摘要
目的了解非p53依赖通路突变型与野生型c-myc基因对p14^ARF基因表达调控及细胞凋亡的诱导功能。方法分别用携带突变型与野生型c-myc基因的慢病毒载体感染p53缺失型乳腺癌HCC1937细胞并得到二者过表达的稳定细胞株,未感染组及感染不携带c-myc基因的慢病毒细胞作空白对照和感染对照,采用反转录(RT)-PCR、Western印迹分别检测突变型与野生型c-myc及p14^ARF基因mRNA和蛋白表达,采用MTT、原位末端标记(TUNEL)检测突变型与野生型c-myc基因过表达乳腺癌HCC1937细胞增殖与凋亡情况。结果RT-PCR和Western印迹均显示突变型组与野生型组c-myc基因mRNA和蛋白过表达,突变型与野生型组c-myc蛋白[c-myc/β-肌动蛋白(actin)]表达量分别为0.560±0.010、0.651±0.010,与两对照组相比差异具有统计学意义(P〈0.05);野生型组p14^ARF蛋白(p14^ARF/β-actin)表达量为0.382±0.013,与两对照组相比差异均有统计学意义(均P〈0.05);突变型组p14^ARF蛋白(p14^ARF/β-actin)表达量为0.154±0.011,与两对照组相比,差异均无统计学意义(均P〉0.05)。突变型组细胞增殖活性显著高于野生型组(P〈0.05),突变型组与两对照组细胞凋亡率比较差异均无统计学意义(均P〉0.05),野生型组细胞凋亡率显著高于突变型组与两对照组(空白对照组为3.8%±0.5%,感染对照组为3.8%±0.4%,突变型组为3.2%±0.4%,野生型组为7.1%±0.7%)(均P〈0.05)。结论非p53依赖性通路中,野生型c-myc基因过表达能明显上调p14^ARF基因表达、诱导细胞凋亡,其促进增殖功能与诱导凋亡功能保持平衡,而突变后此作用丧失,致瘤性增加。
Objective To explore the regulation of p14^ARF expression and induction of cell apoptosis with the mutant and wild-type c-myc genes in a p53-independent pathway of signal transduction. Methods The mutant and wild-type c-myc genes were transfected by lentivirus into HCC1937 to form the stable overexpression cell lines. Uninfected cells and lentivirus-infected ones carrying no c-myc gene acted as blank and infection controls respectively. And c-myc and p14^ARF mRNA and protein, proliferation and apoptosis in HCC1937 with mutant and wild-type c-myc were detected by reverse transcription (RT)-PCR, Western blotting, thiazolyl blue tetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase mediated XdUTP nick end labeling (TUNEL) respectively. Results After the lentivirus-mediated gene transfer, cmyc mRNA and protein expression increased in the mutant and wild-type groups, p14^ARF mRNA and protein increased in the wild-type group and the mutant group and there were significant difference between them with blank and infection controls (mutant groups:0. 560±0. 010,0. 154 ± 0. 011, wild-type groups :0. 651± 0. 010, 0. 382 ± 0. 013, both P 〈 0. 05 ). The group of mutant and wild-type c-myc could promote the proliferation of cell growth. And c-myc was more effective to induce apoptosis in the wild-type group as compared with the mutant group (7.1% ± 0. 7% vs 3.2% ± 0.4%, P 〈 0. 05 ). Conclusions In a p53independent pathway, the over-expression of wild-type c-myc obviously up-regulates the expression of p14^ARF. And cell apoptosis may be induced through the regulation of pl4^ARF-related gene, keep balance of proliferative promotion and apoptosis induction. When there is a loss-of-function of mutant c-myc, tumorigenicity increases via a disturbed balance of proliferative promotion and apoptosis induction.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2012年第30期2140-2143,共4页
National Medical Journal of China
基金
山东省自然科学基金(Y2007C134)