Faipan Athitaya, Sitthirak Sirinya, Wangwiwatsin Arporn, Namwat Nisana, Klanrit Poramate, Titapun Attapol, Jareanrat Apiwat, Thanasukarn Vasin, Khuntikeo Natcha, Boulter Luke, Dokduang Hasaya, Loilome Watcharin
Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen, Thailand.
PLoS One. 2025 Jun 9;20(6):e0323844. doi: 10.1371/journal.pone.0323844. eCollection 2025.
Cholangiocarcinoma (CCA) is a diverse collection of malignant tumors that originate in the bile ducts. Mitochondria, the energy converters in eukaryotic cells, contain circular mitochondrial DNA (mtDNA) which has a greater mutation rate than nuclear DNA. Heteroplasmic variations in mtDNA may suggest an increased risk of cancer-related mortality, serving as a potential prognostic marker. In this study, we investigated the mtDNA variations of five CCA cell lines, including KKU-023, KKU-055, KKU-100, KKU213A, and KKU-452 and compared them to the non-tumor cholangiocyte MMNK-1 cell line. We used Oxford Nanopore Technologies (ONT), a long-read sequencing technology capable of synthesizing the whole mitochondrial genome, which facilitates enhanced identification of complicated rearrangements in mitogenomics. The analysis revealed a high frequency of SNVs and INDELs, particularly in the D-loop, MT-RNR2, MT-CO1, MT-ND4, and MT-ND5 genes. Significant mutations were detected in all CCA cell lines, with particularly notable non-synonymous SNVs such as m.8462T > C in KKU-023, m.9493G > A in KKU-055, m.9172C > A in KKU-100, m.15024G > C in KKU-213A, m.12994G > A in KKU-452, and m.13406G > A in MMNK-1, which demonstrated high pathogenicity scores. The presence of these mutations suggests the potential for mitochondrial dysfunction and CCA progression. Analysis of mtDNA structural variants (SV) revealed significant variability among the cell lines. We identified 208 SVs in KKU-023, 185 SVs in KKU-055, 231 SVs in KKU-100, 69 SVs in KKU-213A, 172 SVs in KKU-452, and 217 SVs in MMNK-1. These SVs included deletions, duplications, and inversions, with the highest variability observed in KKU-100 and the lowest in KKU-213A. Our results underscore the diverse mtDNA mutation landscape in CCA cell lines, highlighting the potential impact of these mutations on mitochondrial function and CCA cell line progression. Future research is required to investigate the functional impacts of these variants, their interactions with nuclear DNA in CCA, and their potential as targets for therapeutic intervention.
胆管癌（CCA）是起源于胆管的多种恶性肿瘤的统称。线粒体是真核细胞中的能量转换器，包含环状线粒体DNA（mtDNA），其突变率高于核DNA。mtDNA的异质性变异可能提示癌症相关死亡率增加，可作为一种潜在的预后标志物。在本研究中，我们调查了5种CCA细胞系（包括KKU - 023、KKU - 055、KKU - 100、KKU213A和KKU - 452）的mtDNA变异，并将它们与非肿瘤胆管细胞MMNK - 1细胞系进行比较。我们使用了牛津纳米孔技术（ONT），这是一种能够合成整个线粒体基因组的长读长测序技术，有助于增强对有丝分裂基因组学中复杂重排的识别。分析显示单核苷酸变异（SNV）和插入缺失（INDEL）的频率很高，特别是在D环、MT - RNR2、MT - CO1、MT - ND4和MT - ND5基因中。在所有CCA细胞系中均检测到显著突变，特别是显著的非同义SNV，如KKU - 023中的m.8462T＞C、KKU - 055中的m.9493G＞A、KKU - 100中的m.9172C＞A、KKU - 213A中的m.15024G＞C、KKU - 452中的m.12994G＞A以及MMNK - 1中的m.13406G＞A，这些均显示出高致病性评分。这些突变的存在提示线粒体功能障碍和CCA进展的可能性。对mtDNA结构变异（SV）的分析显示细胞系之间存在显著差异。我们在KKU - 023中鉴定出208个SV，在KKU - 055中鉴定出185个SV，在KKU - 100中鉴定出231个SV，在KKU - 213A中鉴定出69个SV，在KKU - 452中鉴定出172个SV，在MMNK - 1中鉴定出217个SV。这些SV包括缺失、重复和倒位，其中KKU - 100中观察到的变异性最高，KKU - 213A中最低。我们 的结果强调了CCA细胞系中多样的mtDNA突变格局，突出了这些突变对线粒体功能和CCA细胞系进展的潜在影响。未来需要开展研究来调查这些变异的功能影响、它们在CCA中与核DNA的相互作用以及它们作为治疗干预靶点的潜力。
