Experimental study of human amniotic mesenchymal stem cell exosome promoting fibroblasts migration through microRNA-135a_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

CHEN Tao , GAO Shaoying , HAO Yi , ZHANG Feifei , TANG Xiujun , WEI Zairong , WANG Dali ,  QI Jianping

Department of Burn and Plastic Surgery, Affiliated Hospital of Zunyi Medical University, Zunyi Guizhou, 563000, P.R.China;

Corresponding author：

QI Jianping, Email: ybwblseslss@sina.com

Keywords：

Human amniotic mesenchymal stem cell; exosome; microRNA-135a; fibroblasts

DOI：

10.7507/1002-1892.201907136

Video：

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Objective To investigate the effect of microRNA-135a (miR-135a) in human amnion mesenchymal stem cell exosome (hAMSC-Exo) on the migration of fibroblasts.Methods The hAMSC-Exo was extracted with exosomes separation kit and identified, the effect of hAMSC-Exo on fibroblasts migration was detected by scratch test. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the relative expression of miR-135a gene in hAMSC-Exo after overexpression of miR-135a. Scratch test was used to detect the effect of hAMSC-Exo on the migration of fibroblasts after overexpression and knockdown of miR-135a. Western blot was used to detect the migration related proteins of fibroblasts [large tumor suppressor 2 (LATS2), E-cadherin, N-cadherin, and α smooth muscle actin (α-SMA)] after overexpression and knockdown of miR-135a. The 293T cell exosomes and hAMSC-Exo were used as control.Results hAMSC-Exos were extracted successfully. Scratch test results showed that hAMSC group had the strongest ability to promote fibroblasts migration, and GW4869 (exosome inhibitor) treatment group had reduced ability to promote fibroblasts migration. qRT-PCR test showed that the relative expression of miR-135a gene in hAMSC-Exo increased significantly after over expression of miR-135a. Scratch test results showed that after over expression of miR-135a, hAMSC-Exo enhanced the migration ability of fibroblasts, while after knockdown of miR-135a, hAMSC-Exo weakened the migration ability of fibroblasts. Western blot results showed that the expressions of E-cadherin, N-cadherin, LATS2 were down regulated and α-SMA was up regulated in each hAMSC-Exo treatment group when compared with 293T cell exosomes group; after over expression of miR-135a, hAMSC-Exo decreased the expressions of E-cadherin, N-cadherin, LATS2 and increased the expression of α-SMA; while after knockdown of miR-135a, the ability of hAMSC-Exo was weakened.Conclusion miR-135a in hAMSC-Exo can promote fibroblasts’ migration, inhibit the expressions of E-cadherin, N-cadherin, LATS2, and promote the expression of α-SMA.

Citation： CHEN Tao, GAO Shaoying, HAO Yi, ZHANG Feifei, TANG Xiujun, WEI Zairong, WANG Dali, QI Jianping. Experimental study of human amniotic mesenchymal stem cell exosome promoting fibroblasts migration through microRNA-135a. Chinese Journal of Reparative and Reconstructive Surgery, 2020, 34(2): 234-239. doi: 10.7507/1002-1892.201907136 Copy

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2.	Schertl P, Volk J, Perduns R, et al. Impaired angiogenic differentiation of dental pulp stem cells during exposure to the resinous monomer triethylene glycol dimethacrylate. Dent Mater, 2019, 35(1): 144-155.
3.	Shojaee A, Parham A, Ejeian F, et al. Equine adipose mesenchymal stem cells (eq-ASCs) appear to have higher potential for migration and musculoskeletal differentiation. Res Vet Sci, 2019, 125: 235-243.
4.	熊佳超, 宋建星. 脂肪来源干细胞治疗难愈性创面的研究进展. 中国修复重建外科杂志, 2018, 32(4): 457-461.
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7.	Zhang X, Liu J, Yu B, et al. Effects of mesenchymal stem cells and their exosomes on the healing of large and refractory macular holes. Graefes Arch Clin Exp Ophthalmol, 2018, 256(11): 2041-2052.
8.	Yang L, Niu F, Yao H, et al. Exosomal miR-9 released from HIV tat stimulated astrocytes mediates microglial migration. J Neuroimmune Pharmacol, 2018, 13(3): 330-344.
9.	Chen Y, Zhang J, Wang H, et al. miRNA-135a promotes breast cancer cell migration and invasion by targeting HOXA10. BMC Cancer, 2012, 12: 111.
10.	Hiwatashi N, Bing R, Kraja I, et al. Stem cell-mediated paracrine signaling alters fibroplasia in human vocal fold fibroblasts in vitro. Ann Otol Rhinol Laryngol, 2017, 126(8): 581-588.
11.	Yao Z, Qiao Y, Li X, et al. Exosomes exploit the virus entry machinery and pathway to transmit alpha interferon-induced antiviral activity. J Virol, 2018, 92(24): pii: 1578-1518.
12.	Hormozi M, Baharvand P. Achillea biebersteinni Afan may inhibit scar formation: In vitro study. Mol Genet Genomic Med, 2019, 7(5): e640.
13.	Dangwal S, Stratmann B, Bang C, et al. Impairment of wound healing in patients with type 2 diabetes mellitus influences circulating microRNA patterns via inflammatory cytokines. Arterioscler Thromb Vasc Biol, 2015, 35(6): 1480-1488.
14.	Liu SC, Chuang SM, Hsu CJ, et al. CTGF increases vascular endothelial growth factor-dependent angiogenesis in human synovial fibroblasts by increasing miR-210 expression. Cell Death Dis, 2014, 5: e1485.
15.	Shi K, Qiu X, Zheng W, et al. MiR-203 regulates keloid fibroblast proliferation, invasion, and extracellular matrix expression by targeting EGR1 and FGF2. Biomed Pharmacother, 2018, 108: 1282-1288.
16.	Aunin E, Broadley D, Ahmed MI, et al. Exploring a role for regulatory miRNAs in wound healing during ageing: involvement of miR-200c in wound repair. Sci Rep, 2017, 7(1): 3257.
17.	Zeng YB, Liang XH, Zhang GX, et al. miRNA-135a promotes hepatocellular carcinoma cell migration and invasion by targeting forkhead box O1. Cancer Cell Int, 2016, 16: 63.
18.	Mohamedi Y, Fontanil T, Cobo T, et al. Antitumor potential of fibulin-5 in breast cancer cells depends on its RGD cell adhesion motif. Cell Physiol Biochem, 2019, 53(1): 87-100.

