Yuan Yang, Wang Weixing, Li Huizhong, Yu Yongwei, Tao Jin, Huang Shengdong, Zeng Zhiyong
Department of Cardiothoracic Surgery, Changhai Hospital, Shanghai, P R China.
Department of Medical Imaging, Changhai Hospital, Shanghai, P R China.
BMC Cancer. 2015 May 2;15:346. doi: 10.1186/s12885-015-1349-z.
Previous study showed that mitochondrial ND6 (mitND6) gene missense mutation resulted in NADH dehydrogenase deficiency and was associated with tumor metastasis in several mouse tumor cell lines. In the present study, we investigated the possible role of mitND6 gene nonsense and missense mutations in the metastasis of human lung adenocarcinoma.
The presence of mitND6 gene mutations was screened by DNA sequencing of tumor tissues from 87 primary lung adenocarcinoma patients and the correlation of the mutations with the clinical features was analyzed. In addition, we constructed cytoplasmic hybrid cells with denucleared primary lung adenocarcinoma cell as the mitochondria donor and mitochondria depleted lung adenocarcinoma A549 cell as the nuclear donor. Using these cells, we studied the effects of mitND6 gene nonsense and missense mutations on cell migration and invasion through wounding healing and matrigel-coated transwell assay. The effects of mitND6 gene mutations on NADH dehydrogenase activity and ROS production were analyzed by spectrophotometry and flow cytometry.
mitND6 gene nonsense and missense mutations were detected in 11 of 87 lung adenocarcinoma specimens and was correlated with the clinical features including age, pathological grade, tumor stage, lymph node metastasis and survival rate. Moreover, A549 cell containing mitND6 gene nonsense and missense mutation exhibited significantly lower activity of NADH dehydrogenase, higher level of ROS, higher capacity of cell migration and invasion, and higher pAKT and pERK1/ERK2 expression level than cells with the wild type mitND6 gene. In addition, NADH dehydrogenase inhibitor rotenone was found to significantly promote the migration and invasion of A549 cells.
Our data suggest that mitND6 gene nonsense and missense mutation might promote cell migration and invasion in lung adenocarcinoma, probably by NADH dehydrogenase deficiency induced over-production of ROS.
先前的研究表明，线粒体ND6（mitND6）基因错义突变导致NADH脱氢酶缺乏，并与几种小鼠肿瘤细胞系中的肿瘤转移相关。在本研究中，我们调查了mitND6基因无义突变和错义突变在人肺腺癌转移中的可能作用。
通过对87例原发性肺腺癌患者肿瘤组织进行DNA测序，筛查mitND6基因突变的存在情况，并分析突变与临床特征的相关性。此外，我们构建了以去核原发性肺腺癌细胞为线粒体供体、线粒体缺失的肺腺癌A549细胞为核供体的细胞质杂交细胞。利用这些细胞，通过划痕愈合实验和基质胶包被的Transwell实验，研究mitND6基因无义突变和错义突变对细胞迁移和侵袭的影响。通过分光光度法和流式细胞术分析mitND6基因突变对NADH脱氢酶活性和活性氧（ROS）产生的影响。
在87例肺腺癌标本中的11例中检测到mitND6基因无义突变和错义突变，且与年龄、病理分级、肿瘤分期、淋巴结转移和生存率等临床特征相关。此外，含有mitND6基因无义突变和错义突变的A549细胞，与野生型mitND6基因的细胞相比，表现出显著更低的NADH脱氢酶活性、更高的ROS水平、更高的细胞迁移和侵袭能力，以及更高的pAKT和pERK1/ERK2表达水平。此外，发现NADH脱氢酶抑制剂鱼藤酮可显著促进A549细胞的迁移和侵袭。
我们的数据表明，mitND6基因无义突变和错义突变可能通过NADH脱氢酶缺乏诱导的ROS过量产生促进肺腺癌细胞的迁移和侵袭。
