Effects of Fibulin-5 gene silencing on proliferation and migration of human bladder cancer cell line 5637_West China Medical Journal

Authors：

 CAO Guihua ^1,2 , YAN Yongji ¹ , CHEN Jian ¹ , ZHANG Haiyan ¹ , WANG Guang ¹ , LI Pei ¹ , SHI Xin ¹

1. Department of Urology, the Second Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650101, P. R. China;
2. Department of Urology, People’s Hospital of Leshan, Leshan, Sichuan 614000. P. R. China;

Corresponding?author：

CAO Guihua, Email: cgh75@sina.com

Keywords：

Bladder cancer cells; Fibulin-5 gene; Proliferative activity; Migration

DOI：

10.7507/1002-0179.201701037

Video：

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ObjectiveTo establish human bladder cancer cell line with silenced Fibulin-5 gene and observe the effects and mechanism of Fibulin-5 gene silencing on the proliferation activity and migration of the bladder cancer cells.MethodsThe human bladder cancer cells 5637 were divided into group F5 and group NC, and the cells in group F5 were infected with Fibulin-5 RNA interference (RNAi) lentivirus while the cells in group NC were infected with negative-control virus. Then the expression of Fibulin-5 mRNA was detected by real-time quantitative polymerase chain reaction, the cell proliferation activity was detected by MTT, the migration rate was detected by wound healing method, and the expression levels of proteins in receptor tyrosine kinase (RTK) pathway were detected by PathScan RTK Signaling Antibody Array Kit.ResultsThe Fibulin-5 mRNA expression decreased significantly by Fibulin-5 RNAi lentivirus (0.067±0.013 in group F5 vs. 1.001±0.000 in group NC), and the gene silencing efficiency reached 93.3%, so the Fibulin-5 silencing cell line was established successfully. Comparing with group NC, the relative absorbance value and migration rate of cell 5637 in group F5 decreased significantly (P<0.01); in addition, the expression levels of anaplastic lymphoma kinase, Axl, p44/42 mitogen activated protein kinase, and Src protein were up-regulated in group F5 (P<0.05).ConclusionFibulin-5 may play a role in the proliferation and migration of bladder cancer cells, and may have an inhibitory effect on extracellular signal-regulated kinase and its signaling pathway proteins.

Citation： CAO Guihua, YAN Yongji, CHEN Jian, ZHANG Haiyan, WANG Guang, LI Pei, SHI Xin. Effects of Fibulin-5 gene silencing on proliferation and migration of human bladder cancer cell line 5637. West China Medical Journal, 2018, 33(4): 417-422. doi: 10.7507/1002-0179.201701037 Copy

