下一代测序揭示了结直肠癌患者血浆细胞外囊泡中的线粒体基因组多样性。
Next-generation sequencing reveals mitogenome diversity in plasma extracellular vesicles from colorectal cancer patients.
机构信息
Department of Oncology, Akershus University Hospital, P.O. Box 1000, 1478, Lørenskog, Norway.
Department of Physics, Norwegian University of Science and Technology, Trondheim, Norway.
出版信息
BMC Cancer. 2023 Jul 12;23(1):650. doi: 10.1186/s12885-023-11092-x.
BACKGROUND
Recent reports have demonstrated that the entire mitochondrial genome can be secreted in extracellular vesicles (EVs), but the biological attributes of this cell-free mitochondrial DNA (mtDNA) remain insufficiently understood. We used next-generation sequencing to compare plasma EV-derived mtDNA to that of whole blood (WB), peripheral blood mononuclear cells (PBMCs), and formalin-fixed paraffin-embedded (FFPE) tumor tissue from eight rectal cancer patients and WB and fresh-frozen (FF) tumor tissue from eight colon cancer patients.
METHODS
Total DNA was isolated before the mtDNA was enriched by PCR with either two primer sets generating two long products or multiple primer sets (for the FFPE tumors), prior to the sequencing. mtDNA diversity was assessed as the total variant number, level of heteroplasmy (mutant mtDNA copies mixed with wild-type copies), variant distribution within the protein-coding genes, and the predicted functional effect of the variants in the different sample types. Differences between groups were compared by paired Student's t-test or ANOVA with Dunnett's multiple comparison tests when comparing matched samples from patients. Mann-Whitney U test was used when comparing differences between the cancer types and patient groups. Pearson correlation analysis was performed.
RESULTS
In both cancer types, EV mtDNA presented twice as many variants and had significantly more low-level heteroplasmy than WB mtDNA. The EV mtDNA variants were clustered in the coding regions, and the proportion of EV mtDNA variants that were missense mutations (i.e., estimated to moderately affect the mitochondrial protein function) was significantly higher than in WB and tumor tissues. Nonsense mutations (i.e., estimated to highly affect the mitochondrial protein function) were only observed in the tumor tissues and EVs.
CONCLUSION
Taken together, plasma EV mtDNA in CRC patients exhibits a high degree of diversity.
TRIAL REGISTRATION
ClinicalTrials.gov: NCT01816607 . Registered 22 March 2013.
背景
最近的报告表明，整个线粒体基因组可以在外泌体（EVs）中分泌，但这种细胞外游离线粒体 DNA（mtDNA）的生物学特性还了解不足。我们使用下一代测序技术比较了 8 例直肠癌患者血浆 EV 衍生 mtDNA 与全血（WB）、外周血单核细胞（PBMCs）和福尔马林固定石蜡包埋（FFPE）肿瘤组织，以及 8 例结肠癌患者 WB 和新鲜冷冻（FF）肿瘤组织的 mtDNA。
方法
在使用两种引物组产生两个长产物或多种引物组（用于 FFPE 肿瘤）对 mtDNA 进行 PCR 富集之前，先从总 DNA 中分离 mtDNA，然后进行测序。通过总变异数、异质性水平（突变 mtDNA 拷贝与野生型拷贝混合）、蛋白编码基因内变异分布以及不同样本类型中变异的预测功能效应，评估 mtDNA 多样性。比较患者匹配样本时，使用配对 Student's t 检验或方差分析与 Dunnett 多重比较检验比较组间差异。比较癌症类型和患者组之间的差异时，使用 Mann-Whitney U 检验。进行 Pearson 相关性分析。
结果
在两种癌症类型中，EV mtDNA 的变异数是 WB mtDNA 的两倍，且低水平异质性显著更高。EV mtDNA 变异聚类在编码区，EV mtDNA 变异中错义突变（即估计中度影响线粒体蛋白功能）的比例明显高于 WB 和肿瘤组织。无义突变（即估计高度影响线粒体蛋白功能）仅在肿瘤组织和 EV 中观察到。
结论
总之，CRC 患者的血浆 EV mtDNA 表现出高度的多样性。
试验注册
ClinicalTrials.gov：NCT01816607。2013 年 3 月 22 日注册。
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