microRNA-203 INDUCING DIFFERENTIATION OF HUMAN EPIDERMAL STEM CELLS INTO SWEAT GLAND CELLS IN VITRO_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

SONGZhifang ¹ ,  LIUDewu ² , PENGYan ² , LIJin ² , ZHANGZhiwei ² , NINGPu ² , LIUYifeng ²

1. Department of Cardiovascular Medicine, the First Affiliated Hospital of Nanchang University, Nanchang Jiangxi, 330006, P. R. China;
2. Department of Burns Center, the First Affiliated Hospital of Nanchang University, Nanchang Jiangxi, 330006, P. R. China;

Corresponding author：

LIUDewu, Email: dewuliu@126.com

Keywords：

Epidermal stem cells; Sweat gland cells; Differentiation; microRNA-203; P63

DOI：

10.7507/1002-1892.20150073

Video：

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Objective To observe the possibility and mechanism of microRNA (miRNA)-203 inducing the human epidermal stem cells to differentiate into sweat gland cells. Methods Five normal human foreskin tissues were harvested to prepare a single cell suspension by 0.25% trypsin-EDTA digestion method, then the human epidermal stem cells were isolated and cultured by type IV collagen differential adherent method. The cell morphology was observed by inverted phase contrast microscope. The monoclonal antibodies of integrin β1 (ITGB1), cytokeratin19 (CK19), CK1, CK10, CK18, and carcinoembryonic antigen (CEA) were used for identification by immunocytochemical staining. Double stranded mimics of has-miR-203 were transfected into the human epidermal stem cells with Lipofectamine 2000 (experimental group) and the human epidermal stem cells transfected with nonsense miRNA mimics served as control group. The monoclonal antibodies of ITGB1, CK19, CK1, CK10, CK18, and CEA were used for identifying the cells after transfection by immunocytochemical staining; the mRNA relative expressions of miRNA-203, P63, ITGB1, CK19, CK1, CK10, CK18, and CEA were detected by real-time fluorescence quantitative RT-PCR before transfected and at 72 hours after transfected. The protein relative expressions of P63, ITGB1, CK19, CK1, CK10, CK18, and CEA were detected by Western blot. The mRNA expression of miRNA-203 and the mRNA and protein expressions of P63 were analyzed respectively with Pearson correlation. Results The CK19 and ITGB1 were positively expressed before transfection, but CK1, CK10, CK18, and CEA were expressed positively after transfection. The mRNA relative expression of miRNA-203 after transfection in experimental group was significantly higher than that before transfection (P<0.05). The mRNA and protein relative expressions of CK1, CK10, CK18, and CEA after transfection in experimental group were significantly higher than those before transfection and control group (P<0.05), while the mRNA and protein relative expressions of P63, CK19, and ITGB1 were significantly lower than those before transfection and control group (P<0.05). These indicators showed no significant difference between the control group and before transfection (P>0.05). The expression level of miRNA-203 was negatively correlated with the mRNA and protein relative expressions of P63 before and after transfection, the correlation coefficients before transfection were -0.91 (t=3.862, P=0.042) and -0.96 (t=5.971, P=0.009) respectively; the correlation coefficients after transfection were -0.92 (t=4.283, P=0.031) and -0.95 (t=5.842, P=0.011) respectively. Conclusion miRNA-203 can induce epidermal stem cells to differentiate into sweat gland cells by targeting inhibition of P63 probably.

Citation： SONGZhifang, LIUDewu, PENGYan, LIJin, ZHANGZhiwei, NINGPu, LIUYifeng. microRNA-203 INDUCING DIFFERENTIATION OF HUMAN EPIDERMAL STEM CELLS INTO SWEAT GLAND CELLS IN VITRO. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(3): 343-350. doi: 10.7507/1002-1892.20150073 Copy

