摘要
目的:研究miRNA干扰质粒对胰岛素样生长因子Ⅱ(IGF-Ⅱ)在肝细胞癌表达的抑制作用,探讨IGF-Ⅱ在肝细胞癌治疗中的价值.方法:以人IGF-Ⅱ基因序列设计并合成4条miRNA,将miRNA插入质粒构建pcDNATM6.2-GW/EmGFP miR1-4干扰载体;筛选、转染HepG2细胞,以荧光定量PCR分析靶向肝癌IGF-Ⅱ基因表达的干扰效果;以ELISA法比较转染前后IGF-Ⅱ蛋白表达水平.结果:测序证实,成功构建了真核IGF-Ⅱ干扰质粒MR-IGF-Ⅱ-1-4,将干扰质粒转染至HepG2细胞,镜下显示转染效率50%;经荧光定量PCR扩增,沉默IGF-Ⅱ基因表达效率MR-IGF-Ⅱ-1为33%、MR-IGF-Ⅱ-2为43%、MR-IGF-Ⅱ-3为0%、MR-IGF-Ⅱ-4为3%.将其中MR-IGF-Ⅱ-2转染至HepG2细胞,在转录和蛋白水平上对IGF-Ⅱ具有较高干扰效率,分别达43.0%和43.5%.结论:成功构建了人miRNA干扰质粒,他能有效抑制HepG2细胞IGF-Ⅱ的表达.
AIM：To investigate inhibitory effect of transfection of eukaryotic expression plasmids carrying specific microRNA（miRNA）on insulin-like growth factor II（IGF-II）gene and protein expression in human HepG2 cells.METHODS：Four miRNAs targeting the IGFII gene were synthesized and inserted into the pcDNA TM 6.2-GW/EmGFPmiR vector.The recombinant plasmids were identified and transiently transfected into HepG2 cells.Their inhibitory efficiency on IGF-II expression was determined at gene level by real time-PCR and at protein level by ELISA.RESULTS：Four eukaryotic expression plasmids carrying miRNA targeting IGF-II were constructed successfully and confirmed by sequencing.HepG2 cells were then transfected with these plasmids（MR-IGF-II-1 to 4）.The highest transfection efficiency was up to 50%.The reduced rates of IGF-II gene expression were 33%,43%,0%and 3%in cells transfected with MRIGF-II-1 to 4,respectively.Transfection of the MR-IGF-II-2 plasmid for 72 h reduced IGF-II protein expression by 44%in HepG2 cells.CONCLUSION：The expression plasmids carrying miRNA targeting IGF-II have been successfully constructed.Transfection of these plamsids can efficiently inhibit IGF-II expression in HepG2 cells.
出处
《世界华人消化杂志》
CAS
北大核心
2011年第10期1015-1021,共7页
World Chinese Journal of Digestology
基金
江苏省自然科学基金资助项目
No.BK2008187
南通大学自然科学基金资助项目
No.09ZY013~~
关键词
肝细胞癌
胰岛素样生长因子Ⅱ
微小RNA
质粒构建
基因沉默
Hepatocellular carcinoma
Insulin-like growth factor 2
microRNA
Plasmid construction
Gene silencing
