Effects of over-expressing smad7 gene on keloid fibroblasts_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

CHENZhenwei , LIUYin , XIAOMinqin , XIAOHong , QIUWei ,  ZHAOYanan

Department of Plastic Surgery, Second Affiliated Hospital, Kunming Medical University, Kunming Yunnan, 650101, P. R. China;

Corresponding?author：

ZHAOYanan, Email: yzhao4@hotmail.com

Keywords：

Smad7; Keloids fibroblasts; Signaling pathway; Apoptosis

DOI：

10.7507/1002-1892.20160177

Video：

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Objective To explore if Smad7 protein can inhibit growth of keloids by observing the gene and protein expressions of Smad7, collagen type Ⅰ, and collagen type Ⅲ and cell proliferation after over-expression vectors of Smad7 transfecting keloid fibroblasts (KFb). Methods Fibroblasts were acquired from 10 male patient with keloids at the age of 20 to 25 years. After in vitro culture, KFb were divided into 3 groups: untransfected group (group A), pcDNA3.1 (-) transfected group (group B), and pcDNA3.1 (-)-smad7 transfected group (group C). The mRNA and protein expression levels of Smad7, collagen type Ⅰ, and collagen type Ⅲ were detected by real-time fluorescence quantitative PCR and Western blot at 48 hours after transfection. The cell proliferation ability was detected by MTT assay at 24 hours after transfection. Results The relative expression levels of mRNA and protein of Smad7 in group C were significantly higher than those in group A and group B (P < 0.01). The relative expression levels of mRNA and protein of collagen type Ⅰ and collagen type Ⅲ in group C were significantly lower than those in group A and group B (P < 0.01). The relative expression levels of mRNA of collagen type Ⅰ and collagen type Ⅲ in group B were significantly higher than those in group A (P < 0.01); and the relative expression levels of proteins of Smad7, collagen type Ⅰ, and collagen type Ⅲ were significantly lower than those in group A (P < 0.01). The cell proliferation ability in group C was significantly lower than that in group A and group B at each time point by MTT assay (P < 0.05), but no difference was found between group A and group B (P>0.05). Conclusion Gene expressions of collagen type Ⅰ, and collagen type Ⅲ and cell proliferation will be inhibited after KFb are transfected by over-expression vector of Smad7.

Citation： CHENZhenwei, LIUYin, XIAOMinqin, XIAOHong, QIUWei, ZHAOYanan. Effects of over-expressing smad7 gene on keloid fibroblasts. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(7): 871-875. doi: 10.7507/1002-1892.20160177 Copy

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2.	Mauviel A. Transforming growth factor-beta: a key mediator of fibrosis. Methods Mol Med, 2005, 117: 69-80.
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4.	夏鵬, 童文祥, 喻永敏, 等.大鼠皮膚切創愈合過程中VEGF, TGF-β蛋白的表達.重慶醫科大學學報, 2010, 35(8): l167-1171.
5.	Liang CJ, Yen YH, Hung LY, et al. Thalidomide inhibits fibronectin production in TGF-β1-treated normal and keloid fibroblasts via inhibition of the p38/Smad3 pathway. Biochem Pharmacol, 2013, 85(11): 1594-1602.
6.	Wu CS, Wu PH, Fang AH, et al. FK506 inhibits the enhancing effects of transforming growth factor (TGF)-β1 on collagen expression and TGF-β/Smad signalling in keloid fibroblasts: implication for new therapeutic approach. Br J Dermatol, 2012, 167(3): 532-541.
7.	Kottler UB, Jünemann AG, Aigner T, et al. Comparative effects of TGF-beta 1 and TGF-beta 2 on extracellular matrix production, proliferation, migration, and collagen contraction of human Tenon's capsule fibroblasts in pseudoexfoliation and primary open-angle glaucoma. Exp Eye Res, 2005, 80(1): 121-134.
8.	Chang Z, Kishimoto Y, Hasan A, et al. TGF-β3 modulates the inflammatory environment and reduces scar formation following vocal fold mucosal injury in ruts. Dis Model Mech, 2013, 7(1): 83-91.
9.	Falk P, Angenete E, Bergstrom M, et al. TGF-β1 promotes transition of mesothelial cells into fibroblast phenotype in response to peritoneal injury in a cell culture model. Int J Surg, 2013, 11(9): 977-982.
10.	Attisanno L, Wrana JL. Signal transduction by the TGF-beta superfamily. Science, 2002, 296(5573): 1646-1647.
11.	ten Dijke P, Miyazono K, Heldin CH. Signaling inputs converge on nuclear effectors in TGF-βsignaling. Trends Biochem Sci, 2000, 25(2): 64-70.
12.	李春輝, 潘理會, 張德利.胃癌組織TGF-β、Smad7與ki-67 mRNA表達及其相關性分析.實用醫學雜志, 2014, 30(5): 751-754.
13.	Yu F, Guo Y, Chen B, et al. MicroRNA-17-5p activates hepatic stellate cells through targeting of Smad7. Lab Invest, 2015, 95(7): 781-789.
14.	Brodin G, ten Dijke P, Funa K, et al. Increased Smad expression and activation is associated with apoptosis in normal and malignant prostate after castration. Cancer Res, 1999, 59(11): 2731-2738.
15.	Landstr?m M, Heldin NE, Bu S, et al. Smad7 mediates apoptosis induced by transforming growth factorβin prostatic carcinoma cells. Curr Biol, 2000, 10(9): 535-538.
16.	Kamato D, Burch M, Piva TJ, et al. Transforming growth factor-βsignalling:Role and consequences of Smad linker region phosphorylation. Cells Signal, 2013, 25(10): 2017-2024.
17.	Ladin DA, Hou L, Patel D, et al. P53 and apoptosis alterations in keloids and keloid fibroblasts. Wound Repair Regen, 1998, 6(1): 28-37.
18.	Appleton I, Brown NJ, Willoughby DA. Apoptosis, necrosis, and proliferation:possible implications in the etiology of keloid. Am J Pathol, 1996, 149(5): 1441-1447.
19.	Crowston JG, Chang LH, Constable PH, et al. Apoptosis gene expression and death receptor signaling in mitomycin-C-treated human tenon capsule fibroblasts. Invest Ophthalmol Vis Sci, 2002, 43(3): 692-699.
20.	劉永波, 高建華, 段紅杰, 等.瘢痕疙瘩p53基因突變高發區基因結構的研究.中華整形外科雜志, 2003, 19(4): 258-260.

