Cheng Tong, Zhang Tao, Zhang Yali, Wei Lihua, Xia Dezhen, Wang Yingbin, Zhang Jun, Xia Ningshao
National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Sciences, Xiamen University, Xiamen 361005, China.
Sheng Wu Gong Cheng Xue Bao. 2010 Jan;26(1):63-73.
RNA interference (RNAi) has exhibited huge potentials on anti-HIV-1 therapy research. The obtainment of RNAi element targeting to HIV-1 highly effectively and specifically was crucial for relevant research. Recent reports had described that microRNAs (miRNAs) posses more characteristics of inhibition and expression mechanisms than small interfering RNAs (siRNAs). In this study we explored the construction of artificial miRNA targeting to HIV-1 effectively and specifically. Sixteen siRNAs sequences were selected based on the conserved regions in the HIV-1 pol gene. ShRNA expression vectors were co-transfected with HIV-1 clone pNL4-3 to evaluate the abilities of siRNAs to inhibit HIV-1 expression. The pol1026 sequence was selected from candidates. The target sequence in the stem-loop structure of the well-characterized native miR-30a was replaced with pol1026 sequences, and the artificial miRNA expression vectors were co-transfected with the HIV-1 clone pNL4-3, results showed that HIV-1 can be effectively inhibited by miR-1026E. Target specificity of miR-1026E was confirmed by co-transfection assay with reporter plasmids containing different target sequences. The miR-1026E expression element was then inserted into Lentivirus which was used as a vector to transduce the MT-4 cells, MT-4-miR1026E expressing miR-1026E stably was cloned from transduced cells. The MT-4-miR1026E cell effectively inhibited HIV-1 replication in vitro. And the intracellular miR-181 and miR-16 expression levels and statl mRNA levels were not affected by the expression of miR-1026E in MT-4-miR1026E cells. miR-1026E is a promising candidate for future research.
RNA干扰（RNAi）在抗HIV-1治疗研究中展现出了巨大潜力。高效且特异性地获得靶向HIV-1的RNAi元件对于相关研究至关重要。近期报道称，与小干扰RNA（siRNA）相比，微小RNA（miRNA）具有更多的抑制特性和表达机制。在本研究中，我们探索了有效且特异性靶向HIV-1的人工miRNA的构建。基于HIV-1 pol基因的保守区域选择了16条siRNA序列。将短发夹RNA（shRNA）表达载体与HIV-1克隆pNL4-3共转染，以评估siRNA抑制HIV-1表达的能力。从候选序列中选择了pol1026序列。将特征明确的天然miR-30a茎环结构中的靶序列替换为pol1026序列，并将人工miRNA表达载体与HIV-1克隆pNL4-3共转染，结果表明miR-1026E可有效抑制HIV-1。通过与含有不同靶序列的报告质粒共转染实验证实了miR-1026E的靶标特异性。然后将miR-1026E表达元件插入慢病毒中，用作转导MT-4细胞的载体，从转导细胞中克隆出稳定表达miR-1026E的MT-4-miR1026E细胞。MT-4-miR1026E细胞在体外有效抑制了HIV-1复制。并且MT-4-miR1026E细胞中miR-1026E的表达未影响细胞内miR-181和miR-16的表达水平以及statl mRNA水平。miR-1026E是未来研究的一个有前景的候选对象。
