title: Optimization of a cationic liposome-based gene delivery system for the application of miR-145 in anticancer therapeutics.
abstract: 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.
authors.affiliation: College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou, Zhejiang 310032, P.R. China.
authors.forename: Jin
authors.initials: J
authors.lastname: Tao
authors.affiliation: Medical Center Laboratory, Affiliated Hospital of Nantong University, Nantong, Jiangsu 226000, P.R. China.
authors.forename: Wei-Feng
authors.initials: WF
authors.lastname: Ding
authors.affiliation: Division of Medicine and Pharmacology, Ningbo Institute of Medical Sciences, Ningbo University, Ningbo, Zhejiang 315020, P.R. China.
authors.forename: Xiao-Hang
authors.initials: XH
authors.lastname: Che
authors.affiliation: Division of Medicine and Pharmacology, Ningbo Institute of Medical Sciences, Ningbo University, Ningbo, Zhejiang 315020, P.R. China.
authors.forename: Yi-Chen
authors.initials: YC
authors.lastname: Chen
authors.affiliation: College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou, Zhejiang 310032, P.R. China.
authors.forename: Fang
authors.initials: F
authors.lastname: Chen
authors.affiliation: Division of Medicine and Pharmacology, Ningbo Institute of Medical Sciences, Ningbo University, Ningbo, Zhejiang 315020, P.R. China.
authors.forename: Xiao-Dong
authors.initials: XD
authors.lastname: Chen
authors.affiliation: Division of Medicine and Pharmacology, Ningbo Institute of Medical Sciences, Ningbo University, Ningbo, Zhejiang 315020, P.R. China.
authors.forename: Xiao-Lei
authors.initials: XL
authors.lastname: Ye
authors.affiliation: College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou, Zhejiang 310032, P.R. China.
authors.forename: Su-Bin
authors.initials: SB
authors.lastname: Xiong
mesh_terms: D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006801:Humans; D008081:Liposomes / Q000737:chemistry / Q000633:toxicity; D035683:MicroRNAs / Q000008:administration & dosage / Q000235:genetics*; D053758:Nanoparticles; D010316:Particle Size; D034622:RNA Interference; D014162:Transfection
chemical_list: D008081:Liposomes; C541010:MIRN145 microRNA, human; D035683:MicroRNAs