摘要
目的构建SREBP-1c基因的miRNA真核表达载体,观察其对内质网应激状态下L02和HepG2肝细胞脂质沉积的影响。方法设计并构建SREBP-1c基因的3个miRNA干扰载体,经测序鉴定miRNA载体构建成功后转染肝细胞,荧光显微镜下观察绿色荧光蛋白表达。Western blot检测转染后SREBP-1c的表达;甘油三酯测定和油红O染色检测细胞内脂质沉积;Western blot检测SREBP-1c下游脂代谢相关基因脂肪酸合成酶(fatty acid synthase,FAS)和乙酰辅酶A羧化酶(acetyl CoA carboxylase,ACC1)的表达。结果靶向干扰SREBP-1c的miRNA真核表达载体构建成功。Westernblot结果显示,转染SREBP-1c-1miRNA和SREBP-1c-3miRNA载体后,L02和HepG2细胞内SREBP-1c蛋白水平表达均明显减低(P<0.05),SREBP-1c-1miRNA的干扰效果最好(P<0.05)。与对照组相比,转染SREBP-1c-1miRNA载体后L02和HepG2肝细胞内甘油三酯含量均明显降低(P<0.05),细胞内脂滴明显减少;FAS和ACC1蛋白的表达明显降低(P<0.05)。结论 SREBP-1c基因沉默通过FAS/ACC1降低了内质网应激状态下肝细胞脂质合成。
determine the effect Objective To construct miRNA eukaryotic expression vectors targeting SREBP-lc and of SREBP-lc gene knockdown on lipid accumulation in L02 and HepG2 hepatocytes under endoplasmic reticulum stress. Methods Three miRNA targeting SREBP-lc gene, named SREBP-lc-I-miRNA, SREBP-1 c-2-miRNA and SREBP-1 c-3-miRNA, and a non-homologous negative control expression vector （HK- miRNA） were designed and constructed which were identified by DNA sequencing analysis. After 3 miRNA ex- pression vectors were transiently transfected into hepatocytes, fluorescence microscopy was applied to observe EGFP to confirm the transfection. Western blotting was used to determine expression of SREBP-1 c. Lipid depo- sition in each group was observed by triglyceride content detection and oil red O staining. Western blotting was also applied to measure the protein levels of fatty acid synthase （FAS） and acetyl CoA carboxylase （ACC1）. Results Four miRNA expression vectors were constructed successfully. After 24 hours＇ transfection, EGFP expression was visible under fluorescence microscope. The protein level of SREBP-lc was decreased significantly after transfected with SREBP-lc-lmiRNA and SREBP-lc-3miRNA, when compared with control group （P 〈 0. 05 ）. SREBP-1 c-1 miRNA was identified with most powerful interference （ P 〈 0.05 ）. Compared with the control group, TG content （P 〈 0. 05 ） and lipid droplet were significantly decreased, and FAS and ACC1 proteins were also inhibited in SREBP-lc inference group （P 〈0. 05）. Conclusion SREBP-le gene interference suppresses lipid synthesis through down-regulating FAS and ACC1 in L02 and HepG2 hepatocytes under endoplasmic reticulum stress.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2013年第6期513-517,共5页
Journal of Third Military Medical University
基金
国家自然科学基金青年科学基金(81000357)~~
关键词
非酒精性脂肪肝
SREBP-1C
FAS
内质网应激
RNA干扰
non-alcoholic fatty liver disease
SREBP-lc
fatty acid synthase
endoplasmic reticulumstress
RNA interference
