microRNA-210 MODIFIED HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS INDUCE CAPILLARY FORMATION_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LOU Yuanlei ¹ , GAO Faliang ² , XIE An ¹ , GUO Fei ¹ , DENG Zhifeng ² , WANG Yang. ¹

1Institute of Urology, Nanchang University,Nanchang Jiangxi, 330006, P.R.China;;
2Department of Neurosurgery, the Second Affiliated Hospital, Nanchang University.;

Corresponding author：

Keywords：

microRNA-210 Human umbilical vein endothelial cell Angiogenesis Lentiviral vector

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Objective To construct human recombinant lentiviral expression vector of microRNA-210 (miR-210)
and to explore the over-expression of miR-210 on the capillary formation in human umbilical vein endothelial cells 12 (HUVE-
12). Methods The recombinant lentiviral expression vector of pGCSIL-green fluorescent protein (GFP)-pre-miR-210 was
constructed by molecular cloning and transfected to HUVE-12 (LV-miR-210-GFP group), only pGCSIL-GFP was transfected
as control group (LV-GFP group). The miR-210 expression activity was evaluated by GFP reporter through fluorescence
detection and real-time fluorescent quantitative PCR. The ephrinA3 protein expression was measured by flow cytometry. The
concentration of vascular endothelial growth factor (VEGF) in culture supernatant was determined by ELISA. The cells were
cultured in 96-well culture plate coated with Matrigel to assess the abil ity of capillary formation. Results The recombinant
plasmid pGCSIL-GFP-pre-miR-210 was confirmed by restriction endonuclease analysis and DNA sequencing. Fluorescence
detection showed that the fluorescence intensity of GFP was highest between 48 and 72 hours after transfection. Real-time
fluorescent quantitative PCR showed that the miR-210 expression of LV-miR-210-GFP group was 9.72 times higher than that
in LV-GFP group (t= —11.10，P=0.00). Flow cytometry analysis showed that the positive cell rate of enphrinA3 in LV-miR-
210-GFP group (12.52% ± 0.67%) was significantly lower than that in LV-GFP group (73.22% ± 1.45%) (t= —66.12，P=0.00).
The concentration of VEGF in supernatant in LV-miR-210-GFP group was significantly higher than that in LV-GFP group
[(305.29 ± 16.52) pg/mL vs. (42.52 ± 3.11) pg/mL, t= —27.06，P=0.00]. In vitro capillary-l ike formation assay showed that the
number of capillaries was significantly larger in LV-miR-210-GFP group than in LV-GFP group (17.33 ± 6.33 vs. 6.33 ± 2.33,
t= —2.83，P=0.04). Conclusion The recombinant lentiviral expression vector of miR-210 is constructed successfully and
HUVE-12 over-expressing miR-210 can significantly increase the capillary formation, which facil itates further study on the
molecular functions of miR-210 in angiogenesis.

Citation： LOU Yuanlei,GAO Faliang,XIE An,GUO Fei,DENG Zhifeng,WANG Yang.. microRNA-210 MODIFIED HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS INDUCE CAPILLARY FORMATION. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(5): 587-591. doi: Copy

1.	Carmeliet P, Jain RK. Molecular mechanisms and clinical applications of angiogenesis. Nature, 2011, 473(7347): 298-307.
2.	Ribatti D. Novel angiogenesis inhibitors: addressing the issue of. redundancy in the angiogenic signaling pathway. Cancer Treat Rev, 2011, 37(5): 344-352.
3.	Liu D, Krueger J, Le Noble F. The role of blood flow and microRNAs in blood vessel development. Int J Dev Biol, 2011, 55(4-5): 419-429.
4.	van Solingen C, de Boer HC, Bijkerk R, et al. MicroRNA-126 modulates endothelial SDF-1 expression and mobilization of Sca-1 (+) /Lin (-) progenitor cells in ischaemia. Cardiovasc Res, 2011, 92(3): 449-455.
5.	Caporali A, Emanueli C. MicroRNA regulation in angiogenesis. Vascul Pharmacol, 2011, 55(4): 79-86.
6.	Anand S, Cheresh DA. MicroRNA-mediated regulation of the angiogenic Switch. Curr Opin Hematol, 2011, 18(3): 171-176.
7.	Tie J, Fan D. Big roles of microRNAs in tumorigenesis and tumor development. Histol Histopathol, 2011, 26(10): 1353-1361.
8.	Sato F, Tsuchiya S, Meltzer SJ, et al. MicroRNAs and epigenetics. FEBS J, 2011, 278(10): 1598-1609.
9.	Fasanaro P, D’alessandra Y, Di Stefano V, et al. MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3. J Biol Chem, 2008, 283(23): 15878-15883.
10.	Pillai RS, Bhattacharyya SN, Filipowicz W. Repression of protein synthesis by miRNAs: how many mechanisms? Trends Cell Biol, 2007, 17(3): 118-126.
11.	Lim LP, Lau NC, Garrett-Engele P, et al. Microarray analysis shows that some microRNAs downregulate large numbers of-target mRNAs. Nature, 2005, 433(7027): 769-773.
12.	Stark A, Brennecke J, Bushati N, et al. Animal MicroRNAs confer robustness to gene expression and have a significant impact on 3’UTR evolution. Cell, 2005, 123(6): 1133-1146.
13.	Ivan M, Harris AL, Martelli F, et al. Hypoxia response and microRNAs: no longer two separate worlds. J Cell Mol Med, 2008, 12(5A): 1426-1431.
14.	Kelly TJ, Souza AL, Clish CB, et al. A hypoxia-induced positive feedback loop promotes hypoxia-inducible factor 1alpha stability through miR-210 suppression of glycerol-3-phosphate dehydrogenase 1-like. Mol Cell Biol, 2011, 31(13): 2696-2706.
15.	Mutharasan RK, Nagpal V, Ichikawa Y, et al. microRNA-210 is upregulated in hypoxic cardiomyocytes through Akt- and p53-dependent pathways and exerts cytoprotective effects. Am J Physiol Heart Circ Physiol, 2011, 301(4): H1519-H1530.
16.	Shibuya M. Vascular endothelial growth factor-dependent and -independent regulation of angiogenesis. BMB Rep, 2008, 41(4): 278-286.
17.	Kuijper S, Turner CJ, Adams RH. Regulation of angiogenesis by Eph-ephrin interactions. Trends Cardiovasc Med, 2007, 17(5): 145-151.
18.	Byrne AM, Bouchier-Hayes DJ, Harmey JH. Angiogenic and cell survival functions of vascular endothelial growth factor (VEGF). J cell Mol Med, 2005, 9(4): 777-794.
19.	Hicklin DJ, Ellis LM. Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol, 2005, 23(5): 1011-1027.
20.	Kume T. Specification of arterial, venous, and lymphatic endothelial cells during embryonic development. Histol Histopathol, 2010, 25(5): 637-646.