1. Kim HD, Hwang SM, Lim KR, et al. Recurrent auricular keloids during pregnancy. Arch Plast Surg, 2013, 40(1): 70-72．.
2. Mauviel A. Transforming growth factor-beta: a key mediator of fibrosis. Methods Mol Med, 2005, 117: 69-80.
3. 陳勇, 戴和平, 龍志高, 等.人皮膚成纖維細胞的分離和體外培養.湖南醫科大學學報, 2002, 27(5): 477-478.
4. 夏鵬, 童文祥, 喻永敏, 等.大鼠皮膚切創愈合過程中VEGF, TGF-β蛋白的表達.重慶醫科大學學報, 2010, 35(8): l167-1171.
5. Liang CJ, Yen YH, Hung LY, et al. Thalidomide inhibits fibronectin production in TGF-β1-treated normal and keloid fibroblasts via inhibition of the p38/Smad3 pathway. Biochem Pharmacol, 2013, 85(11): 1594-1602.
6. Wu CS, Wu PH, Fang AH, et al. FK506 inhibits the enhancing effects of transforming growth factor (TGF)-β1 on collagen expression and TGF-β/Smad signalling in keloid fibroblasts: implication for new therapeutic approach. Br J Dermatol, 2012, 167(3): 532-541.
7. Kottler UB, Jünemann AG, Aigner T, et al. Comparative effects of TGF-beta 1 and TGF-beta 2 on extracellular matrix production, proliferation, migration, and collagen contraction of human Tenon's capsule fibroblasts in pseudoexfoliation and primary open-angle glaucoma. Exp Eye Res, 2005, 80(1): 121-134.
8. Chang Z, Kishimoto Y, Hasan A, et al. TGF-β3 modulates the inflammatory environment and reduces scar formation following vocal fold mucosal injury in ruts. Dis Model Mech, 2013, 7(1): 83-91.
9. Falk P, Angenete E, Bergstrom M, et al. TGF-β1 promotes transition of mesothelial cells into fibroblast phenotype in response to peritoneal injury in a cell culture model. Int J Surg, 2013, 11(9): 977-982.
10. Attisanno L, Wrana JL. Signal transduction by the TGF-beta superfamily. Science, 2002, 296(5573): 1646-1647.
11. ten Dijke P, Miyazono K, Heldin CH. Signaling inputs converge on nuclear effectors in TGF-βsignaling. Trends Biochem Sci, 2000, 25(2): 64-70.
12. 李春輝, 潘理會, 張德利.胃癌組織TGF-β、Smad7與ki-67 mRNA表達及其相關性分析.實用醫學雜志, 2014, 30(5): 751-754.
13. Yu F, Guo Y, Chen B, et al. MicroRNA-17-5p activates hepatic stellate cells through targeting of Smad7. Lab Invest, 2015, 95(7): 781-789.
14. Brodin G, ten Dijke P, Funa K, et al. Increased Smad expression and activation is associated with apoptosis in normal and malignant prostate after castration. Cancer Res, 1999, 59(11): 2731-2738.
15. Landstr?m M, Heldin NE, Bu S, et al. Smad7 mediates apoptosis induced by transforming growth factorβin prostatic carcinoma cells. Curr Biol, 2000, 10(9): 535-538.
16. Kamato D, Burch M, Piva TJ, et al. Transforming growth factor-βsignalling:Role and consequences of Smad linker region phosphorylation. Cells Signal, 2013, 25(10): 2017-2024.
17. Ladin DA, Hou L, Patel D, et al. P53 and apoptosis alterations in keloids and keloid fibroblasts. Wound Repair Regen, 1998, 6(1): 28-37.
18. Appleton I, Brown NJ, Willoughby DA. Apoptosis, necrosis, and proliferation:possible implications in the etiology of keloid. Am J Pathol, 1996, 149(5): 1441-1447.
19. Crowston JG, Chang LH, Constable PH, et al. Apoptosis gene expression and death receptor signaling in mitomycin-C-treated human tenon capsule fibroblasts. Invest Ophthalmol Vis Sci, 2002, 43(3): 692-699.
20. 劉永波, 高建華, 段紅杰, 等.瘢痕疙瘩p53基因突變高發區基因結構的研究.中華整形外科雜志, 2003, 19(4): 258-260.

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Effects of over-expressing smad7 gene on keloid fibroblasts

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