1.	Rink M, Ehdaie B, Cha EK, et al. Stage-specific impact of tumor location on oncologic outcomes in patients with upper and lower tract urothelial carcinoma following radical surgery. Eur Urol, 2012, 62(4): 677-684.
2.	Roupret M, Zigeuner R, Palou J, et al. European guidelines for the diagnosis and management of upper urinary tract urothelial cell carcinomas: 2011 update. Eur Urol, 2011, 59(4): 584-594.
3.	Chromecki TF, Bensalah K, Remzi M, et al. Prognostic factors for upper urinary tract urothelial carcinoma. Nat Rev Urol, 2011, 8(8): 440-447.
4.	Yanagisawa H, Schluterman MK, Brekken RA. Fibulin-5, an integrin-binding matricellular protein: its function in development and disease. J Cell Commun Signal, 2009, 3(3/4): 337-347.
5.	Furie N, Shteynberg D, Elkhatib R, et al. Fibulin-5 regulates keloid-derived fibroblast-like cells through integrin beta-1. Int J Cosmet Sci, 2016, 38(1): 35-40.
6.	Ohara H, Akatsuka S, Nagai H, et al. Stage-specific roles of fibulin-5 during oxidative stress-induced renal carcinogenesis in rats. Free Radic Res, 2011, 45(2): 211-220.
7.	Schiemann WP, Blobe GC, Kalume DE, et al. Context-specific effects of fibulin-5 (DANCE/EVEC) on cell proliferation, motility, and invasion. Fibulin-5 is induced by transforming growth factor-beta and affects protein kinase cascades. J Biol Chem, 2002, 277(30): 27367-27377.
8.	Wlazlinski A, Engers R, Hoffmann MJ, et al. Downregulation of several fibulin genes in prostate cancer. Prostate, 2007, 67(16): 1770-1780.
9.	Hu Z, Ai Q, Xu H, et al. Fibulin-5 is down-regulated in urothelial carcinoma of bladder and inhibits growth and invasion of human bladder cancer cell line 5637. Urol Oncol, 2011, 29(4): 430-435.
10.	Winship AL, Rainczuk K, Ton A, et al. Fibulin-5 localisation in human endometrial cancer shifts from epithelial to stromal with increasing tumour grade, and silencing promotes endometrial epithelial cancer cell proliferation. Oncol Lett, 2016, 12(1): 651-657.
11.	Yue W, Sun Q, Landreneau R, et al. Fibulin-5 suppresses lung cancer invasion by inhibiting matrix metalloproteinase-7 expression. Cancer Res, 2009, 69(15): 6339-6346.
12.	M?ller HD, Ralfkj?r U, Cremers N, et al. Role of fibulin-5 in metastatic organ colonization. Mol Cancer Res, 2011, 9(5): 553-563.
13.	Chen X, Song X, Yue W, et al. Fibulin-5 inhibits Wnt/β-catenin signaling in lung cancer. Oncotarget, 2015, 6(17): 15022-15034.
14.	Topalovski M, Hagopian M, Wang M, et al. Hypoxia and transforming growth factor β cooperate to induce fibulin-5 expression in pancreatic cancer. J Biol Chem, 2016, 291(42): 22244-22252.
15.	Lee YH, Albig AR, Regner MA, et al. Fibulin-5 initiates epithelial-mesenchymal transition (EMT) and enhances EMT induced by TGF-beta in mammary epithelial cells via a MMP-dependent mechanism. Carcinogenesis, 2008, 29(12): 2243-2251.
16.	Tsai HT, Chen CH. Dysregulation of fibulin-5 correlates with poor prognosis of nasopharyngeal carcinoma and promotes the metastasis of nasopharyngeal cells through the AKT signaling pathway. Biomark Genom Med, 2013, 5(3): 120.
17.	Wang M, Topalovski M, Toombs JE, et al. Fibulin-5 blocks microenvironmental ROS in pancreatic cancer. Cancer Res, 2015, 75(23): 5058-5069.
18.	Shi XY, Wang L, Cao CH, et al. Effect of fibulin-5 on cell proliferation and invasion in human gastric cancer patients. Asian Pac J Trop Med, 2014, 7(10): 787-791.
19.	Hiratsuka S, Nakamura K, Iwai S, et al. MMP9 induction by vascular endothelial growth factor receptor-1 is involved in lung-specific metastasis. Cancer Cell, 2002, 2(4): 289-300.
20.	Gonzalez DM, Medici D. Signaling mechanisms of the epithelial-mesenchymal transition. Sci Signal, 2014, 7(344): re8.
21.	文燦, 董世武, 余匯洋. 人胃癌組織中 fibulin-5 的表達及其臨床意義. 消化外科, 2006, 5(6): 463-465.