1. Carmeliet P, Jain RK. Molecular mechanisms and clinical applications of angiogenesis. Nature, 2011, 473(7347): 298-307.
2. Ribatti D. Novel angiogenesis inhibitors: addressing the issue of. redundancy in the angiogenic signaling pathway. Cancer Treat Rev, 2011, 37(5): 344-352.
3. Liu D, Krueger J, Le Noble F. The role of blood flow and microRNAs in blood vessel development. Int J Dev Biol, 2011, 55(4-5): 419-429.
4. van Solingen C, de Boer HC, Bijkerk R, et al. MicroRNA-126 modulates endothelial SDF-1 expression and mobilization of Sca-1 (+) /Lin (-) progenitor cells in ischaemia. Cardiovasc Res, 2011, 92(3): 449-455.
5. Caporali A, Emanueli C. MicroRNA regulation in angiogenesis. Vascul Pharmacol, 2011, 55(4): 79-86.
6. Anand S, Cheresh DA. MicroRNA-mediated regulation of the angiogenic Switch. Curr Opin Hematol, 2011, 18(3): 171-176.
7. Tie J, Fan D. Big roles of microRNAs in tumorigenesis and tumor development. Histol Histopathol, 2011, 26(10): 1353-1361.
8. Sato F, Tsuchiya S, Meltzer SJ, et al. MicroRNAs and epigenetics. FEBS J, 2011, 278(10): 1598-1609.
9. Fasanaro P, D’alessandra Y, Di Stefano V, et al. MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3. J Biol Chem, 2008, 283(23): 15878-15883.
10. Pillai RS, Bhattacharyya SN, Filipowicz W. Repression of protein synthesis by miRNAs: how many mechanisms? Trends Cell Biol, 2007, 17(3): 118-126.
11. Lim LP, Lau NC, Garrett-Engele P, et al. Microarray analysis shows that some microRNAs downregulate large numbers of-target mRNAs. Nature, 2005, 433(7027): 769-773.
12. Stark A, Brennecke J, Bushati N, et al. Animal MicroRNAs confer robustness to gene expression and have a significant impact on 3’UTR evolution. Cell, 2005, 123(6): 1133-1146.
13. Ivan M, Harris AL, Martelli F, et al. Hypoxia response and microRNAs: no longer two separate worlds. J Cell Mol Med, 2008, 12(5A): 1426-1431.
14. Kelly TJ, Souza AL, Clish CB, et al. A hypoxia-induced positive feedback loop promotes hypoxia-inducible factor 1alpha stability through miR-210 suppression of glycerol-3-phosphate dehydrogenase 1-like. Mol Cell Biol, 2011, 31(13): 2696-2706.
15. Mutharasan RK, Nagpal V, Ichikawa Y, et al. microRNA-210 is upregulated in hypoxic cardiomyocytes through Akt- and p53-dependent pathways and exerts cytoprotective effects. Am J Physiol Heart Circ Physiol, 2011, 301(4): H1519-H1530.
16. Shibuya M. Vascular endothelial growth factor-dependent and -independent regulation of angiogenesis. BMB Rep, 2008, 41(4): 278-286.
17. Kuijper S, Turner CJ, Adams RH. Regulation of angiogenesis by Eph-ephrin interactions. Trends Cardiovasc Med, 2007, 17(5): 145-151.
18. Byrne AM, Bouchier-Hayes DJ, Harmey JH. Angiogenic and cell survival functions of vascular endothelial growth factor (VEGF). J cell Mol Med, 2005, 9(4): 777-794.
19. Hicklin DJ, Ellis LM. Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol, 2005, 23(5): 1011-1027.
20. Kume T. Specification of arterial, venous, and lymphatic endothelial cells during embryonic development. Histol Histopathol, 2010, 25(5): 637-646.

Chinese Journal of Reparative and Reconstructive Surgery

microRNA-210 MODIFIED HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS INDUCE CAPILLARY FORMATION

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