Abstract
Purpose
Geminin has been correlated with vascular smooth muscle cell (VSMC) proliferation, but its mechanism is unclear. We selectively silenced the geminin gene of rat VSMCs by using RNAi technology and examined how geminin regulated VSMC proliferation.
Methods
By using RNA interference in A10 cells and flow cytometry, 3H-thymidine and 5-ethynyl-2’-deoxyuridine (EdU) measurements were used to detect VSMC proliferation. We performed a Western blot, polymerase chain reaction, and immunohistochemistry to detect the expression and location of geminin and cyclin-dependent kinase-1 (CDK1) in VSMCs.
Results
Silencing geminin significantly increased 3H-thymidine and EdU incorporation in VSMCs. We observed a significant increase in 3H-thymidine incorporation 24 h after a serum challenge in the geminin-RNAi-lentiviral vector group (4401.38 ± 438.39 cpm/mg), versus the non-targeting geminin-lentiviral vector (2836.88 ± 476.18 cpm/mg) and control groups (3069.50 ± 508.18 cpm/mg; P < 0.05). In the geminin-RNAi-lentiviral vector group, the EdU-positive cell rate was significantly increased (0.75 ± 0.03; P < 0.05), versus the non-targeting geminin-lentiviral vector (0.41 ± 0.0) or control group (0.40 ± 0.03). Geminin promoted VSMC proliferation, accelerating G0/G1-S cell-cycle progression (G0/G1 cells, 10 % decrease; S-phase cells, approximate 6 % increase) 12 h after serum withdrawal. Both CDK1 protein and mRNA expression were significantly increased in the positive group versus the controls. The immunofluorescence and co-immunoprecipitation results revealed a close interaction existed between CDK1 and the geminin gene in VSMC proliferation.
Conclusions
Geminin gene inhibition could augment VSMC proliferation by increasing CDK1 expression; thus, geminin may be a potential target for treating vascular diseases, specifically VSMCs.
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Acknowledgments
This study was supported by the National Natural Science Foundation of China (No. 30971228). The funder had no role in the study design, data collection, analysis, decision to publish, or preparation of the manuscript. We declare there was no commercial, proprietary, or financial interest in the products or companies described in this article.
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The authors declare that they have no conflict of interest.
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Yuanyuan Zhang and Zhouqin Jiang contributed equally to this work.
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Zhang, Y., Jiang, Z., Li, L. et al. Geminin Interference Facilitates Vascular Smooth Muscle Cell Proliferation by Upregulation of CDK-1. Cardiovasc Drugs Ther 28, 407–414 (2014). https://doi.org/10.1007/s10557-014-6550-9
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DOI: https://doi.org/10.1007/s10557-014-6550-9