Jiang Xiao-Hua, Xie Yu-Tao, Jiang Bo, Tang Meng-Ying, Ma Tao, Peng Hua
Department of Infectious Diseases, Xiangya Hospital of Central South University, Changsha, Hunan 410087, P.R. China.
Department of Infectious Diseases, The First Affiliated Hospital of The University of South China, Hengyang, Hunan 421001, P.R. China.
Mol Med Rep. 2015 Aug;12(2):1905-13. doi: 10.3892/mmr.2015.3571. Epub 2015 Mar 31.
The present study aimed to evaluate the silencing effect of artificial microRNAs (amiRNAs) against the hepatitis C virus (HCV) 1b (HCV1b) genotype core (C) and non-structural protein 4B (NS4B) genes. pDsRed-monomer-Core and pDsRed-monomer-NS4B plasmids, containing the target genes were constructed. A total of eight artificial micro RNA (amiRNA)-expressing plasmids, namely, pmiRE-C-mi1 to -mi4 and pmiRE-NS4B-mi1 to -mi4, were designed and constructed to interfere with various sites of the core and NS4B genes, and the amiRNA interfering plasmid and the corresponding target gene-expressing plasmid were co-transfected into HepG2 cells. At 48 h after transfection, HCV core and NS4B gene expression levels were detected using fluorescence microscopy, flow cytometry, reverse transcription quantitative polymerase chain reaction and western blot analysis. Fluorescence microscopy revealed that the target gene-transfected cells expressed red fluorescent protein, whereas the interfering plasmid-transfected cells exhibited expression of green fluorescent protein. The percentage of red fluorescent proteins and mean fluorescence intensity, as well as protein expression levels of the core and NS4B genes within the cells, which were co-transfected by the amiRNA interfering plasmid and the target gene, were significantly decreased. The results of the present study confirmed that amiRNAs may effectively and specifically inhibit the expression of HCV1b core and NS4B genes in HepG2 cells, potentially providing a novel therapeutic strategy for the treatment of HCV.
本研究旨在评估人工微小RNA（amiRNA）对丙型肝炎病毒（HCV）1b型（HCV1b）核心（C）基因和非结构蛋白4B（NS4B）基因的沉默作用。构建了包含靶基因的pDsRed-单体-Core和pDsRed-单体-NS4B质粒。设计并构建了总共8种表达人工微小RNA（amiRNA）的质粒，即pmiRE-C-mi1至-mi4和pmiRE-NS4B-mi1至-mi4，以干扰核心基因和NS4B基因的各个位点，并将amiRNA干扰质粒和相应的靶基因表达质粒共转染至HepG2细胞中。转染后48小时，使用荧光显微镜、流式细胞术、逆转录定量聚合酶链反应和蛋白质印迹分析检测HCV核心基因和NS4B基因的表达水平。荧光显微镜显示，转染靶基因的细胞表达红色荧光蛋白，而转染干扰质粒的细胞则表达绿色荧光蛋白。amiRNA干扰质粒和靶基因共转染的细胞内红色荧光蛋白的百分比和平均荧光强度，以及核心基因和NS4B基因的蛋白质表达水平均显著降低。本研究结果证实，amiRNA可有效且特异性地抑制HepG2细胞中HCV1b核心基因和NS4B基因的表达，可能为丙型肝炎的治疗提供一种新的治疗策略。
