Liu Zheng, Tian Jinhai, Peng Fuhong, Wang Jiang
Department of Oncology, People's hospital of Dongxihu District, Wuhan, Hubei 430040, P.R.China.
Department of Orthopedics, People's hospital of Dongxihu District, Wuhan, Hubei 430040, P.R.China.
J Cancer. 2022 Jan 1;13(1):304-312. doi: 10.7150/jca.62278. eCollection 2022.
Kidney cancers including clear cell carcinoma (RCC) are identified with very vulnerable mitochondria DNA (mtDNA) and frequent epigenetic aberrations. Bone metastasis from RCC is prevalent and destructive. Bone marrow contains a quite hypoxic microenvironment that usually insitigate 50% of hypermethylation events in conferring a selective advantage for tumor growth. We hypothesized that hypermethylation of mtDNA in RCC cells would significantly contribute to bone metastatic tumor progression. Methylation-specific polymerase chain reaction assay (MSP) was adopted to measure the methylation status of D-loop region of mtDNA in 15 pairs of bone metastatic and primary RCC as well as tumor adjescent normal kidney tissues. mtDNA copy number was examined by the real-time quantitative polymerase chain reaction (qPCR). Western blotting analysis was used to measure the accumulation of several DNA methyltransferases (DNMTs) in the mitochondria and nucleus fractions of bone metastatic RCC cells. mRNA expression of mitochondria encoded genes was examined by RT-PCR. Reactive oxygen species (ROS), mitochondrial membrane potential and ATP content were measured using cells treated with de-methylation drug 5-Azacytidine (5-Aza). Non-invasive bioluminescent imaging was performed to monitor tumor occurrence in skeleton in mice. Our results showed that the D-loop region in bone metastatic tumor cells was markedly hypermethylated than those in primary RCC tumor cells, that is associated with a decreased mtDNA copy number and accumulation of DNMT1 in the mitochondria. The bone-tropism tumor colonization and progression of RCC cells was significantly suppressed by demethylating the D-loop region of mtDNA and reducing the intracellular level of ROS and ATP by 5-Aza treatment. In conclusion, our study provided a direct association between hypermethylation of mtDNA in RCC with bone metastastic tumor growth.
包括透明细胞癌（肾细胞癌）在内的肾癌具有非常脆弱的线粒体DNA（mtDNA），且频繁出现表观遗传异常。肾细胞癌的骨转移很常见且具有破坏性。骨髓含有相当低氧的微环境，通常会引发50%的高甲基化事件，从而为肿瘤生长提供选择性优势。我们假设肾癌细胞中mtDNA的高甲基化会显著促进骨转移性肿瘤的进展。采用甲基化特异性聚合酶链反应分析（MSP）来检测15对骨转移性和原发性肾细胞癌以及肿瘤邻近正常肾组织中mtDNA D环区域的甲基化状态。通过实时定量聚合酶链反应（qPCR）检测mtDNA拷贝数。蛋白质免疫印迹分析用于测量骨转移性肾癌细胞线粒体和细胞核部分中几种DNA甲基转移酶（DNMTs）的积累。通过逆转录聚合酶链反应（RT-PCR）检测线粒体编码基因的mRNA表达。使用经去甲基化药物5-氮杂胞苷（5-Aza）处理的细胞测量活性氧（ROS）、线粒体膜电位和ATP含量。进行非侵入性生物发光成像以监测小鼠骨骼中的肿瘤发生情况。我们的结果表明，骨转移性肿瘤细胞中的D环区域甲基化程度明显高于原发性肾细胞癌肿瘤细胞，这与mtDNA拷贝数减少和线粒体中DNMT1的积累有关。通过对mtDNA的D环区域去甲基化以及用5-Aza处理降低细胞内ROS和ATP水平，显著抑制了肾癌细胞的骨嗜性肿瘤定植和进展。总之，我们的研究提供了肾细胞癌中mtDNA高甲基化与骨转移性肿瘤生长之间的直接关联。
