ROLE OF Smad4 ON FIBROSIS OF TENDON DERIVED FIBROBLASTS INDUCED BY TRANSFORMING GROWTH FACTOR β<sub>1</sub> BY TARGETED REGULATION OF miRNA219-5P_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

RUANHongjiang , FANDapeng , CHENShuai , LIXujun ,  FANCunyi

Department of Orthopaedics, the Sixth Affiliated People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200233, P. R. China;

Corresponding author：

FANCunyi, Email: fancunyi888@163.com

Keywords：

Tendon adhesion; Tendon derived fibroblasts; miRNA219-5P; Smad4; Fibrosis

DOI：

10.7507/1002-1892.20160128

Video：

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Abstract Full text Figures/Tables Video References Cited by

Objective To investigate the effect of Smad4 on the fibrosis of tendon derived fibroblasts (TDFs) induced by transforming growth factor β₁(TGF-β₁) by targeted regulation of miRNA219-5P (miR219-5P). Methods The tendons donated by the volunteers were harvested to isolate and culture TDFs. The 3rd generation cells were used for experiment. Chemically synthesized miR219-5P mimics, miR219-5P inhibitor, and negative control sequences were transfected into TDFs. The gene expression of miR219-5P in TDFs was detected by real-time PCR, and the protein expression of Smad4 in TDFs was detected by Western blot at 48 hours after transfection. The combining sites of miR219-5P and Smad4 in 3'UTR district were predicted by informatics software. Wild type and mutant type reporter gene expression vectors were constructed and then targeted verification was carried out by the luciferase reporter gene test. Transfected TDFs were then induced by TGF-β₁. The proliferation activity of the cells were measured by the cell counting kit 8 after culturing for 24, 48, and 72 hours. The expressions of fibrosis related proteins in TDFs were detected by Western blot at 72 hours. Results After TDFs were transfected by miR219-5P mimics, miR219-5P expression was significantly up-regulated, but the expressions of Smad4 was decreased subsequently (P<0.05). Intracellular expression of miR219-5P was inhibited by miR219-5P mimics inhibitor, however, the protein expression of Smad4 was significantly increased (P<0.05). Luciferase reporter gene test showed that luciferase activities were significantly decreased in pGL3-WT-Smad4+mimics group, but were significantly increased in pGL3-WT-Smad4+inhibitor group when compared with pGL3-WT-Smad4 transfected group (P<0.05), but no significant difference was found between GL3-MT-Smad4+mimics and pGL3-MT-Smad4+inhibitor groups (P>0.05). Cell proliferation and the fibrosis related proteins were increased in TGF-β₁ induced TDFs, however, decreased in TGF-β₁ induced TDFs after transfected by miR219-5P inhibitor (P<0.01). Conclusion miR219-5P can significantly inhibit fibrosis of TDFs induced by TGF-β₁ by down-regulating Smad4 expression.

Citation： RUANHongjiang, FANDapeng, CHENShuai, LIXujun, FANCunyi. ROLE OF Smad4 ON FIBROSIS OF TENDON DERIVED FIBROBLASTS INDUCED BY TRANSFORMING GROWTH FACTOR β₁ BY TARGETED REGULATION OF miRNA219-5P. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(5): 641-646. doi: 10.7507/1002-1892.20160128 Copy

