非p53依赖通路c-myc基因对p14^ARF基因的调控及细胞凋亡的诱导

Regulation of p14ARF expression and induction of cell apoptosis with c-myc in a p53-independentpathway

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.