Feasibility analysis of using MPC30-DEA70 to transfect microRNA-21 AS-ODN into cardiac fibroblasts

QU Xiao-ya; LI Yan-ru; ZHANG Qiong; ZHANG Zhuo-qi

doi:10.3969/j.issn.1002-266X.2015.25.001

Abstract：Objective To investigate whether a cationic phosphorylcholine polymer MPC 30-DEA70 ( PC polymer ) can transfect the microRNA-21 antisense oligonucleotide ( AS-ODN) into the rat cardiac fibroblasts in vitro successfully , and then to observe the effects of MPC30-DEA70/microRNA-21ODN complexes on the expression of pre-microRNA-21 and the inhibitory effects on downstream products in rat cardiac fibroblasts after transfection .Methods MPC30-DEA70 was com-plexed with microRNA-21ODN at different N∶P ratios.Cardiac fibroblasts from neonatal Sprague Dawley (SD) rats were i-solated by trypsin digestion and cultured by differential attachment methods .The distribution of MPC 30-DEA70/microRNA-21ODN complexes in the cells was observed by fluorescence microscopy ( FM) and confocal laser scanning microscopy (CLSM).The expression of ERK-MAP kinase and collagenⅠproteins and pre-microRNA-21 was detected by Western blot-ting and RT-PCR.Results Under the inverted microscope , the primary cultured cardiac fibroblasts appeared irregular tri-angle and polygon, and oval nuclei in the middle.The transfection efficiency of MPC30-DEA70/microRNA-21ODN comple-xes was high in the cardiac fibroblasts, and was in a dosage-dependent manner (all P<0.05).From the CLSM images, most of the complexes located around the cell nucleus and a few were in inside of the nucleus .When MPC30-DEA70 trans-fected the microRNA-21ODN into the cardiac fibroblasts , the expression of pre-microRNA-21, ERK-MAP kinase and colla-genⅠproteins was significantly down-regulated (all P<0.05).Conclusions MPC30-DEA70 may transfect the microRNA-21ODN into the cardiac fibroblasts in vitro successfully , and the expression of microRNA-21, ERK-MAP kinase and colla-genⅠproteins is significantly down-regulated.Thus, MPC30-DEA70 can work as an effective transgenic vector .