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2.	Ruan H, Liu S, Li F, et al. Prevention of tendon adhesions by ERK2 small interfering RNAs. Int J Mol Sci, 2013, 14(2):4361-4371.
3.	阮洪江, 李来锋, 范存义, 等. Smad7在肌腱粘连组织中的表达及对肌腱成纤维细胞纤维化过程中的作用. 中国矫形外科杂志, 2016, 24(5):448-452.
4.	Mehra A, Wrana JL. TGF-beta and the Smad signal transduction pathway. Biochem Cell Biol, 2002, 80(5):605-622.
5.	Moustakas A, Souchelnytskyi S, Heldin CH. Smad regulation in TGF-beta signal transduction. J Cell Sci, 2001, 114(Pt 24):4359-4369.
6.	Dennier S, Itoh S, Vivien D, et al. Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of humanp lasminogen activator inhibitor-type 1 gene. EMBO J, 1998, 17(11):3091-3100.
7.	Feng XH, Derynck R. Specificity and versatility in tgf-beta signaling through Smads. Annu Rev Cell Dev Biol, 2005, 21:659-693.
8.	姜士超, 刘珅, 范存义. 肌腱粘连机制及预防的研究进展. 中国修复重建外科杂志, 2013, 27(5):633-636.
9.	夏长所, 杨选影, 韩迎秋, 等. TGF-β₁中和抗体对TGF-β诱导的肌腱胶原产生和术后粘连形成的影响. 中国修复重建外科杂志, 2009, 23(6):698-703.
10.	Juneja SC, Schwarz EM, O'Keefe RJ, et al. Cellular and molecular factors in flexor tendon repair and adhesions:a histological and gene expression analysis. Connect Tissue Res, 2013, 54(3):218-226.
11.	Loiselle AE, Yukata K, Geary MB, et al. Development of antisense oligonucleotide (ASO) technology against Tgf-β signaling to prevent scarring during flexor tendon repair. J Orthop Res, 2015, 33(6):859-866.
12.	Zhou Y, Zhang L, Zhao W, et al. Nanoparticle-mediated delivery of TGF-β₁ miRNA plasmid for preventing flexor tendon adhesion formation. Biomaterials, 2013, 34(33):8269-8278.
13.	Zhou Y, Zhu C, Wu YF, et al. Effective modulation of transforming growth factor-β₁ expression through engineered microRNA-based plasmid-loaded nanospheres. Cytotherapy, 2015, 17(3):320-329.
14.	Chen Q, Lu H, Yang H. Chitosan inhibits fibroblasts growth in Achilles tendon via TGF-β₁/Smad3 pathway by miR-29b. Int J Clin Exp Pathol, 2014, 7(12):8462-8470.
15.	Ambros V. The functions of animal microRNAs. Nature, 2004, 431(7006):350-355.
16.	Jia J, Feng X, Xu W, et al. MiR-17-5p modulates osteoblastic differentiation and cell proliferation by targeting SMAD7 in nontraumatic osteonecrosis. Exp Mol Med, 2014, 46:e107.
17.	Butz H, Rácz K, Hunyady L, et al. Crosstalk between TGF-β signaling and the microRNA machinery. Trends Pharmacol Sci, 2012, 33(7):382-393.
18.	Hao J, Zhang S, Zhou Y, et al. MicroRNA 483-3p suppresses the expression of DPC4/Smad4 in pancreatic cancer. FEBS Lett, 2011, 585(1):207-213.
19.	Hao J, Zhang S, Zhou Y, et al. MicroRNA 421 suppresses DPC4/Smad4 in pancreatic cancer. Biochem Biophys Res Commun, 2011, 406(4):552-557.
20.	He Y, Huang C, Sun X, et al. MicroRNA-146a modulates TGFbeta1-induced hepatic stellate cell proliferation by targeting SMAD4. Cell Signal, 2012, 24(10):1923-1930.

