SNVs of KKU-023 cell line.

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（Cholangiocarcinoma, CCA）是一类起源于胆管的异质性恶性肿瘤集合。线粒体是真核细胞的能量转换器，其含有环状线粒体DNA（mitochondrial DNA, mtDNA），该DNA的突变率高于核DNA。线粒体DNA的异质性变异可能预示癌症相关死亡风险升高，可作为潜在的预后标志物。本研究针对5株胆管癌细胞系（KKU-023、KKU-055、KKU-100、KKU213A、KKU-452）的线粒体DNA变异展开分析，并以非肿瘤性胆管细胞系MMNK-1作为对照。本研究采用牛津纳米孔技术（Oxford Nanopore Technologies, ONT）——一种可完整拼接线粒体基因组的长读长测序技术，该技术可提升线粒体基因组复杂重排的识别效率。分析结果显示，单核苷酸变异（Single Nucleotide Variants, SNVs）与插入缺失变异（Insertions and Deletions, INDELs）出现频率较高，尤其集中于D-loop、MT-RNR2、MT-CO1、MT-ND4及MT-ND5基因区域。所有胆管癌细胞系均检测到显著突变，其中尤为值得关注的非同义单核苷酸变异包括：KKU-023的m.8462T>C、KKU-055的m.9493G>A、KKU-100的m.9172C>A、KKU-213A的m.15024G>C、KKU-452的m.12994G>A，以及MMNK-1的m.13406G>A，上述变异均表现出较高的致病性评分。此类突变的存在提示线粒体功能障碍与胆管癌进展存在潜在关联。对线粒体DNA结构变异（Structural Variants, SV）的分析显示，各细胞系间存在显著差异。本研究在KKU-023中鉴定出208个结构变异，KKU-055中185个，KKU-100中231个，KKU-213A中69个，KKU-452中172个，MMNK-1中217个。此类结构变异包含缺失、重复与倒位，其中KKU-100的变异程度最高，KKU-213A最低。本研究结果凸显了胆管癌细胞系中线粒体DNA突变谱的多样性，并强调了此类突变对线粒体功能及胆管癌细胞系进展的潜在影响。未来仍需开展相关研究，以阐明这些变异的功能效应、其与胆管癌中核DNA的相互作用，以及其作为治疗干预靶点的潜在价值。