1. Powers JG, Higham C, Broussard K, et al. Wound healing and treating wounds: Chronic wound care and management. J Am Acad Dermatol, 2016, 74(4): 607-625.
2. Schertl P, Volk J, Perduns R, et al. Impaired angiogenic differentiation of dental pulp stem cells during exposure to the resinous monomer triethylene glycol dimethacrylate. Dent Mater, 2019, 35(1): 144-155.
3. Shojaee A, Parham A, Ejeian F, et al. Equine adipose mesenchymal stem cells (eq-ASCs) appear to have higher potential for migration and musculoskeletal differentiation. Res Vet Sci, 2019, 125: 235-243.
4. 熊佳超, 宋建星. 脂肪来源干细胞治疗难愈性创面的研究进展. 中国修复重建外科杂志, 2018, 32(4): 457-461.
5. 尹刚, 刘蔡钺, 林耀发, 等. 脂肪干细胞来源外泌体对周围神经损伤后再生作用的实验研究. 中国修复重建外科杂志, 2018, 32(12): 1592-1596.
6. Melzer C, Rehn V, Yang Y, et al. Taxol-loaded MSC-derived exosomes provide a therapeutic vhicle to target metastatic breast cancer and other carcinoma cells. Cancers (Basel), 2019, 11(6): pii: E798.
7. Zhang X, Liu J, Yu B, et al. Effects of mesenchymal stem cells and their exosomes on the healing of large and refractory macular holes. Graefes Arch Clin Exp Ophthalmol, 2018, 256(11): 2041-2052.
8. Yang L, Niu F, Yao H, et al. Exosomal miR-9 released from HIV tat stimulated astrocytes mediates microglial migration. J Neuroimmune Pharmacol, 2018, 13(3): 330-344.
9. Chen Y, Zhang J, Wang H, et al. miRNA-135a promotes breast cancer cell migration and invasion by targeting HOXA10. BMC Cancer, 2012, 12: 111.
10. Hiwatashi N, Bing R, Kraja I, et al. Stem cell-mediated paracrine signaling alters fibroplasia in human vocal fold fibroblasts in vitro. Ann Otol Rhinol Laryngol, 2017, 126(8): 581-588.
11. Yao Z, Qiao Y, Li X, et al. Exosomes exploit the virus entry machinery and pathway to transmit alpha interferon-induced antiviral activity. J Virol, 2018, 92(24): pii: 1578-1518.
12. Hormozi M, Baharvand P. Achillea biebersteinni Afan may inhibit scar formation: In vitro study. Mol Genet Genomic Med, 2019, 7(5): e640.
13. Dangwal S, Stratmann B, Bang C, et al. Impairment of wound healing in patients with type 2 diabetes mellitus influences circulating microRNA patterns via inflammatory cytokines. Arterioscler Thromb Vasc Biol, 2015, 35(6): 1480-1488.
14. Liu SC, Chuang SM, Hsu CJ, et al. CTGF increases vascular endothelial growth factor-dependent angiogenesis in human synovial fibroblasts by increasing miR-210 expression. Cell Death Dis, 2014, 5: e1485.
15. Shi K, Qiu X, Zheng W, et al. MiR-203 regulates keloid fibroblast proliferation, invasion, and extracellular matrix expression by targeting EGR1 and FGF2. Biomed Pharmacother, 2018, 108: 1282-1288.
16. Aunin E, Broadley D, Ahmed MI, et al. Exploring a role for regulatory miRNAs in wound healing during ageing: involvement of miR-200c in wound repair. Sci Rep, 2017, 7(1): 3257.
17. Zeng YB, Liang XH, Zhang GX, et al. miRNA-135a promotes hepatocellular carcinoma cell migration and invasion by targeting forkhead box O1. Cancer Cell Int, 2016, 16: 63.
18. Mohamedi Y, Fontanil T, Cobo T, et al. Antitumor potential of fibulin-5 in breast cancer cells depends on its RGD cell adhesion motif. Cell Physiol Biochem, 2019, 53(1): 87-100.

Chinese Journal of Reparative and Reconstructive Surgery

Experimental study of human amniotic mesenchymal stem cell exosome promoting fibroblasts migration through microRNA-135a

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