1. Border WA, Noble NA. Transforming growth factor beta in tissue fibrosis. N Engl J Med, 1994, 331(9):1286-1292.
2. Ruan H, Liu S, Li F, et al. Prevention of tendon adhesions by ERK2 small interfering RNAs. Int J Mol Sci, 2013, 14(2):4361-4371.
3. 阮洪江, 李来锋, 范存义, 等. Smad7在肌腱粘连组织中的表达及对肌腱成纤维细胞纤维化过程中的作用. 中国矫形外科杂志, 2016, 24(5):448-452.
4. Mehra A, Wrana JL. TGF-beta and the Smad signal transduction pathway. Biochem Cell Biol, 2002, 80(5):605-622.
5. Moustakas A, Souchelnytskyi S, Heldin CH. Smad regulation in TGF-beta signal transduction. J Cell Sci, 2001, 114(Pt 24):4359-4369.
6. Dennier S, Itoh S, Vivien D, et al. Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of humanp lasminogen activator inhibitor-type 1 gene. EMBO J, 1998, 17(11):3091-3100.
7. Feng XH, Derynck R. Specificity and versatility in tgf-beta signaling through Smads. Annu Rev Cell Dev Biol, 2005, 21:659-693.
8. 姜士超, 刘珅, 范存义. 肌腱粘连机制及预防的研究进展. 中国修复重建外科杂志, 2013, 27(5):633-636.
9. 夏长所, 杨选影, 韩迎秋, 等. TGF-β₁中和抗体对TGF-β诱导的肌腱胶原产生和术后粘连形成的影响. 中国修复重建外科杂志, 2009, 23(6):698-703.
10. Juneja SC, Schwarz EM, O'Keefe RJ, et al. Cellular and molecular factors in flexor tendon repair and adhesions:a histological and gene expression analysis. Connect Tissue Res, 2013, 54(3):218-226.
11. Loiselle AE, Yukata K, Geary MB, et al. Development of antisense oligonucleotide (ASO) technology against Tgf-β signaling to prevent scarring during flexor tendon repair. J Orthop Res, 2015, 33(6):859-866.
12. Zhou Y, Zhang L, Zhao W, et al. Nanoparticle-mediated delivery of TGF-β₁ miRNA plasmid for preventing flexor tendon adhesion formation. Biomaterials, 2013, 34(33):8269-8278.
13. Zhou Y, Zhu C, Wu YF, et al. Effective modulation of transforming growth factor-β₁ expression through engineered microRNA-based plasmid-loaded nanospheres. Cytotherapy, 2015, 17(3):320-329.
14. Chen Q, Lu H, Yang H. Chitosan inhibits fibroblasts growth in Achilles tendon via TGF-β₁/Smad3 pathway by miR-29b. Int J Clin Exp Pathol, 2014, 7(12):8462-8470.
15. Ambros V. The functions of animal microRNAs. Nature, 2004, 431(7006):350-355.
16. Jia J, Feng X, Xu W, et al. MiR-17-5p modulates osteoblastic differentiation and cell proliferation by targeting SMAD7 in nontraumatic osteonecrosis. Exp Mol Med, 2014, 46:e107.
17. Butz H, Rácz K, Hunyady L, et al. Crosstalk between TGF-β signaling and the microRNA machinery. Trends Pharmacol Sci, 2012, 33(7):382-393.
18. Hao J, Zhang S, Zhou Y, et al. MicroRNA 483-3p suppresses the expression of DPC4/Smad4 in pancreatic cancer. FEBS Lett, 2011, 585(1):207-213.
19. Hao J, Zhang S, Zhou Y, et al. MicroRNA 421 suppresses DPC4/Smad4 in pancreatic cancer. Biochem Biophys Res Commun, 2011, 406(4):552-557.
20. He Y, Huang C, Sun X, et al. MicroRNA-146a modulates TGFbeta1-induced hepatic stellate cell proliferation by targeting SMAD4. Cell Signal, 2012, 24(10):1923-1930.

Chinese Journal of Reparative and Reconstructive Surgery

ROLE OF Smad4 ON FIBROSIS OF TENDON DERIVED FIBROBLASTS INDUCED BY TRANSFORMING GROWTH FACTOR β₁ BY TARGETED REGULATION OF miRNA219-5P

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ROLE OF Smad4 ON FIBROSIS OF TENDON DERIVED FIBROBLASTS INDUCED BY TRANSFORMING GROWTH FACTOR β1 BY TARGETED REGULATION OF miRNA219-5P

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ROLE OF Smad4 ON FIBROSIS OF TENDON DERIVED FIBROBLASTS INDUCED BY TRANSFORMING GROWTH FACTOR β₁ BY TARGETED REGULATION OF miRNA219-5P