Noxa基因转染乳腺癌MCF-7细胞的增殖抑制和促凋亡作用

doi:10.3971/j.issn.1000-8578.2010.10.003

Noxa基因转染乳腺癌MCF-7细胞的增殖抑制和促凋亡作用

Noxa Gene Inhibited Proliferation and Induced Apoptosis in Human Breast Cancer Cell Line MCF-7

摘要

摘要: 目的探讨Noxa基因转染乳腺癌MCF-7细胞的增殖抑制和促凋亡作用。方法利用脂质体将真核表达载体pIRES2-EGFP-Noxa瞬时转染至乳腺癌MCF-7细胞，通过RT-PCR检测转染后Noxa基因mRNA的表达，Western blot检测转染后蛋白的表达，MTT 比色法测定细胞增殖的抑制，流式细胞仪检测细胞的凋亡和细胞周期的变化，Hoechst 33342染色检测细胞的凋亡情况。结果 Noxa基因转染后在乳腺癌MCF-7细胞中成功表达。转染后mRNA及蛋白表达持续上升，其转染后24h、48h、72h mRNA的相对灰度值分别为(0.347±0.031)、(0.703±0.041)、(1.044±0.033)，差异具有统计学意义（P<0.05）。转染24h、48h、72h后蛋白表达的相对灰度值为(1.171±0.086)、(1.013±0.088)、(0.886±0.063)，差异具有统计学意义（P<0.05）。Noxa基因的表达使得乳腺癌MCF-7细胞出现增殖抑制，其24h、48h、72h抑制率分别为（23.9±4.2）%、（36.6±3.0）%、（47.0±3.3）%，差异具有统计学意义（P<0.05）。流式细胞仪检测显示MCF-7细胞DNA合成受到抑制，细胞周期主要抑制在G0/G1 期。其转染24h、48h、72h的G0/G1期分别为（68.1±2.5）%、（72.6±1.5）%、（75.6±0.9）%，与阴性对照组相比，差异具有统计学意义（P<0.05）；其24h、48h、72h凋亡率分别为（11.5±0.9）%、（19.6±0.8）%、（25.4±0.7）%,组间差异有统计学意义（P<0.05）。Hoechst 33342染色显示Noxa基因转染后细胞出现凋亡，其24h、48h、72h凋亡率分别为（7.3±4.1）%、（16.8±3.3）%、（23.8±2.3）%，与阴性对照组相比，其差异具有统计学意义（P<0.05）。结论 Noxa基因转染乳腺癌MCF-7细胞后能够抑制细胞增殖并促进其凋亡。

Abstract: Objective To explore the effect of Noxa gene on proliferation and apoptosis of human breast cancer cell line MCF-7. Methods The recombinant eukaryotic expression plasmid pIRES2-EGFP-Noxa was transiently transfected into human breast cancer cell line MCF-7 with lipofectamine. Both Noxa mRNA and protein were detected by RT-PCR and Western blot respectively. The inhibition of cell proliferation was evaluated by MTT assay. Changes in cell cycle were detected by flow cytometry(FCM).The cell apoptosis was studied by Hoechst 33342 staining. Results Exotic Noxa gene was expressed successfully in MCF-7 cells after transfected. The expression of Noxa mRNA and protein of MCF-7 cells was significantly increasing up-regulated after transfected 24h, 48h and 72h(0.347±0.031,0.703±0.041 and 1.044±0.033 for mRNA and 0.886±0.063,1.013±0.088 and 1.171±0.086 for protein,P<0.05). The Noxa expression inhibited MCF-7 cells proliferation with a time-dependent inhibition rate of (23.9%±4.2%),(36.6%±3.0%) and (47.0%±3.3%) for 24h, 48h and 72h, respectively. In Noxa overexpressed in MCF-7 cells the DNA synthesis was inhibited and arrested in G0/G1 phrase according to FCM. The percent of G0/G1 after transfected 24h、48h and 72h was (68.1%±2.5%),(72.6%±1.5%) and (75.6%±0.9%), respectively,and apoptosis rate was (11.5%±0.9%), (19.6%±0.8%), (25.4%±0.7%),respectively. It was significantly different compared with the control group(P<0.05). Apoptosis rate after transfected 24h, 48h and 72h was (7.3%±4.1%), (16.8%±3.3%) and (23.8%±2.3%), respectively.There were significant difference between Noxa and the control group(P<0.05). Conclusion Noxa gene overexpressed was able to effectively inhibit the proliferation and promote the apoptosis of breast cancer cells.

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