1.	Tao R, Sun TJ, Han YQ, et al. Epimorphin-induced differentiation of human umbilical cord mesenchymal stem cells into sweat gland cells. Eur Rev Med Pharmacol Sci, 2014, 18(9):1404-1410.
2.	宋志芳, 刘德伍. 微小RNA在皮肤发育再生及创面愈合中的作用. 中国修复重建外科杂志, 2014, 28(7):913-918.
3.	Suh HN, Han HJ. Sonic hedgehog increases the skin wound-healing ability of mouse embryonic stem cells through the microRNA200 family. Br J Pharmacol, 2015, 172(3):815-828.
4.	Yi R, Poy MN, Stoffel M, et al. A skin microRNA promotes differentiation by repressing ‘stemness’. Nature, 2008, 452(7184):225-229.
5.	Wei T, Orfanidis K, Xu N, et al. The expression of microRNA-203 during human skin mor phogenesis. Exp Dermatol, 2010, 19(9):854-856.
6.	Nissan X, Denis JA, Saidani M, et al. miR-203 modulates epithelial differentiation of human embryonic stem cells towards epidermal stratification. Dev Biol, 2011, 356(2):506-515.
7.	Yang RH, Xie JL, Shu B, et al. An improved method for the isolation and culture of rat epidermal stem cells. Int J Clin Exp Pathol, 2013, 6(11):2529-2534.
8.	Plichta JK, Droho S, Curtis BJ, et al. Local burn injury impairs epithelial permeability and antimicrobial peptide barrier function in distal unburned skin. Crit Care Med, 2014, 42(6):e420-431.
9.	Stollery N. Sebaceous and sweat gland disorders. Practitioner, 2013, 257(1757):32-33.
10.	Chuong CM, Randall VA, Widelitz RB, et al. Physiological regeneration of skin appendages and implications for regenerative medicine. Physiology (Bethesda), 2012, 27(2):61-72.
11.	Wang Y, Liu ZY, Zhao Q, et al. Future application of hair follicle stem cells:capable in differentiation into sweat gland cells. Chin Med J (Engl), 2013, 126(18):3545-3552.
12.	Yang S, Ma K, Feng C, et al. Capacity of human umbilical cord-derived mesenchymal stem cells to differentiate into sweat gland-like cells:a preclinical study. Front Med, 2013, 7(3):345-353.
13.	Gkegkes ID, Aroni K, Agrogiannis G, et al. Expression of caspase-14 and keratin-19 in the human epidermis and appendages during fetal skin development. Arch Dermatol Res, 2013, 305(5):379-387.
14.	Bu P, Yang P. MicroRNA-203 inhibits malignant melanoma cell migration by targeting versican. Exp Ther Med, 2014, 8(1):309-315.
15.	Andl T, Murchison EP, Liu F, et al. The miRNA-processing enzyme dicer is essential for the morphogenesis and maintenance of hair follicles. Curr Biol, 2006, 16(10):1041-1049.
16.	Yi R, Pasolli HA, Landthaler M, et al. DGCR8-dependent microRNA biogenesis is essential for skin development. Proc Natl Acad Sci USA, 2009, 106(2):498-502.
17.	Pan X, Hobbs RP, Coulombe PA. The expanding significance of keratin intermediate filaments in normal and diseased epithelia. Curr Opin Cell Biol, 2013, 25(1):47-56.
18.	Homberg M, Magin TM. Beyond expectations:novel insights into epidermal keratin function and regulation. Int Rev Cell Mol Biol, 2014, 311:265-306.
19.	Gao Y, Li M, Zhang X, et al. Isolation, culture and phenotypic characterization of human sweat gland epithelial cells. Int J Mol Med, 2014, 34(4):997-1003.
20.	Fatt MP, Cancino GI, Miller FD, et al. p63 and p73 coordinate p53 function to determine the balance between survival, cell death, and senescence in adult neural precursor cells. Cell Death Differ, 2014, 21(10):1546-1559.
21.	Orzol P, Nekulova M, Vojtesek B, et al. P63-an important player in epidermal and tumour development. Klin Onkol, 2012, 25 Suppl 2:2S11-15.
22.	Yao JY, Chen JK. Roles of p63 in epidermal development and tumorigenesis. Biomed J, 2012, 35(6):457-463.
23.	Candi E, Amelio I, Agostini M, et al. MicroRNAs and p63 in epithelial stemness. Cell Death Differ, 2015, 22(1):12-21.

