卷曲螺旋结构域蛋白137在骨肉瘤组织中高表达并促进细胞增殖、迁移及血管新生

Expression of coiled-coil domain containing 137 in osteosarcoma and its effect on osteosarcoma cell proliferation,migration and angiogenesis

目的探讨卷曲螺旋结构域蛋白137(CCDC137)在骨肉瘤组织中的表达水平,及对骨肉瘤细胞增殖、迁移及血管新生的影响。方法免疫印记及实时定量PCR实验用于检测CCDC137在肿瘤组织中的表达水平及小干扰RNA(siRNA)下调CCDC137后的沉默效率验证;噻唑蓝(MTT)及细胞计数试剂盒(CCK-8)实验用于检测CCDC137对骨肉瘤细胞增殖的影响。划痕实验及管状结构形成实验用于检测CCDC137对骨肉瘤细胞迁移及血管新生的影响;免疫荧光实验检测CCDC137对骨肉瘤细胞极化的影响,组间比较采用t检验。结果CCDC137在骨肉瘤组织(1.56±0.45)表达高于癌旁组织(0.99±0.28),差异有统计学意义(t=4.854,P<0.01);下调CCDC137后抑制骨肉瘤细胞的增殖,两条siRNA抑制CCDC137表达后490 nm波长处吸光度值(0.22±0.18、0.23±0.16)低于对照组(0.46±0.16),差异均有统计学意义(t_(1)=2.956,t_(2)=3.025,P<0.01),CCK-8实验抑制CCDC137表达后吸光度值(0.16±0.12、0.18±0.14)低于对照组(0.37±0.18),差异均有统计学意义(t_(1)=2.843,t_(2)=2.361,P<0.01);下调CCDC137后抑制内皮细胞迁移,两条siRNA抑制CCDC137表达后迁移能力(62.67±7.51、54.67±2.52)低于对照组(81.33±6.11),差异均有统计学意义(t_(1)=3.341,t_(2)=6.990,P<0.01);两条siRNA沉默CCDC137后血管新生能力(2.33±0.40、2.63±0.32)低于对照组(5.73±0.55),差异均有统计学意义(t_(1)=8.621,t_(2)=8.420,P<0.01)。下调CCDC137后骨肉瘤细胞极化(52.67±4.04)细胞数目低于对照组(83.33±4.51),差异均有统计学意义(t=8.772,P<0.01);角度(35.27±10.56)低于对照组(64.27±12.66),差异均有统计学意义(t=6.812,P<0.01)。结论CCDC137能够促进骨肉瘤细胞增殖、迁移、血管新生及细胞极化。

Objective To investigate the expression level of coiled-coil domain containing 137(CCDC137)in osteosarcoma and its effect on osteosarcoma cell proliferation,migration and angiogenesis.Methods Immunoimprinting and real-time quantitative PCR were used to detect the expression level of CCDC137 in tumor tissues and verify the silencing efficiency of CCDC137 down-regulated by small interfering RNA(siRNA).Methyl thiazolyl tetrazolium(MTT)and cell counting kit-8(CCK-8)assays were used to detect the effect of CCDC137 on osteosarcoma cell proliferation.The effects of CCDC137 on osteosarcoma cell migration and angiogenesis were investigated by scratch test and tubular structure formation test.The effect of CCDC137 on the polarization of osteosarcoma cells was detected by immunofluorescence assay.The t-test is used to determine statistical significance.Results CCDC137 expression was significantly increased in osteosarcoma tissues(1.56±0.45)compared with paracancer tissues(0.99±0.28),and the difference was statistically significant(t=4.854,P<0.01).The proliferation of osteosarcoma cells was inhibited by downregulation of CCDC137,and the absorbance value at 490 nm wavelength(0.22±0.18,0.23±0.16)was significantly lower than that of the control group(0.46±0.16),with statistical significance(t_(1)=2.956,t_(2)=3.025,P<0.01),the absorbance value(0.16±0.12,0.18±0.14)of CCK-8 significantly decreased compared with the control group(0.37±0.18),the differences were statistically significant(t_(1)=2.843,t_(2)=2.361,P<0.01).Down-regulation of CCDC137 inhibited the migration of endothelial cells,and the migration ability of CCDC137 inhibited by two sirnas(62.67±7.51,54.67±2.52)was significantly lower than that of the control group(81.33±6.11),with statistical significance(t_(1)=3.341,t_(2)=6.990,P<0.01);The angiogenesis of CCDC137 silenced by two siRNA groups(2.33±0.40,2.63±0.32)was significantly lower than that of the control group(5.73±0.55),and the differences were statistically significant(t_(1)=8.621,t_(2)=8.420,P<0.01).After CCDC137 down-regulation,the number of osteosarcoma cell polarization(52.67±4.04)decreased significantly compared with the control group(83.33±4.51),with statistical significance(t=8.772,P<0.01).The Angle(35.27±10.56)was significantly lower than that of the control group(64.27±12.66),with statistical significance(t=6.812,P<0.01).Conclusion We believe that CCDC137 can promote the proliferation,migration,angiogenesis and cell polarization of osteosarcoma cells.