国际肿瘤学杂志

国际肿瘤学杂志 ›› 2019, Vol. 46 ›› Issue (4): 193-198.doi: 10.3760/cma.j.issn.1673-422X.2019.04.001

• 论著 •    下一篇

质膜型唾液酸酶NEU3活性对骨肉瘤MG-63细胞增殖与凋亡的影响

杨晓1,李思2,彭锦3,王琳4,吴衣论4,冯颖2   

  1. 1四川省医学科学院·四川省人民医院骨科，成都  610072； 2四川大学华西基础医学与法医学院，成都  610041； 3四川大学华西临床医学院，成都  610041； 4四川大学华西第二医院出生缺陷与相关妇儿疾病教育部重点实验室，成都  610041
  • 收稿日期:2019-01-10 修回日期:2019-02-19 出版日期:2019-04-08 发布日期:2019-05-29
  • 通讯作者: 冯颖，Email： yingfeng27@scu.edu.cn E-mail:yingfeng27@scu.edu.cn
  • 基金资助:

    四川省科技计划项目(2019YFS0318);四川省卫生和计划生育委员会课题（16PJ434）

Effect of plasma membrane-associated sialidase NEU3 activity on the proliferation and apoptosis of osteosarcoma MG-63 cells

Yang Xiao1, Li Si2, Peng Jin3, Wang Lin4, Wu Yilun4, Feng Ying2   

  1. 1Department of Orthopedics, Sichuan Academy of Medical Sciences & Sichuan Provincial People′s Hospital, Chengdu 610072, China; 2West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu 610041, China; 3West China School of Medicine, Sichuan University, Chengdu 610041, China; 4Key Laboratory of Birth Defects and Related Diseases of Women and Children, Ministry of Education, West China Second University Hospital, Sichuan University, Chengdu 610041, China
  • Received:2019-01-10 Revised:2019-02-19 Online:2019-04-08 Published:2019-05-29
  • Contact: Feng Ying, Email: yingfeng27@scu.edu.cn E-mail:yingfeng27@scu.edu.cn
  • Supported by:

    Science and Technology Planning Project of Sichuan Province of China (2019YFS0318); Health and Family Planning Commission of Sichuan Province of China (16PJ434)

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摘要： 目的  探讨质膜型唾液酸酶（NEU3）活性对骨肉瘤MG-63细胞在体外增殖及凋亡的调控作用。方法  体外培养MG-63细胞，以抗NEU3抗体免疫荧光染色，显示NEU3在MG-63细胞中的定位；以0 nmol/L唾液酸酶活性抑制剂（DANA）处理组或0 μg/ml抗NEU3抗体处理组为空白对照组，以10、20、50 nmol/L DANA或0.5、1.0、2.0 μg/ml抗NEU3抗体处理24 h 或48 h后，采用CCK-8比色法、流式细胞术分别检测细胞增殖抑制率和细胞凋亡率，Western blotting法检测癌基因相关蛋白Ras蛋白和Bcl-2蛋白的表达水平。结果  免疫荧光染色结果显示，NEU3表达定位于MG-63细胞质中。作用48 h后，0、10、20、50 nmol/L DANA处理组细胞增殖抑制率分别为0、15.10%±3.23%、41.46%±2.31%、64.68%±4.12%，差异具有统计学意义（F=99.90，P＜0.001），不同DANA浓度组间两两比较，差异均具有统计学意义（均P＜0.05）；0、0.5、1.0、2.0 μg/ml抗NEU3抗体封闭处理48 h后，MG-63细胞增殖抑制率分别为0、9.34%±1.53%、19.66%±4.18%、42.50%±5.68%，差异具有统计学意义（F=25.67，P＜0.001），不同抗体浓度组间两两比较，除0.5 μg/ml与1.0 μg/ml组比较差异无统计学意义外（P>0.05），其余组间差异均具有统计学意义（均P＜0.05）。采用不同浓度DANA（0、10、20、50 nmol/L）处理MG-63细胞24 h后，凋亡率分别为4.05%±0.07%、4.15%±0.23%、12.85%±1.48%、8.29%±0.86%，组间差异具有统计学意义（F=23.21，P<0.001），不同DANA浓度组间两两比较，除0 nmol/L与10 nmol/L、20 nmol/L与50 nmol/L处理组间差异无统计学意义外（P>0.05），其余组间比较差异均有统计学意义（均P<0.05）；以不同浓度NEU3抗体（0、0.5、1.0、2.0 μg/ml）处理MG-63细胞24 h后，细胞凋亡率分别为4.05%±0.07%、20.13%±2.97%、20.29%±2.82%、20.58%±0.70%，组间差异有统计学意义（F=15.36，P=0.001），不同抗体浓度组间两两比较，0 μg/ml与各处理组间差异有统计学意义（均P<0.05），而各处理组间差异无统计学意义（P>0.05）。Western blotting结果显示随DANA和NEU3抗体浓度升高，Bcl-2蛋白和Ras蛋白的表达逐渐降低。结论  抑制NEU3酶活性可抑制骨肉瘤细胞MG-63的存活，促进该细胞的凋亡，其作用机制可能是通过下调癌基因相关蛋白Ras和Bcl-2的表达实现的，提示NEU3可能是治疗骨肉瘤的潜在药物靶点。

