Department of Gastroenterological Surgery, Graduate School of Medicine, Osaka University, Osaka, Japan.
PLoS One. 2018 Feb 15;13(2):e0193159. doi: 10.1371/journal.pone.0193159. eCollection 2018.
Alterations in mitochondrial DNA (mtDNA) copy numbers in various human cancers have been studied, but any such changes in esophageal squamous cell carcinoma (ESCC) are not established. In the present study, we investigated the correlation of mtDNA copy number with clinicopathologic features, prognosis, and malignant potential of ESCC. MtDNA copy numbers of resected specimens from 80 patients treated with radical esophagectomy were measured by quantitative real-time PCR analyses. Human ESCC cells, TE8 and TE11, were cultured, and depletion of mtDNA content was induced by knockdown of mitochondrial transcription factor A expression or treatment with ethidium bromide. The mRNA and protein expression, proliferation, invasion, and cell cycle were investigated. The results showed that the mtDNA copy number of cancerous portions was 56.0 (37.4-234.5) percent that of non-cancerous parts and significantly lower (p<0.01). Low mtDNA copy number in resected cancerous tissues was significantly correlated with pathological depth of tumor invasion (p = 0.045) and pathological stage (p = 0.025). Patients with lower mtDNA copy number had significantly poorer 5-year overall survival compared to patients with higher levels (p<0.01). The mtDNA-depleted TE8 and TE11 cells had morphological changes and proliferated more slowly than control cells under normoxia but proliferated at almost the same rate under hypoxic conditions. In mtDNA-depleted cells, E-cadherin mRNA expression was decreased, and N-cadherin, vimentin, zeb-1, and cd44 mRNA expression was increased. Immunoblotting and flow cytometry analysis also showed downregulated E-cadherin and upregulated N-cadherin and CD44 protein in mtDNA-depleted cells. Moreover, mtDNA-depleted cells had enhanced invasion, migration, and sphere formation abilities, and the cell cycle arrest at G0/G1 phase was induced in these cells. These results suggested that mtDNA-depleted ESCC cells had mesenchymal characteristics, cancer stemness, and tolerance to hypoxia, which played important role in cancer progression. In conclusion, a low copy number of mtDNA is associated with tumor progression in ESCC.
线粒体 DNA（mtDNA）拷贝数在各种人类癌症中的改变已经被研究过，但食管癌（ESCC）中是否存在任何此类变化尚未确定。在本研究中，我们调查了 mtDNA 拷贝数与 ESCC 的临床病理特征、预后和恶性潜能的相关性。通过定量实时 PCR 分析测量了 80 例接受根治性食管切除术治疗的患者切除标本中的 mtDNA 拷贝数。培养人 ESCC 细胞 TE8 和 TE11，并通过敲低线粒体转录因子 A 的表达或用溴化乙锭处理来诱导 mtDNA 含量耗竭。研究了 mRNA 和蛋白质表达、增殖、侵袭和细胞周期。结果表明，癌组织部分的 mtDNA 拷贝数为非癌组织部分的 56.0（37.4-234.5）%，显著降低（p<0.01）。切除癌组织中 mtDNA 拷贝数低与肿瘤侵袭的病理深度（p=0.045）和病理分期（p=0.025）显著相关。mtDNA 拷贝数较低的患者与 mtDNA 拷贝数较高的患者相比，5 年总生存率显著降低（p<0.01）。在常氧条件下，mtDNA 耗竭的 TE8 和 TE11 细胞形态发生变化，增殖速度较慢，但在低氧条件下增殖速度几乎相同。在 mtDNA 耗竭的细胞中，E-钙粘蛋白 mRNA 表达减少，而 N-钙粘蛋白、波形蛋白、zeb-1 和 cd44 mRNA 表达增加。免疫印迹和流式细胞术分析也显示 mtDNA 耗竭细胞中 E-钙粘蛋白下调和 N-钙粘蛋白和 CD44 蛋白上调。此外，mtDNA 耗竭细胞的侵袭、迁移和球体形成能力增强，并且这些细胞的细胞周期在 G0/G1 期停滞。这些结果表明，mtDNA 耗竭的 ESCC 细胞具有间充质特征、癌症干性和对缺氧的耐受性，这在癌症进展中发挥了重要作用。总之，mtDNA 低拷贝数与 ESCC 中的肿瘤进展有关。
