去甲斑蝥素对神经母细胞瘤细胞增殖的影响

Influence of norcantharidin on neuroblastoma cell proliferation

目的 探讨去甲斑蝥素(norcantharidin, NCTD)对神经母细胞瘤细胞增殖的影响及其对C-MYC基因表达的调控作用。方法 采用实时荧光定量PCR法检测人神经母细胞瘤细胞SK-N-BE、SK-SY5Y、CHLA-119、SK-N-AS及人星形胶质细胞NHA c-Myc mRNA相对表达量,选择与NHA细胞c-Myc mRNA相对表达量差异最大的SK-N-AS细胞进行后续试验。对数生长期SK-N-AS细胞分为NCTD组(给予50μmol/L NCTD处理)和对照组(加入等体积细胞培养液),采用MTS法检测2组培养第1~5天细胞增殖的吸光度值,采用BrdU摄入实验检测2组培养第1天细胞BrdU摄入指数,采用实时荧光定量PCR法检测2组培养第4天c-Myc mRNA相对表达量,采用Western blot法检测2组培养第4天细胞c-Myc蛋白相对表达量。构建C-MYC基因的慢病毒过表达质粒和空白对照慢病毒质粒,将对数生长期SK-N-AS细胞分为NCTD+c-Myc组(加入50μmol/L NCTD及转染c-Myc过表达慢病毒质粒)、NCTD对照组(加入50μmol/L NCTD)、空白对照组(转染空白对照慢病毒质粒),采用MTS法检测3组培养第1~5天细胞增殖的吸光度值,采用Western blot法检测3组培养第4天细胞c-Myc蛋白相对表达量。结果 SK-N-BE、SK-SY5Y、CHLA-119、SK-N-AS细胞c-Myc mRNA相对表达量(2.04±0.34、2.34±0.55、2.70±0.76、4.54±0.58)均高于NHA细胞(1.02±0.23)(P<0.05),SK-N-AS细胞c-Myc mRNA相对表达量与NHA细胞差异最大,选择SK-N-AS细胞进行后续实验。培养第3~5天,NCTD组细胞增殖的吸光度值均高于对照组(P<0.05);培养第1天,NCTD组细胞BrdU摄入指数(0.56±0.12)低于对照组(1.11±0.25)(t=3.429,P=0.027);培养第4天,NCTD组细胞c-Myc mRNA(0.53±0.19)和c-Myc蛋白(0.11±0.00)相对表达量均低于对照组(0.96±0.16、1.00±0.01)(t=3.086,P=0.003;t=133.900,P<0.001)。培养第3~5天,空白对照组和NCTD+c-Myc组细胞增殖的吸光度值高于NCTD对照组(P<0.05),NCTD+c-Myc组与空白对照组比较差异无统计学意义(P>0.05);培养第4天,NCTD+c-Myc组和空白对照组细胞c-Myc蛋白相对表达量(0.79±0.12、0.83±0.05)均高于NCTD对照组(0.44±0.23)(P<0.05),空白对照组与NCTD+c-Myc组比较差异无统计学意义(P>0.05)。结论 NCTD通过抑制C-MYC基因的表达抑制神经母细胞瘤细胞增殖。

Objective To investigate the influences of norcantharidin(NCTD) on the proliferation of neuroblastoma and the regulation of C-MYC gene expression. Methods The relative expressions of c-Myc mRNA in human neuroblastoma cells SK-N-BE, SK-SY5 Y, CHLA-119 and SK-N-AS as well as human astrocytes NHA were detected by real-time fluorescence quantitative PCR. The SK-N-AS cells, with the largest difference from the relative expression of c-Myc mRNA in NHA cells, were selected for subsequent experiments. The SK-N-AS cells in logarithmic growth phase were divided into NCTD group(50 μmol/L NCTD treatment) and control group(equivalent volume culture). MTS method was used to detect the optical density(OD) of cell proliferation after 1-to 5-day culture in two groups, BrdU labeling index was detected by BrdU uptake assay after 1-day culture, real-time fluorescence quantitative PCR and Western blot were used to detect the relative expressions of c-Myc mRNA and protein after 4-day culture. The c-Myc overexpressed lentiviral plasmid and blank control lentiviral plasmid were constructed. The SK-N-AS cells in logarithmic growth phase were divided into NCTD+c-Myc group(50 μmol/L NCTD+transfecting c-Myc-overexpressed lentiviral plasmid), NCTD control group(50 μmol/L NCTD treatment) and blank control group(transfecting blank control lentiviral plasmid). MTS method was used to detect the OD of cell proliferation in three groups after 1-to 5-day culture, and Western blot was used to detect the relative expression of c-Myc protein after 4-day culture. Results The relative expression of c-Myc mRNA was higher in SK-N-BE, SK-SY5 Y, CHLA-119 and SK-N-AS cells(2.04±0.34, 2.34±0.55, 2.70±0.76, 4.54±0.58) than that in NHA cells(1.02±0.23), respectively(P<0.05). The relative expression of c-Myc mRNA in SK-N-AS cells was mostly different from that in NHA cells, and the SK-N-AS cells were selected for subsequent experiments. After 3-to 5-day culture, the OD value was higher in NCTD group than that in control group(P<0.05). After 1-day culture, the BrdU labeling index was lower in NCTD group(0.56±0.12) than that in control group(1.11±0.25)(t=3.429, P=0.027). After 4-day culture, the relative expressions of c-Myc mRNA and protein were lower in NCTD group(0.53±0.19,0.11±0.00)than those in control group(0.96±0.16,1.00±0.01)(t=3.086,P=0.003;t=133.900,P<0.001).After 3-to 5-day culture,the OD value was higher in blank control group and NCTD+c-Myc group than that in NCTD control group(P<0.05),and showed no significant difference between blank control group and NCTD+c-Myc group(P>0.05).After 4-day culture,the relative expression of c-Myc protein was higher in NCTD+c-Myc group(0.79±0.12)and blank control group(0.83±0.05)than that in NCTD control group(0.44±0.23)(P<0.05),and showed no significant difference between blank control group and NCTD+c-Myc group(P>0.05).Conclusion NCTD suppresses the proliferation of neuroblastoma cells by regulating the expression of C-MYCgene.