关键词: 神经氨酸酶, 骨肉瘤, 细胞增殖, 细胞凋亡, MG-63细胞

Abstract: Objective To investigate the effect of plasma membrane-associated sialidase 3 (NEU3) activity on the proliferation and apoptosis of osteosarcoma MG-63 cells in vitro. Methods  MG-63 cells were cultured in vitro. Anti-NEU3 antibody (Ab) immunofluorescent staining was used to indicate the cellular localization of NEU3 in MG-63 cells. The cells treated with 0 nmol/L 2-deoxy-2,3didehydro-N-acetyl neuraminic acid (DANA) or 0 μg/ml anti-NEU3 Ab were used as blank control groups. The cells were treated with 10, 20, 50 nmol/L DANA, or 0.5, 1.0, 2.0 μg/ml antiNEU3 Ab for 24 h or 48 h, respectively. The inhibition rates of the cell proliferation and cell apoptosis rates were measured with CCK-8 and flow cytometry. The expression levels of oncogene-related proteins, Ras protein and Bcl-2 protein, were detected by Western blotting. Results  The immunofluorescence result showed that NEU3 was located in the cytoplasm of MG-63 cell. After treating with 0, 10, 20, 50 nmol/L DANA for 48 h, the inhibition rates of cell proliferation were 0, 15.10%±3.23%, 41.46%±2.31%, 64.68%±4.12%, with significant statistical difference (F=99.90, P<0.001), and the following contrast between each two groups met the statistical significance (all P<0.05). After treating with 0, 0.5, 1.0, 2.0 μg/ml anti-NEU3 Ab for 48 h, the inhibition rates of cell proliferation were 0, 9.34%±1.53%, 19.66%±4.18%, 42.50%±5.68%, and the difference was statistically significant (F=25.67, P<0.001), and the following contrast between each two groups met the statistical significance (P<0.05), except the difference between 0.5 and 1.0 μg/ml groups (P>0.05). When the MG-63 cells were treated with 0, 10, 20, 50 nmol/L DANA for 24 h, the cell apoptosis rates were 4.05%±0.07%, 4.15%±0.23%, 12.85%±1.48%, 8.29%±0.86%, respectively, and the difference was statistically significant (F=23.21, P<0.001). And the following contrast between each two groups met the statistical significance (P<0.05), except the differences between 0 nmol/L and 10 nmol/L, 20 nmol/L and 50 nmol/L groups (P>0.05). When the MG-63 cells were treated with 0, 0.5, 1.0, 2.0 μg/ml anti-NEU3 Ab for 24 h, the cell apoptosis rates were 4.05%±0.07%, 20.13%±2.97%, 20.29%±2.82%, 20.58%±0.70%, with statistical significant difference (F=15.36, P=0.001). And the following contrast between each two groups showed that the differences between 0 μg/ml and each treated group were statistically significant (P<0.05), while the differences between two treated groups were not statistically significant (P>0.05). Western blotting results showed that the expression levels of Ras and Bcl-2 decreased with the increasing concentrations of DANA and anti-NEU3. Conclusion  Inhibition of NEU3 enzyme activity can suppress the survival rate of MG63 cells and increase the cell apoptosis. The possible mechanism may be related to the declined expression of oncogene-related proteins Ras and Bcl-2, which suggests that NEU3 may be a possible target for treating osteosarcoma.

Key words: Neuraminidase, Osteosarcoma, Cell proliferation, Apoptosis, MG-63 cells