Chen Yong, Mahato Ram I
Huaian 4th People's Hospital, Jiangsu, PR China.
J Drug Target. 2008 Feb;16(2):140-8. doi: 10.1080/10611860701878750.
The main objective was to determine whether a pool of small interfering RNAs (siRNAs) targeting different regions of hepatitis B virus surface antigen (HBsAg) efficiently inhibits hepatitis B virus (HBV) infection. siRNAs targeting different regions of HBsAg were transfected into HBV-producing HepG2.2.15 cells and at 72 h post-transfection, the culture medium was collected for ELISA to determine HBsAg, while total RNA was isolated from the cells for real-time PCR. Three siRNA sequences that efficiently inhibited HBV infection were converted into small hairpin RNAs (shRNAs) and then cloned into a single plasmid psiSTRIKE driven by a single U6 promoter. These shRNA expressing plasmids were tested for HBsAg gene silencing in HepG2.2.15 cells. A pool of siRNAs targeting HBsAg efficiently inhibited HBV replication and antigen expression when transfected into HepG2.2.15 cells, compared with the use of single siRNA. Similarly, the plasmid encoding three different shRNAs driven by a single U6 promoter was more effective in silencing HBsAg at DNA, mRNA and protein levels compared with the plasmid encoding single shRNA. No apoptotic change was observed in the cells when the plasmid was transfected at a dose of 0.5-2 microg/1 x 10(6) cells after complex formation with Lipofectamine LTX. Furthermore, transfection with siRNA or shRNA did not increase interferon-gamma (IFNs-gamma) release, suggesting no induction of IFN response. In conclusion, a pool of chemically synthesised siRNAs as well as the shRNA expression plasmid encoding multiple shRNAs targeting different regions of HBsAg showed high gene silencing in HepG2.2.15 cells.
主要目的是确定一组靶向乙型肝炎病毒表面抗原（HBsAg）不同区域的小干扰RNA（siRNA）是否能有效抑制乙型肝炎病毒（HBV）感染。将靶向HBsAg不同区域的siRNA转染到产生HBV的HepG2.2.15细胞中，转染后72小时，收集培养基进行ELISA以测定HBsAg，同时从细胞中分离总RNA用于实时PCR。将三个有效抑制HBV感染的siRNA序列转化为小发夹RNA（shRNA），然后克隆到由单个U6启动子驱动的单个质粒psiSTRIKE中。检测这些表达shRNA的质粒在HepG2.2.15细胞中对HBsAg基因的沉默作用。与使用单个siRNA相比，一组靶向HBsAg的siRNA转染到HepG2.2.15细胞后能有效抑制HBV复制和抗原表达。同样，与编码单个shRNA的质粒相比，由单个U6启动子驱动的编码三种不同shRNA的质粒在DNA、mRNA和蛋白质水平上对HBsAg的沉默效果更显著。当质粒与Lipofectamine LTX形成复合物后以0.5 - 2μg/1×10⁶细胞的剂量转染时，未观察到细胞凋亡变化。此外，用siRNA或shRNA转染不会增加干扰素-γ（IFN-γ）的释放，表明未诱导IFN反应。总之，一组化学合成的siRNA以及编码靶向HBsAg不同区域的多个shRNA的shRNA表达质粒在HepG2.2.15细胞中显示出高效的基因沉默作用。
