抑癌基因NDRG2对人结肠癌细胞系DLD-1丙酮酸脱氢酶(PDH)的表达和有氧氧化代谢的调节  

Tumor suppressor gene NDRG2 regulates the expression of pyruvate dehydrogenase( PDH) and oxidative phosphorylation in human colorectal carcinoma cell line DLD- 1

作　　者：徐欣元[1] 雷世雄 沈岚[1] 

机构地区：[1]第四军医大学生物化学和分子生物学教研室,陕西西安710032 [2]第四军医大学唐都医院普外科,陕西西安710038

出　　处：《现代肿瘤医学》2015年第5期581-585,共5页Journal of Modern Oncology

基　　金：国家自然科学基金资助项目(编号:31171112;81230043)

摘　　要：目的:采用重组慢病毒系统,通过过表达和RNA干涉的方法分别增加和降低NDRG2(N-Myc downstream regulated gene 2)基因后,检测人结肠癌细胞系DLD-1中丙酮酸脱氢酶(PDH)的表达水平及氧消耗率(OCR)。方法:分别采用表达NDRG2慢病毒载体p Lenti6-NDRG2和NDRG2 shRNA慢病毒载体p LKO.1-sh NDRG2制备病毒载体并感染DLD-1细胞,经两周耐药筛选,获得稳定感染的细胞株。采用Real-time PCR和Western blot的方法明确NDRG2在稳定感染细胞株中的表达情况,再使用上述方法检测稳定感染细胞株中PDH的表达水平,最后,通过Seahorse能量呼吸代谢仪,检测稳定感染细胞株中的OCR。结果:Realtime PCR和Western blot结果表明,在慢病毒p Lenti6-NDRG2稳定感染的DLD-1细胞中,NDRG2表达上调,PDH表达上调;p LKO.1-sh NDRG2稳定感染的DLD-1细胞中,NDRG2的表达下调,PDH表达下调。Seahorse结果表明,NDRG2过表达细胞株中细胞的OCR上升,而NDRG2 RNA干涉细胞株中细胞的OCR明显下降。结论:通过过表达和抑制NDRG2基因,证实了NDRG2作为抑癌候选基因能够有效促进结肠癌细胞系DLD-1中丙酮酸脱氢酶PDH的表达,并促进有氧氧化。Objective：To investigate the effects of over - expression and knockdown of NDRG2（N - Myc down-stream regulated gene 2）gene on the expression of pyruvate dehydrogenase（ PDH）and oxygen consumption rate （OCR）of human colorectal carcinoma DLD - 1 cells by using recombinant lentivirus vectors containing NDRG2 or NDRG2 shRNA. Methods：The lentiviruses containing NDRG2 or NDRG2 shRNA,prepared separately from NDRG2 lentiviral expression vector pLenti6 - NDRG2 or NDRG2 shRNA lentiviral expression vector pLKO. 1 - shNDRG2, were used to infect human colorectal carcinoma cell line DLD - 1. The stable cell lines were obtained by selection with blasticidin or puromycin for 2 weeks. Real - time PCR and Western blot were employed to examine the expression of NDRG2 and PDH in these stable cell lines. Seahorse Extracellular Flux（XF24e ）analyzer was used to determine basal oxygen consumption rate（OCR）of these stable cell lines. Results：The mRNA and protein levels of NDRG2 were up -regulated in DLD - 1 cell lines infected with pLenti6 - NDRG2,and down - regulated in those with pLKO. 1 -shNDRG2. The PDH and OCR were up - regulated in DLD - 1 cell lines stably infected with pLenti6 - NDRG2,and down - regulated in those with pLKO. 1 - shNDRG2. Conclusion：NDRG2,as a candidate tumor suppressor gene, could promote the expression of PDH and boost oxidative phosphorylation.

关 键 词：NDRG2 肿瘤 慢病毒 PDH OCR 

分 类 号：R730.23[医药卫生—肿瘤] R735.35[医药卫生—临床医学]