1. Rink M, Ehdaie B, Cha EK, et al. Stage-specific impact of tumor location on oncologic outcomes in patients with upper and lower tract urothelial carcinoma following radical surgery. Eur Urol, 2012, 62(4): 677-684.
2. Roupret M, Zigeuner R, Palou J, et al. European guidelines for the diagnosis and management of upper urinary tract urothelial cell carcinomas: 2011 update. Eur Urol, 2011, 59(4): 584-594.
3. Chromecki TF, Bensalah K, Remzi M, et al. Prognostic factors for upper urinary tract urothelial carcinoma. Nat Rev Urol, 2011, 8(8): 440-447.
4. Yanagisawa H, Schluterman MK, Brekken RA. Fibulin-5, an integrin-binding matricellular protein: its function in development and disease. J Cell Commun Signal, 2009, 3(3/4): 337-347.
5. Furie N, Shteynberg D, Elkhatib R, et al. Fibulin-5 regulates keloid-derived fibroblast-like cells through integrin beta-1. Int J Cosmet Sci, 2016, 38(1): 35-40.
6. Ohara H, Akatsuka S, Nagai H, et al. Stage-specific roles of fibulin-5 during oxidative stress-induced renal carcinogenesis in rats. Free Radic Res, 2011, 45(2): 211-220.
7. Schiemann WP, Blobe GC, Kalume DE, et al. Context-specific effects of fibulin-5 (DANCE/EVEC) on cell proliferation, motility, and invasion. Fibulin-5 is induced by transforming growth factor-beta and affects protein kinase cascades. J Biol Chem, 2002, 277(30): 27367-27377.
8. Wlazlinski A, Engers R, Hoffmann MJ, et al. Downregulation of several fibulin genes in prostate cancer. Prostate, 2007, 67(16): 1770-1780.
9. Hu Z, Ai Q, Xu H, et al. Fibulin-5 is down-regulated in urothelial carcinoma of bladder and inhibits growth and invasion of human bladder cancer cell line 5637. Urol Oncol, 2011, 29(4): 430-435.
10. Winship AL, Rainczuk K, Ton A, et al. Fibulin-5 localisation in human endometrial cancer shifts from epithelial to stromal with increasing tumour grade, and silencing promotes endometrial epithelial cancer cell proliferation. Oncol Lett, 2016, 12(1): 651-657.
11. Yue W, Sun Q, Landreneau R, et al. Fibulin-5 suppresses lung cancer invasion by inhibiting matrix metalloproteinase-7 expression. Cancer Res, 2009, 69(15): 6339-6346.
12. M?ller HD, Ralfkj?r U, Cremers N, et al. Role of fibulin-5 in metastatic organ colonization. Mol Cancer Res, 2011, 9(5): 553-563.
13. Chen X, Song X, Yue W, et al. Fibulin-5 inhibits Wnt/β-catenin signaling in lung cancer. Oncotarget, 2015, 6(17): 15022-15034.
14. Topalovski M, Hagopian M, Wang M, et al. Hypoxia and transforming growth factor β cooperate to induce fibulin-5 expression in pancreatic cancer. J Biol Chem, 2016, 291(42): 22244-22252.
15. Lee YH, Albig AR, Regner MA, et al. Fibulin-5 initiates epithelial-mesenchymal transition (EMT) and enhances EMT induced by TGF-beta in mammary epithelial cells via a MMP-dependent mechanism. Carcinogenesis, 2008, 29(12): 2243-2251.
16. Tsai HT, Chen CH. Dysregulation of fibulin-5 correlates with poor prognosis of nasopharyngeal carcinoma and promotes the metastasis of nasopharyngeal cells through the AKT signaling pathway. Biomark Genom Med, 2013, 5(3): 120.
17. Wang M, Topalovski M, Toombs JE, et al. Fibulin-5 blocks microenvironmental ROS in pancreatic cancer. Cancer Res, 2015, 75(23): 5058-5069.
18. Shi XY, Wang L, Cao CH, et al. Effect of fibulin-5 on cell proliferation and invasion in human gastric cancer patients. Asian Pac J Trop Med, 2014, 7(10): 787-791.
19. Hiratsuka S, Nakamura K, Iwai S, et al. MMP9 induction by vascular endothelial growth factor receptor-1 is involved in lung-specific metastasis. Cancer Cell, 2002, 2(4): 289-300.
20. Gonzalez DM, Medici D. Signaling mechanisms of the epithelial-mesenchymal transition. Sci Signal, 2014, 7(344): re8.
21. 文燦, 董世武, 余匯洋. 人胃癌組織中 fibulin-5 的表達及其臨床意義. 消化外科, 2006, 5(6): 463-465.

West China Medical Journal

Effects of Fibulin-5 gene silencing on proliferation and migration of human bladder cancer cell line 5637

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