FUNCTION OF MicroRNA-199b-5p IN EWING'S SARCOMA CELL LINES_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIWeihua ,  WANGXiao , ZHANGYongle , GUOJiankuo , PANHuagang

Department of Orthopaedics, Huaihe Hospital, Henan University, Kaifeng Henan, 475000, P. R. China;

Corresponding author：

WANGXiao, Email: wangxiao0622@163.com

Keywords：

Ewing's sarcoma; microRNA-199b-5p; Cell proliferation; Cyclin-L1; c-Kit

DOI：

10.7507/1002-1892.20150129

Video：

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Objective To investigate the function and possible mechanism of microRNA-199b-5p (miR-199b-5p) in Ewing's sarcoma cell lines so as to provide theoretical basis for biological treatment in the future. Methods A673 cells and TC252 cells were adopted as Ewing's sarcoma cell lines in vitro. miR-199b-5p oligonucleotide fragments (mimic) and scramble control (mimic control) were used to transfect TC252 cells and A673 cells, respectively. The expression of miR-199b-5p was measured in 2 cell lines by real-time quantitative-PCR and compared with that in mesenchymal stem cells (MSCs). The cell proliferation was examined by cell counting kit 8. The cell cycle and apoptosis were detected by flow cytometry. The possible targets of miR-199b-5p were determined by luciferase assays. The protein expressions of possible targets were measured by Western blot. Results The expression of miR-199b-5p in control group was significantly down-regulated in A673 cells and TC252 cells when compared with that in MSCs (P<0.05); and the expression of miR-199b-5p in experimental group was significantly up-regulated when compared with that in control group (P<0.05). G₁ phase cells increased obviously and S phase cells decreased significantly compared with corresponding cells in control group (P<0.05); but no significant difference was found in G₂/M phase cells between 2 groups (P>0.05). The apoptosis rate increased significantly in experimental group when compared with that in control group (P<0.05). The possible targets of miR-199b-5p were cyclin-L1 (CCNL1) and c-Kit by luciferase assays. Western blot results showed that the CCNL1 and c-Kit protein expression levels in experimental group were significantly lower than those in control group (P<0.05). Conclusion miR-199b-5p can suppress the cell proliferation, block the cell cycle, and promote the cell apoptosis, so miR-199b-5p inhibits Ewing's sarcoma cell lines by targeting CCNL1 and c-Kit.

Citation： LIWeihua, WANGXiao, ZHANGYongle, GUOJiankuo, PANHuagang. FUNCTION OF MicroRNA-199b-5p IN EWING'S SARCOMA CELL LINES. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(5): 595-599. doi: 10.7507/1002-1892.20150129 Copy

1.	Windsor R, Strauss S, Seddon B, et al. Experimental therapies in Ewing's sarcoma. Expert Opin Invest Drugs, 2009, 18(2):143-159.
2.	Teicher BA, Bagley RG, Rouleau C, et al. Characteristics of human Ewing/PNET sarcoma models. Ann Saudi Med, 2011, 31(2):174-182.
3.	Zhang Z, Huang L, Yu Z, et al. Let-7 a functions as a tumor suppressor in Ewing's sarcoma cell lines partly by targeting cyclin-dependent kinase 6. DNA Cell Biol, 2014, 33(3):136-147.
4.	Borinstein SC, Beeler N, Block JJ, et al. A Decade in Banking Ewing Sarcoma:a Report from the Children's Oncology Group. Front Oncol, 2013, 3:57.
5.	Nilsen TW. Mechanisms of microRNA-mediated gene regulation in animal cells. Trends Genet, 2007, 23(5):243-249.
6.	Robin TP, Smith A, McKinsey E, et al. EWS/FLI1 regulates EYA3 in Ewing sarcoma via modulation of microRNA-708, resulting in increased cell survival and chemoresistance. Mol Cancer Res, 2012, 10(8):1098-1108.
7.	Dylla L, Jedlicka P. Growth-promoting role of the miR-106a~363 cluster in Ewing sarcoma. PLoS One, 2013, 8(4):e63032.
8.	Dylla L, Moore C, Jedlicka P. MicroRNAs in Ewing Sarcoma. Front Oncol, 2013, 3:65.
9.	徐凌, 郭传勇. MicroRNAs在胃癌中的研究进展. 中华肿瘤防治杂志, 2010, 17(21):92-94.
10.	Iorio MV, Croce CM. MicroRNA dysregulation in cancer:diagnostics, monitoring and therapeutics. A comprehensive review. EMBO Mol Med, 2012, 4(3):143-159.
11.	Liu MX, Siu MK, Liu SS, et al. Epigenetic silencing of microRNA-199b-5p is associated with acquired chemoresistance via activation of JAG₁-Notch1 signaling in ovarian cancer. Oncotarget, 2014, 5(4):944-958.
12.	Fang C, Zhao Y, Guo B. MiR-199b-5p targets HER2 in breast cancer cells. J Cell Biochem, 2013, 114(7):1457-1463.
13.	Brennecke J, Stark A, Russell RB, et al. Principles of microRNA-target recognition. PLoS Biol, 2005, 3(3):e85.
14.	Won KY, Kim YW, Kim HS, et al. MicroRNA-199b-5p is involved in the Notch signaling pathway in osteosarcoma. Hum Pathol, 2013, 44(8):1648-1655.
15.	Muller D, Millon R, Théobald S, et al. Cyclin L1 (CCNL1) gene alterations in human head and neck squamous cell carcinoma. Br J Cancer, 2006, 94(7):1041-1044.
16.	Sticht C, Hofele C, Flechtenmacher C, et al. Amplification of Cyclin L1 is associated with lymph node metastases in head and neck squamous cell carcinoma (HNSCC). Br J Cancer, 2005, 92(4):770-774.
17.	Li Y, Bai H, Zhang Z, et al. The up-regulation of miR-199b-5p in erythroid differentiation is associated with GATA-1 and NF-E2. Mol Cells, 2014, 37(3):213-219.
18.	Phung B, Sun J, Schepsky A, et al. C-KIT signaling depends on microphthalmia-associated transcription factor for effects on cell proliferation. PLoS One, 2011, 6(8):e24064.
19.	Landuzzi L, De Giovanni C, Nicoletti G, et al. The metastatic ability of Ewing's sarcoma cells is modulated by stem cell factor and by its receptor c-kit. Am J Pathol, 2000, 157(6):2123-2131.
20.	Garzia L, Andolfo I, Cusanelli E, et al. MicroRNA-199b-5p impairs cancer stem cells through negative regulation of HES1 in medulloblastoma. PLoS One, 2009, 4(3):e4998.
21.	Ikeda AK, Judelson DR, Federman N, et al. ABT-869 inhibits the proliferation of Ewing Sarcoma cells and suppresses platelet-derived growth factor receptor beta and c-KIT signaling pathways. Mol Cancer Ther, 2010, 9(3):653-660.

