PRDX3（Thioredoxin-dependent peroxide reductase）作为存在于线粒体的过氧化物还原酶，能够调节胞内的氧化还原状态。线粒体DNA相比细胞核DNA，由于其靠近电子传递链，更容易受到氧化损伤。UNG1（Isoform 1 of Uracil-DNA glycosylase）也是一个主要存在于线粒体中的碱基修复酶，主要负责启动碱基切除修复，修复线粒体DNA中由于氧化或突变导致的尿嘧啶。实验室前期研究中发现UNG1过表达细胞（UNG1（+））相比空载细胞（UNG1（-））更能抵抗氧化应激。此外我们发现在氧化应激过程中，PRDX3主要结合UNG1，并且UNG1和PRDX3可以相互IP下来。我们也进一步证明，在过氧化氢条件下，含有尿嘧啶的oligo DNA能够特异性结合PRDX3 和UNG1复合体，这表明PRDX3可能通过与UNG1相互作用，来清除ROS，从而保护线粒体DNA完整性。UNG1能够招募PRDX3行使保护UNG1不受氧化损伤的功能。另一方面，ROS能够诱导A549wt细胞和UNG1（+）中UNG1的降解，而在氧化应激条件下，UNG1的mRNA水平却有增加。ROS介导的UNG1的降解，不依赖于泛素蛋白酶体途径，而是主要由线粒体里特有的蛋白酶LonP1介导。有报道称PRDX3在肝细胞癌（Hepatocellular carcinoma，HCC）中过表达。但PRDX3如何参与肝细胞癌的发生发展过程并没有很好的阐述。在本研究中，我们在HepG2细胞中建立了PRDX3稳定表达和沉默细胞系。我们发现沉默PRDX3后，HepG2细胞的生长速率降低，线粒体DNA氧化增加。通过定量蛋白质组分析了PRDX3敲低和对照组细胞系，鉴定475个差异表达的蛋白。这些蛋白参与了抗氧化活性、血管生成、细胞粘附、细胞生长，ATP合成，核酸结合，氧化还原和分子伴侣等功能。敲低PRDX3的表达，导致ATP合成酶的下调，导致细胞内ATP水平的下降，从而影响了细胞的生长速度。此外，沉默PRDX3后下调了基质金属蛋白酶抑制剂1（Tissue inhibitors of metalloproteinase 1，TIMP-1）的表达，而增加胞外基质（Extracellular matrix，ECM）的降解，促进肿瘤细胞的侵袭。综上所述，本研究结果首次证明ROS通过Lonp1介导UNG1降解，并揭示UNG1的一种新功能，即能够招募PRDX3到线粒体DNA上，保护与UNG1结合的线粒体DNA免受 ROS的损伤，提高细胞抗氧化应激的能力。而沉默PRDX3能抑制肿瘤细胞的生长，增加肿瘤细胞的迁移能力。
The mitochondrial DNA (mtDNA) is more susceptible to oxidative damage than the nuclear DNA due to its close proximity to the respiration chain. Thioredoxin dependent peroxide reductase (PRDX3) is a mitochondrial peroxide reductase that regulates cellular redox state. Isoform 1 of Uracil-DNA glycosylase (UNG1) is the major protein responsible for initiating base-excision repair in mitochondria DNA repair. Effects of oxidative stress on regulation and function of PRDX3-UNG1 have not been well characterized. Preliminary works showed that UNG1 mainly binded to PRDX3 under oxidative stress, and the binding of UNG1 and PRDX3 was confirmed by reciprocal IP. We further demonstrated that the uracil-containing double-stranded oligo DNA specifically binded to the complex of UNG1 and PRDX3 formed in the hydrogen peroxide-treated cells, suggesting that PRDX3 protects integrity of mtDNA from reactive oxygen species (ROS) through PRDX3-UNG1 interaction, On the other hand, our results showed that ROS induced degradation of UNG1 in both wild type and UNG1(+) cells, whereas the mRNA expression level of UNG1 was increased under oxidative stress. We also found that the ROS induced degradation of UNG1 was proteasome-independent and LonP1 was responsible for oxidative stress-induced UNG1 degradation. Taken together, these results demonstrated for the first time that ROS mediated degradation of UNG1 by Lonp1, and revealed a novel function of UNG1 in recruitment of PRDX3 to mtDNA, which enables protection of UNG1-bounded DNA from ROS damage and in turn enhances cells’ resistance to oxidative stress. It has been reported that PRDX3 is overexpressed in liver cancer, but how PRDX3 is involved in hepatocellular carcinoma (HCC) tumorigenesis and progression has not been well characterized. In the present study, we established two stable cell lines by overexpressing or knocking down PRDX3 in HepG2 cells. We found that PRDX3 silencing decreased the growth rate of HepG2 cells and increased mtDNA oxidation. Quantitative proteomics identified 475 differentially expressed proteins between the PRDX3 knockdown and the control cells. These proteins were involved in antioxidant activity, angiogenesis, cell adhesion, cell growth, ATP synthesis, nucleic acid binding, redox and chaperones. PRDX3 knockdown led to down-regulation of ATP synthases and the decreased cellular ATP level contributing to slow down of cell growth. Furthermore, silencing PRDX3 enhanced invasive properties of HepG2 cells via TIMP-1 (tissue inhibitors of metalloproteinase 1) down-regulation and the increased extracellular matrix (ECM) degradation. Taken together, our results indicate that PRDX3 promotes HCC growth and mediates cell migration and invasiveness and is a potential therapeutic target for HCC treatment.
