Uncoupling protein 2 variants and cell proliferation and apoptosis of human umbilical vein endothelial cells_Chinese Journal of Ocular Fundus Diseases

Authors：

ShenYinchen , ChenFeng'e , SunTao , GuQing , LiuKun , ZhengZhi , ChenYihui ,  WangNing , XuXun

Department of Ophthalmology, Shanghai First People's Hospital, Shanghai Jiaotong University, Shanghai 200080, China;

Corresponding author：

WangNing, Email: drwangning@126.com

Keywords：

Endothelial cells/pathophysiology; Cell proliferation; Apoptosis; Gene expression; Lentivirus infections

DOI：

10.3760/cma.j.issn.1005-1015.2017.01.014

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Objective To observe the influences of uncoupling protein 2 (UCP-2) rs660339 variants transfection on cell proliferation and apoptosis of human umbilical vein endothelial cell (HUVEC). Methods Two UCP-2 green fluorescent protein (GFP) lentivirus constructs were created with the rs660339 locus carried C or T (UCP-2C or UCP-2T), respectively. HUVEC were cultured after lentiviral infection of UCP-2C or UCP-2T. The expression of UCP-2C or UCP-2T was detected with real time polymerase chain reaction. Cell proliferation and cell apoptosis were compared among negative control (NC) group, UCP-2T group and UCP-2C group using CCK-8 cell viability and flow cytometry. Western blot and immunostaining were employed to examine the expression of Bcl-2 gene. Results The lentivirus constructs were successfully created. ＞80% of the transfected cells were found to express GFP under fluorescent microscope. The mRNA levels of UCP-2 gene were significantly increased (F=29.183,P=0.001) in the UCP-2T group and UCP-2C group. The CCK-8 assay revealed that on day two (F=15.970,P=0.004), day three (F=16.738,P=0.004), day four (F=5.414,P=0.045) post-infection, UCP-2T and UCP-2C group showed significantly greater proliferation than the NC cells. The apoptotic rate in the UCP-2T and UCP-2C group was significantly lower than NC group (F=277.138,P=0.000), and the apoptotic rate of UCP-2T was significantly lower than that of UCP-2C (P=0.003). The protein levels of Bcl-2 in the UCP-2T and UCP-2C group were significantly greater than that in the NC group (F=425.679,P=0.000), and the Bcl-2 expression of UCP-2T was greater than that of UCP-2C (P=0.002). The Bcl-2 density in the UCP-2T and UCP-2C group were greater than that in the NC group (F=11.827,P=0.008), while there was no difference between UCP-2T and UCP-2C group (P=0.404). Conclusion The variants of UCP-2 rs660339 may influence HUVEC proliferation and apoptosis, and UCP-2T showed a stronger effect of inhibiting apoptosis than UCP-2C.

Citation： ShenYinchen, ChenFeng'e, SunTao, GuQing, LiuKun, ZhengZhi, ChenYihui, WangNing, XuXun. Uncoupling protein 2 variants and cell proliferation and apoptosis of human umbilical vein endothelial cells. Chinese Journal of Ocular Fundus Diseases, 2017, 33(1): 52-56. doi: 10.3760/cma.j.issn.1005-1015.2017.01.014 Copy

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1. 王宁, 许迅. 糖尿病视网膜病变相关基因多态性研究进展[J]. 中华眼科杂志, 2006, 6(42): 563-566. DOI: 10.3760/j.issn.0412-4081.2006.06.024.Wang N, Xu X. Recent advances in the studies of genetic polymorphisms in diabetic retinopathy[J].Chin J Ophthalmol,2006, 6(42): 563-566. DOI: 10.3760/j.issn.0412-4081.2006.06.024.
2. Lee HJ, Ryu HJ, Shin HD, et al. Associations between polymorphisms in the mitochondrial uncoupling proteins (UCPs) with T2DM[J]. Clin Chim Acta, 2008, 398(1-2): 27-33. DOI: 10.1016/j.cca.2008.07.029.
3. Skulachev VP. Uncoupling: new approaches to an old problem of bioenergetics[J]. Biochim Biophys Acta, 1998, 1363(2): 100-124.
4. Duval C, Negre-Salvayre A, Dogilo A, et al. Increased reactive oxygen species production with antisense oligonucleotides directed against uncoupling protein 2 in murine endothelial cells[J]. Biochem Cell Biol, 2002, 80(6): 757-764.
5. Yu X, Jacobs DR Jr, Schreiner PJ, et al. The uncoupling protein 2 Ala55Val polymorphism is associated with diabetes mellitus: the CARDIA study[J]. Clin Chem, 2005, 51(8): 1451-1456.
6. Qin LJ, Wen J, Qu YL, et al. Lack of association of functional UCP2 2866G/A and Ala55Val polymorphisms and type 2 diabetes in the Chinese population based on a case-control study and a meta-analysis[J]. Genet Mol Res, 2013, 12(3): 3324-3334. DOI: 10.4238/2013.September.3.9.
7. Oktavianthi S, Trimarsanto H, Febinia CA, et al. Uncoupling protein 2 gene polymorphisms are associated with obesity[J]. Cardiovasc Diabetol, 2012, 11: 41. DOI: 10.1186/1475-2840-11-41.
8. Gioli-Pereira L, Santos PC, Sugaya LS, et al. Association between UCP2 A55V polymorphism and risk of cardiovascular events in patients with multi-vessel coronary arterial disease[J]. BMC Med Genet, 2013, 14: 40. DOI: 10.1186/1471-2350-14-40.
9. Shen Y, Wen Z, Wang N, et al. Investigation of variants in UCP2 in Chinese type 2 diabetes and diabetic retinopathy[J/OL]. PLoS One, 2014, 9(11): 112670 [2014-11-14]. 10.1371/journal.pone.0112670">http://dx.doi.org/10.1371/ journal.pone.0112670. DOI: 10.1371/journal.pone.0112670.
10. 朱宝芹, 韩冠英, 郭斌.人脐静脉内皮细胞的培养与鉴定[J]. 中国公共卫生, 2006, 22(6): 742-743.Zhu BQ, Han GY, Guo B. The culture and identification of human umbilical vein endothelial cells[J]. Chinese Journal of Public Health, 2006, 22(6): 742-743.
11. Dal Monte M, Rezzola S, Cammalleri M, et al. Antiangiogenic effectiveness of the urokinase receptor-derived peptide UPARANT in a model of oxygen-induced retinopathy[J]. Invest Ophthalmol Vis Sci, 2015, 56(4):2392-2407. DOI: 10.1167/iovs.14-16323.
12. Liegl R, Koenig S, Siedlecki J, et al. Temsirolimus inhibits proliferation and migration in retinal pigment epithelial and endothelial cells via mTOR inhibition and decreases VEGF and PDGF expression[J/OL]. PLoS One, 2014, 9(2): 88203 [2014-02-26]. DOI: 10.1371/journal.pone.0088203. http://dx.doi.org/ 10.1371/journal.pone.0088203.
13. He Y, Wang N, Shen Y, et al. Inhibition of high glucose induced apoptosis by uncoupling protein 2 in human umbilical vein endothelial cells[J]. Int J Mol Med, 2014, 33(5): 1275-1281. DOI: 10.3892/ijmm.2014.1676.

Chinese Journal of Ocular Fundus Diseases

Uncoupling protein 2 variants and cell proliferation and apoptosis of human umbilical vein endothelial cells

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