1. Windsor R, Strauss S, Seddon B, et al. Experimental therapies in Ewing's sarcoma. Expert Opin Invest Drugs, 2009, 18(2):143-159.
2. Teicher BA, Bagley RG, Rouleau C, et al. Characteristics of human Ewing/PNET sarcoma models. Ann Saudi Med, 2011, 31(2):174-182.
3. Zhang Z, Huang L, Yu Z, et al. Let-7 a functions as a tumor suppressor in Ewing's sarcoma cell lines partly by targeting cyclin-dependent kinase 6. DNA Cell Biol, 2014, 33(3):136-147.
4. Borinstein SC, Beeler N, Block JJ, et al. A Decade in Banking Ewing Sarcoma:a Report from the Children's Oncology Group. Front Oncol, 2013, 3:57.
5. Nilsen TW. Mechanisms of microRNA-mediated gene regulation in animal cells. Trends Genet, 2007, 23(5):243-249.
6. Robin TP, Smith A, McKinsey E, et al. EWS/FLI1 regulates EYA3 in Ewing sarcoma via modulation of microRNA-708, resulting in increased cell survival and chemoresistance. Mol Cancer Res, 2012, 10(8):1098-1108.
7. Dylla L, Jedlicka P. Growth-promoting role of the miR-106a~363 cluster in Ewing sarcoma. PLoS One, 2013, 8(4):e63032.
8. Dylla L, Moore C, Jedlicka P. MicroRNAs in Ewing Sarcoma. Front Oncol, 2013, 3:65.
9. 徐凌, 郭传勇. MicroRNAs在胃癌中的研究进展. 中华肿瘤防治杂志, 2010, 17(21):92-94.
10. Iorio MV, Croce CM. MicroRNA dysregulation in cancer:diagnostics, monitoring and therapeutics. A comprehensive review. EMBO Mol Med, 2012, 4(3):143-159.
11. Liu MX, Siu MK, Liu SS, et al. Epigenetic silencing of microRNA-199b-5p is associated with acquired chemoresistance via activation of JAG₁-Notch1 signaling in ovarian cancer. Oncotarget, 2014, 5(4):944-958.
12. Fang C, Zhao Y, Guo B. MiR-199b-5p targets HER2 in breast cancer cells. J Cell Biochem, 2013, 114(7):1457-1463.
13. Brennecke J, Stark A, Russell RB, et al. Principles of microRNA-target recognition. PLoS Biol, 2005, 3(3):e85.
14. Won KY, Kim YW, Kim HS, et al. MicroRNA-199b-5p is involved in the Notch signaling pathway in osteosarcoma. Hum Pathol, 2013, 44(8):1648-1655.
15. Muller D, Millon R, Théobald S, et al. Cyclin L1 (CCNL1) gene alterations in human head and neck squamous cell carcinoma. Br J Cancer, 2006, 94(7):1041-1044.
16. Sticht C, Hofele C, Flechtenmacher C, et al. Amplification of Cyclin L1 is associated with lymph node metastases in head and neck squamous cell carcinoma (HNSCC). Br J Cancer, 2005, 92(4):770-774.
17. Li Y, Bai H, Zhang Z, et al. The up-regulation of miR-199b-5p in erythroid differentiation is associated with GATA-1 and NF-E2. Mol Cells, 2014, 37(3):213-219.
18. Phung B, Sun J, Schepsky A, et al. C-KIT signaling depends on microphthalmia-associated transcription factor for effects on cell proliferation. PLoS One, 2011, 6(8):e24064.
19. Landuzzi L, De Giovanni C, Nicoletti G, et al. The metastatic ability of Ewing's sarcoma cells is modulated by stem cell factor and by its receptor c-kit. Am J Pathol, 2000, 157(6):2123-2131.
20. Garzia L, Andolfo I, Cusanelli E, et al. MicroRNA-199b-5p impairs cancer stem cells through negative regulation of HES1 in medulloblastoma. PLoS One, 2009, 4(3):e4998.
21. Ikeda AK, Judelson DR, Federman N, et al. ABT-869 inhibits the proliferation of Ewing Sarcoma cells and suppresses platelet-derived growth factor receptor beta and c-KIT signaling pathways. Mol Cancer Ther, 2010, 9(3):653-660.

Chinese Journal of Reparative and Reconstructive Surgery

FUNCTION OF MicroRNA-199b-5p IN EWING'S SARCOMA CELL LINES

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