靶向HIV-1 pol的高效人工miRNA的构建与体外抗病毒能力评价

Construction and in vitro antiviral avcitvity of highly effective artifical miRNA targeting to HIV-1 pol

摘要:

RNAi技术在抗HIV-1治疗研究中已显示出巨大潜力，获得可高效特异抑制HIV-1的RNAi元件是进行相关研究的重要基础。miRNA在抑制和表达方式上相比siRNA具有更多的优势。本研究即探讨构建可高效特异靶向HIV-1的人工miRNA元件。选择以保守性较好的HIV-1 pol基因为靶区筛选高效保守的RNAi序列，设计了16个可靶向pol区高保守区段的RNAi靶点，构建表达载体与HIV-1感染性克隆进行共转染抑制实验，筛选显示pol1026序列兼具高保守性及高抑制效率特点。以天然miR-30a为基础骨架构建了靶向pol1026靶点的人工miRNA元件，通过与HIV-1感染性克隆质粒的共转染抑制实验验证获得了可有效抑制HIV-1表达的人工miRNA元件 (miR-1026E)。通过与携带靶序列的报告质粒的共转染实验证明miR-1026E具有良好的靶点特异性。本研究进一步构建了携带miR-1026E表达元件的重组慢病毒，转导MT-4细胞并对转导后细胞进行克隆化筛选，获得稳定整合miR-1026E表达元件的MT-4-miR1026E细胞克隆，该细胞在体外攻毒实验中可高效抑制HIV-1的复制，具有显著的抑制HIV-1的能力。同时应用实时RT-PCR方法检测显示，miR-1026E在细胞中不会影响内源性代表miRNA (miR-181与miR-16) 的表达水平和干扰素效应相关基因stat1的表达水平，具有良好的特异性。所获得的可特异高效抑制HIV-1复制的人工miRNA元件可为抗HIV-1研究提供重要参考。

Abstract:

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 stat1 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.