Effect of mineralocorticoid receptor gene silencing by RNA interference on proliferation and apoptosis of murine macrophage cell line RAW 264.7 |
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Authors: | JI Wen-jie HU Dao-chuan CHEN Xue-fen MA Yong-qiang LU Rui-yi ZHOU Xin WEI LU-qing |
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Institution: | 1.Department of Respirology and Critical Care Medicine,Affiliated Hospital, ;2.Armed Police Forces Institute for Cardiovascular Diseases, Logistics University of Chinese People’s Armed Police Forces, Tianjin 300162, China. |
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Abstract: | AIM: To establish stable knockdown of mineralocorticoid receptor (MR) expression through short hairpin RNA (shRNA)-mediated silencing in murine RAW 264.7 macrophages. METHODS: Stable MR silencing in RAW 264.7 cells was achieved by recombinant shRNA plasmid targeting murine MR gene via liposome-mediated transfection, followed by G418 selection. The efficacies of plasmid transfection and MR silencing in G418-resistant cells were verified by immunofluorescent microcopy and real-time PCR, respectively. Proliferative activity of MR-silencing cell line was analyzed by CCK-8 assay. Cell cycle and apoptosis were evaluated by flow cytometry. RESULTS: MR gene expression was down-regulated by 70% compared with the negative control (NC) plasmid transfection. In addition, MR-silencing cells exhibited lower proliferative activity compared with NC and wide type RAW 264.7 cells (P<0.05), along with reduced proliferation index of 31.0%±1.3% (P<0.05), compared with the wide type cells (37.2%±0.5%) and the NC cells (37.5%±1.6%). In resting state, the apoptotic rate in wide type, NC and MR-silencing cells were 2.18%±0.36%, 6.65%±0.81% and 7.70%±1.34%, respectively, and no statistical difference was observed between NC and MR-silencing cells (P>0.05). CONCLUSION: MR gene silencing inhibits the proliferation of RAW 264.7 macrophages, but has no obvious effect on the apoptosis of the resting state cells. |
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Keywords: | Receptors mineralocorticoid RNA interference RAW 264 7 cells Cell proliferation Apoptosis |
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