X Li1, M Lin1, Z Xie1, R Huang1, A F Chen2, W Jiang3. 1. Department of Cardiology, The Third Xiangya Hospital of Central South University, 410013, Changsha, Hunan, China. 2. Department of Cardiology, The Third Xiangya Hospital of Central South University, 410013, Changsha, Hunan, China. afychen@yahoo.com. 3. Department of Cardiology, The Third Xiangya Hospital of Central South University, 410013, Changsha, Hunan, China. jiangweihongdoc@163.com.
Abstract
BACKGROUND: Renalase is a novel secretory amino oxidase expressed in the kidney and heart. To study the protective mechanism of renalase in local heart tissue, we established a low-expression renalase model with lentivirus (LV)-mediated RNA interference technology. MATERIALS AND METHODS: Three renalase-targeting oligonucleotides were designed after analyzing the mRNA of renalase. LV particles were prepared with LV expression systems (using the Trono 3 plasmid component system), after which LV-RNLS-shRNAs and LV-NC-shRNA were transfected into H9C2 cells in different cell culture plates. The optimal oligonucleotide was screened by real-time PCR and Western blot. These techniques were also used to detect renalase gene expression in the heart tissue. RESULTS: In the cell screening experiment, the efficacy of the inhibition of renalase mRNA expression was 93.7 % and that of renalase protein expression was 83.1 % in H9C2 cells. When the oligonucleotide was injected into the pericardial cavities of the SD rats on the 10th day, it inhibited 63.9 % of the expression of renalase protein in the heart tissue. CONCLUSION: LV-RNLS-RNAi (19813-1) can be used to establish an optimal renalase low-expression model for further research on the renalase system.
BACKGROUND:Renalase is a novel secretory amino oxidase expressed in the kidney and heart. To study the protective mechanism of renalase in local heart tissue, we established a low-expression renalase model with lentivirus (LV)-mediated RNA interference technology. MATERIALS AND METHODS: Three renalase-targeting oligonucleotides were designed after analyzing the mRNA of renalase. LV particles were prepared with LV expression systems (using the Trono 3 plasmid component system), after which LV-RNLS-shRNAs and LV-NC-shRNA were transfected into H9C2 cells in different cell culture plates. The optimal oligonucleotide was screened by real-time PCR and Western blot. These techniques were also used to detect renalase gene expression in the heart tissue. RESULTS: In the cell screening experiment, the efficacy of the inhibition of renalase mRNA expression was 93.7 % and that of renalase protein expression was 83.1 % in H9C2 cells. When the oligonucleotide was injected into the pericardial cavities of the SD rats on the 10th day, it inhibited 63.9 % of the expression of renalase protein in the heart tissue. CONCLUSION: LV-RNLS-RNAi (19813-1) can be used to establish an optimal renalase low-expression model for further research on the renalase system.