Effect of attenuated expression of neuraminidase 3 via RNA interference on the proliferation and apoptosis of osteosarcoma MG-63 cells_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

YANG Xiao ¹ , LI Si ² , XIANG Shang ³ , WU Yilun ⁴ , WANG Lin ⁴ , PENG Jin ³ ,  FENG Ying ²

1. Department of Orthopedics, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu Sichuan, 610072, P.R.China;
2. West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu Sichuan, 610041, P.R.China;
3. West China School of Medicine, Sichuan University, Chengdu Sichuan, 610041, P.R.China;
4. Key Laboratory of Birth Defects and Related Diseases of Women and Children, Ministry of Education, West China Second University Hospital, Sichuan University, Chengdu Sichuan, 610041, P.R.China;

Corresponding author：

FENG Ying, Email: yingfeng27@scu.edu.cn

Keywords：

Neuraminidase; MG-63 cells; osteosarcoma; cell proliferation; apoptosis

DOI：

10.7507/1002-1892.201801077

Video：

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ObjectiveTo investigate the effect of attenuated expression of neuraminidase 3 (NEU3) via RNA interference on the proliferation and apoptosis in human osteosarcoma MG-63 cells.MethodsMG-63 cells were immunostained to observe the expression of NEU3. The cells were then divided into 5 groups: MG-63 cells in normal control group (group A) were not treated; MG-63 cells in 30, 50, and 100 nmol/L NEU3 RNA interference groups (groups B, C, and D) were transfected with 30, 50, and 100 nmol/L of NEU3 small interfering RNA (siRNA); negative control group (group E), MG-63 cells were transfected with different species negative siRNA (actin siRNA of mice, 50 nmol/L). The expression level of NEU3 mRNA was measured with real-time fluorescence quantitative PCR (qPCR). The proliferation of the cells was measured by cell counting kit 8 (CCK-8). The cell apoptosis rate was detected by flowcytometry (FCM). The expressions of cell apoptosis related proteins (Ras and Bcl-2) were detected by Western blot assay.ResultsNEU3 expressed in the cytoplasm of MG-63 cells under fluorescence microscope. The qPCR results showed that NEU3 mRNA levels were significantly lower in groups B, C, D than that in groups A and E (P<0.05) after 24 hours of transfection; meanwhile, with the increase of siRNA concentration, NEU3 mRNA levels were significantly decreased (P<0.05). The CCK-8 results showed that with the increase of siRNA concentration, the survival rate of MG-63 cells was significantly suppressed (P<0.05) and the apoptosis rate of MG-63 cells was significantly accelerated (P<0.05) after 48 hours of transfection. FCM results showed that after 24 hours of transfection, the number of live MG-63 cells decreased as that of the dead cells increased in groups B, C, D, and showing significant differences between 3 groups (P<0.05). While the apoptosis rate in groups B, C, and D showed significant difference when compared with that of group A (P<0.05); and when compared with group E, the apoptosis rate in groups C and D were significantly reduced (P<0.05), but there was no significant difference between groups B and E (P>0.05). The results of Western bolt assay showed that the protein levels of Ras and Bcl-2 in groups B and C were not significantly different from groups A and E (P>0.05), while the protein levels of Ras and Bcl-2 were significantly decreased in group D (P<0.05).ConclusionAttenuated expression of NEU3 could inhibit the survival of MG-63 cells and accelerate its apoptosis. The results suggest that NEU3 could be a possible target for treating osteosarcoma.

Citation： YANG Xiao, LI Si, XIANG Shang, WU Yilun, WANG Lin, PENG Jin, FENG Ying. Effect of attenuated expression of neuraminidase 3 via RNA interference on the proliferation and apoptosis of osteosarcoma MG-63 cells. Chinese Journal of Reparative and Reconstructive Surgery, 2018, 32(7): 887-892. doi: 10.7507/1002-1892.201801077 Copy

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17.	Jacobsen A, Bosch LJW, Martens-de Kemp SR, et al. Aurora kinase A (AURKA) interaction with Wnt and Ras-MAPK signalling pathways in colorectal cancer. Sci Rep, 2018, 8(1): 7522.
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20.	Perini GF, Ribeiro GN, Pinto Neto JV, et al. BCL-2 as therapeutic target for hematological malignancies. J Hematol Oncol, 2018, 11(1): 65.

