变性肝细胞中CYP3A4 m RNA和蛋白表达均显著下降(P<0.05),mi R-150表达上升(P<0.05)。经生物信息学分析发现CYP3A4为mi R-150的靶基因,转染质粒和mi R-150模拟物后,荧光活性下降。mi R-150模拟物可使CYP3A4 m RNA表达呈浓度梯度下降(P<0.05),也可使CYP3A4蛋白表达下降;mi R-150抑制剂使CYP3A4 m RNA表达上升(P<0.05)。结论:mi R-150可直接与CYP3A4的3'-UTR结合,进而直接调控CYP3A4的表达。
AIM：To explore the role of mi R-150 in the regulation of CYP3 A4 and the underlying mechanism.METHODS：CYP3 A4 m RNA and protein expression were tested by RT-PCR and Western blot when the Chang liver cells were stimulated by FFA containing fatty acids free 1% BSA.Bioinformatics mi RNA databases were used to identify CYP3 A4-related mi RNAs and the wild-type CYP3 A43＇-UTR plasmids were constructed,mi R-150 and reporter plasmid were co-transfected into the Chang liver cells to test the expression ratio of the reported fluorescence and the correction fluorescence.Then the mi R-150 mimic were transfected into the Chang liver cells,while mi RNA inhibitor were transfected into the steatosis cells.RT-PCR and Western blot were used to detect CYP3 A4 m RNA and protein expression.RESULTS：The CYP3 A4 m RNA and protein level were statistically significantly decreased（P〈0.05）,while the mature mi R-150 levels were increased（P〈0.05） when the Chang liver cells were stimulated after 24 h by 1 mmol/L FFA.CYP3 A4 was found to be the target gene of mi R-150 with a bioinformatics analysis.Then CYP3 A4 m RNA level was significantly decreased（P〈0.05）.Meanwhile,the expression level of CYP3 A4 protein also decreased when the mi R-150 mimic were transfected into the Chang liver cells.But CYP3 A4 m RNA expression has no statistical significance（P = 0.071）in mi R-150 inhibitor group.CONCLUSION：The mi R-150 can combine with CYP3 A4 3＇-UTR directly,and can further regulate the expression of CYP3 A4 m RNA.
作者
刘莉
彭金富
郭成贤
阳喜定
李海刚
阳国平
LIU Li;PENG Jinfu;GUO Chengxian;YANG Xiding;LI Haigang;YANG Guoping(Department of Pharmacy,Changsha Medical University,Changsha 410200,Hunan,China;Department of Pharmacy,the Third Xiangya Hospital,Central South University,Changsha 410013,Hunan,China;Department of Pharmacy,the Second Xiangya Hospital,Central South University,Changsha 410011,Hunan,China)
出处
《中国临床药理学与治疗学》
CAS
CSCD
2018年第7期749-754,共6页
Chinese Journal of Clinical Pharmacology and Therapeutics
基金
湖南省教育厅科学研究项目资助(17C0169)
湖南省教育厅科学研究项目资助(15C0160)
国家自然科学基金(81373476)
新型药物制剂研发湖南省重点实验室培育基地资助(2016TP1029)