1. Tao R, Sun TJ, Han YQ, et al. Epimorphin-induced differentiation of human umbilical cord mesenchymal stem cells into sweat gland cells. Eur Rev Med Pharmacol Sci, 2014, 18(9):1404-1410.
2. 宋志芳, 刘德伍. 微小RNA在皮肤发育再生及创面愈合中的作用. 中国修复重建外科杂志, 2014, 28(7):913-918.
3. Suh HN, Han HJ. Sonic hedgehog increases the skin wound-healing ability of mouse embryonic stem cells through the microRNA200 family. Br J Pharmacol, 2015, 172(3):815-828.
4. Yi R, Poy MN, Stoffel M, et al. A skin microRNA promotes differentiation by repressing ‘stemness’. Nature, 2008, 452(7184):225-229.
5. Wei T, Orfanidis K, Xu N, et al. The expression of microRNA-203 during human skin mor phogenesis. Exp Dermatol, 2010, 19(9):854-856.
6. Nissan X, Denis JA, Saidani M, et al. miR-203 modulates epithelial differentiation of human embryonic stem cells towards epidermal stratification. Dev Biol, 2011, 356(2):506-515.
7. Yang RH, Xie JL, Shu B, et al. An improved method for the isolation and culture of rat epidermal stem cells. Int J Clin Exp Pathol, 2013, 6(11):2529-2534.
8. Plichta JK, Droho S, Curtis BJ, et al. Local burn injury impairs epithelial permeability and antimicrobial peptide barrier function in distal unburned skin. Crit Care Med, 2014, 42(6):e420-431.
9. Stollery N. Sebaceous and sweat gland disorders. Practitioner, 2013, 257(1757):32-33.
10. Chuong CM, Randall VA, Widelitz RB, et al. Physiological regeneration of skin appendages and implications for regenerative medicine. Physiology (Bethesda), 2012, 27(2):61-72.
11. Wang Y, Liu ZY, Zhao Q, et al. Future application of hair follicle stem cells:capable in differentiation into sweat gland cells. Chin Med J (Engl), 2013, 126(18):3545-3552.
12. Yang S, Ma K, Feng C, et al. Capacity of human umbilical cord-derived mesenchymal stem cells to differentiate into sweat gland-like cells:a preclinical study. Front Med, 2013, 7(3):345-353.
13. Gkegkes ID, Aroni K, Agrogiannis G, et al. Expression of caspase-14 and keratin-19 in the human epidermis and appendages during fetal skin development. Arch Dermatol Res, 2013, 305(5):379-387.
14. Bu P, Yang P. MicroRNA-203 inhibits malignant melanoma cell migration by targeting versican. Exp Ther Med, 2014, 8(1):309-315.
15. Andl T, Murchison EP, Liu F, et al. The miRNA-processing enzyme dicer is essential for the morphogenesis and maintenance of hair follicles. Curr Biol, 2006, 16(10):1041-1049.
16. Yi R, Pasolli HA, Landthaler M, et al. DGCR8-dependent microRNA biogenesis is essential for skin development. Proc Natl Acad Sci USA, 2009, 106(2):498-502.
17. Pan X, Hobbs RP, Coulombe PA. The expanding significance of keratin intermediate filaments in normal and diseased epithelia. Curr Opin Cell Biol, 2013, 25(1):47-56.
18. Homberg M, Magin TM. Beyond expectations:novel insights into epidermal keratin function and regulation. Int Rev Cell Mol Biol, 2014, 311:265-306.
19. Gao Y, Li M, Zhang X, et al. Isolation, culture and phenotypic characterization of human sweat gland epithelial cells. Int J Mol Med, 2014, 34(4):997-1003.
20. Fatt MP, Cancino GI, Miller FD, et al. p63 and p73 coordinate p53 function to determine the balance between survival, cell death, and senescence in adult neural precursor cells. Cell Death Differ, 2014, 21(10):1546-1559.
21. Orzol P, Nekulova M, Vojtesek B, et al. P63-an important player in epidermal and tumour development. Klin Onkol, 2012, 25 Suppl 2:2S11-15.
22. Yao JY, Chen JK. Roles of p63 in epidermal development and tumorigenesis. Biomed J, 2012, 35(6):457-463.
23. Candi E, Amelio I, Agostini M, et al. MicroRNAs and p63 in epithelial stemness. Cell Death Differ, 2015, 22(1):12-21.

Chinese Journal of Reparative and Reconstructive Surgery

microRNA-203 INDUCING DIFFERENTIATION OF HUMAN EPIDERMAL STEM CELLS INTO SWEAT GLAND CELLS IN VITRO

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