Tao Jin, Ding Wei-Feng, Che Xiao-Hang, Chen Yi-Chen, Chen Fang, Chen Xiao-Dong, Ye Xiao-Lei, Xiong Su-Bin
College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou, Zhejiang 310032, P.R. China.
Medical Center Laboratory, Affiliated Hospital of Nantong University, Nantong, Jiangsu 226000, P.R. China.
Int J Mol Med. 2016 May;37(5):1345-54. doi: 10.3892/ijmm.2016.2530. Epub 2016 Mar 17.
In order to improve the delivery efficiency of microRNA (miRNA or miR)-145, the present study examined several factors which may affect cationic liposome (CL)-based transfection, including the hydration medium used for the preparation of liposomes, the quantity of the plasmid, the molar ratio of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP)/cholesterol (chol), or DOTAP/chol, and the weight ratio of DOTAP/DNA. In order to enhance the transfection efficiency, protamine was selected as a DNA-condensing agent to form liposome‑protamine‑DNA (LPD) ternary complexes. An agarose gel retardation assay was used to examine the DNA binding affinity of the CLs. Following transfection, GFP fluorescence images were captured and flow cytometry was performed to determine the transfection efficiency. Furthermore, an MTT assay was performed to determine the cytotoxicity of the liposome complexes. The final optimal conditions were as follows: 5% glucose as the hydration medium, a molar ratio of DOTAP/chol at 3:1 for the preparation of CLs, a weight ratio of DOTAP/protamine/DNA of 3:0.5:1, with 8 µg plasmid added for the preparation of the LPD complexes. In vitro, the LPD complexes exhibited an enhanced transfection efficiency and low cytotoxicity, which indicated that the presented LPD vector enhanced the transfection efficiency of the CLs. The HepG2 cells were found to have the lowest expression levels of miR‑145 out of the cell lines tested (A549, BGC-823, HepG2, HeLa, LoVo and MCF-7). Following the transient transfection of the HepG2 cells with miR‑145, the results revealed that the overexpression of miR‑145 inhibited the proliferation of the HepG2 cells and downregulated the expression of cyclin-dependent kinase 6 (CDK6), cyclinD1, c-myc, and Sp1 transcription factor (Sp1). In conclusion, in this study, we optimized a liposome‑based delivery system for the efficient delivery of miR‑145 into cancer cells. This may provide a foundation for further research into the use of miR‑145 in anticancer therapeutics.
为了提高微小RNA（miRNA或miR）-145的递送效率，本研究考察了几个可能影响基于阳离子脂质体（CL）的转染的因素，包括用于制备脂质体的水化介质、质粒的量、N-[1-(2,3-二油酰氧基)丙基]-N,N,N-三甲基氯化铵（DOTAP）/胆固醇（chol）的摩尔比，即DOTAP/chol，以及DOTAP/DNA的重量比。为了提高转染效率，选择鱼精蛋白作为DNA凝聚剂以形成脂质体-鱼精蛋白-DNA（LPD）三元复合物。采用琼脂糖凝胶阻滞试验检测CLs与DNA的结合亲和力。转染后，采集绿色荧光蛋白（GFP）荧光图像并进行流式细胞术检测以确定转染效率。此外，进行MTT试验以确定脂质体复合物的细胞毒性。最终的最佳条件如下：5%葡萄糖作为水化介质，制备CLs时DOTAP/chol的摩尔比为3:1，DOTAP/鱼精蛋白/DNA的重量比为3:0.5:1，制备LPD复合物时加入8 μg质粒。在体外，LPD复合物表现出增强的转染效率和低细胞毒性，这表明所呈现的LPD载体提高了CLs的转染效率。在所测试的细胞系（A549、BGC-823、HepG2、HeLa、LoVo和MCF-7）中，发现HepG2细胞中miR-145的表达水平最低。用miR-145瞬时转染HepG2细胞后，结果显示miR-145的过表达抑制了HepG细胞的增殖，并下调了细胞周期蛋白依赖性激酶6（CDK6）、细胞周期蛋白D1、c-myc和Sp1转录因子（Sp1）的表达。总之，在本研究中，我们优化了一种基于脂质体的递送系统，用于将miR-145高效递送至癌细胞中。这可能为进一步研究miR-145在抗癌治疗中的应用提供基础。