1. Chen X, Varki A. Advances in the biology and chemistry of sialic acids. ACS Chem Biol, 2010, 5(2): 163-176.
2. Bhide GP, Colley KJ. Sialylation of N-glycans: mechanism, cellular compartmentalization and function. Histochem Cell Biol, 2017, 147(2): 149-174.
3. Vajaria BN, Patel PS. Glycosylation: a hallmark of cancer? Glycoconj J, 2017, 34(2): 147-156.
4. Vajaria BN, Patel KR, Begum R, et al. Sialylation: an avenue to target cancer cells. Pathol Oncol Res, 2016, 22(3): 443-447.
5. Li Y, Chen X. Sialic acid metabolism and sialyltransferases: natural functions and applications. Appl Microbiol Biotechnol, 2012, 94(4): 887-905.
6. Monti E, Miyagi T. Structure and function of mammalian sialidases. Top Curr Chem, 2015, 366: 183-208.
7. Miyagi T, Yamaguchi K. Mammalian sialidases: physiological and pathological roles in cellular functions. Glycobiology, 2012, 22(7): 880-896.
8. Miyagi T, Wada T, Yamaguchi K, et al. Plasma membrane-associated sialidase as a crucial regulator of transmembrane signalling. J Biochem, 2008, 144(3): 279-285.
9. Miyagi T, Takahashi K, Shiozaki K, et al. Plasma membrane-associated sialidase confers cancer initiation, promotion and progression. Adv Exp Med Biol, 2015, 842: 139-145.
10. Zheng C, Terreni M, Sollogoub M, et al. Ganglioside GM3 and its role in cancer. Curr Med Chem, 2018. [Epub ahead of print].
11. 陈阳, 吴英良, 山形贞子, 等. 细胞质唾液酸酶 (Neu2) 通过 Cdk2 通路抑制 FBJ-LL 细胞生长. 沈阳药科大学学报, 2010,27(2): 147-151.
12. Sandhu R, Lal H, Kundu ZS, et al. Serum fluoride and sialic acid levels in osteosarcoma. Biol Trace Elem Res, 2011, 144(1-3): 1-5.
13. 郭征. 我国骨肉瘤治疗的现状与问题及发展方向. 中国骨与关节杂志, 2015, 4(5): 338-342.
14. 耿磊, 陈继营, 许猛, 等. 骨肉瘤的治疗进展. 中国矫形外科杂志, 2015, 23(21): 1975-1978.
15. Hata K, Tochigi T, Sato I, et al. Increased sialidase activity in serum of cancer patients: Identification of sialidase and inhibitor activities in human serum. Cancer science, 2015, 106(4): 383-389.
16. Miyagi T, Takahashi K, Hata K, et al. Sialidase significance for cancer progression. Glycoconj J, 2012, 29(8-9): 567-577.
17. Jacobsen A, Bosch LJW, Martens-de Kemp SR, et al. Aurora kinase A (AURKA) interaction with Wnt and Ras-MAPK signalling pathways in colorectal cancer. Sci Rep, 2018, 8(1): 7522.
18. Khan AQ, Kuttikrishnan S, Siveen KS, et al. RAS-mediated oncogenic signaling pathways in human malignancies. Semin Cancer Biol, 2018. [Epub ahead of print].
19. Campbell KJ, Tait SWG. Targeting BCL-2 regulated apoptosis in cancer. Open Biol, 2018, 2(8): 443-460.
20. Perini GF, Ribeiro GN, Pinto Neto JV, et al. BCL-2 as therapeutic target for hematological malignancies. J Hematol Oncol, 2018, 11(1): 65.

Chinese Journal of Reparative and Reconstructive Surgery

Effect of attenuated expression of neuraminidase 3 via RNA interference on the proliferation and apoptosis of osteosarcoma MG-63 cells

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