The Expression And Functional Mechanisms Of Peroxiredoxin 6 In Hepatocellular Carcinoma

Backgrounds:Hepatocellular carcinoma (HCC) is one for the most common malignancy in the digestive system. China has a large population with HBV infection, accounting for over 50% newly-diagnosed HCC in the world. HCC is a highly malignant, fast progessing disease, without obvious manifestation at the beginning. Liver resection and liver transplantation is the common curative remedy for HCC, but the recurrence rate is high. Moreover, only part of the patients are suitable for surgical treatments. Therefore, the prognosis for HCC is not acceptable. Now we are still in lack of effective diagnostic markers or therapeutic targets for HCC.Aim:This study aims to find differently expressed proteins in the HCC tissues and paired peri-tumoral tissues using proteomic techniques. By exploring the expression pattern and mechanisms of the identified protein, we hope to find new effective tumor markers and therapeutic targets.Methods1. We use 2-dimensional fluorescence difference gel electrophoresis (2-DIGE) on 3 pairs of HCC and peri-tumoral tissues (without macro-vascular invasion, single nodule and tumor diameter<5cm) to find differently expressed protein spots, and then use mass spectrometry (MS) to identify the proteins in these spots. This study subsequently employ real-time fluorescence quantitative PCR (qPCR, n=59), Western blot (n=16) and immunohistochemistey (IHC, n=265) to validate the expression pattern of Peroxiredoxin 6 (PRDX6) in HCC and paired peri-tumoral tissues, and also to analyze the correlation of PRDX6 expression and demographical, clinical and pathological features of the HCC patients. Normal liver tissues (n=42) and cirrhotic tissues (n=48) were also included to study the change of PRDX6 expression level in the carcinogenesis of HCC. In the same time, we collected serum samples from 15 healthy subjects and 40 HCC patients to study the PRDX6 changes in the serum of HCC patients.2. We used HepG2 and BEL-7402 cell lines for in-vitro experiments, and used related lentivirus and siRNA to over-express and knock down PRDX6 expressionl in the cells. Colony formation, xenograft tumor model, cell counting kit-8 (CCK8) experiment, cell cycle detection, and cell apoptosis detection were used to study the impact of PRDX6 on cancer cell functions. We also use H2O2 and tumor necrosis factor-α/ Cycloheximide (TNF-a/CHX) to induce cancer cell apoptosis and then study the effect of PRDX6 under these conditions. This study employ the inhibitor (bromoenol lactone, BEL solution) of Ca2+independent phospholipase A2 (iPLA2)to study the effect of iPLA2 activity of PRDX6 on TNF-a/CHX induced cancer cell apopstosis.Results1.2D-DIGE and MS identified 8 proteins that were differently expressed (>3-fold) in paired HCC and peri-tumoral tissues, including PRDX6. Western blot, q-PCR and IHC results validated that PRDX6 was over-expressed in peri-tumoral tissues. PRDX6 level is the lowest in the liver cirrhotic tissues, and the normal liver tissues had higher PRDX6 than cirrhotic tissues but lower than the peri-tumoral tissues. PRDX6 level in HCC tissues correlated with pre-operative serum a-fetoprotein (AFP) and differentation (p<0.05), and its level in peri-tumoral tissues correlated with liver cirrhosis, nodule number, and macrovascular invasion (p<0.05). PRDX6 level in HCC tissues was significantly associated with the prognsis, and is an independent prognostic factor for HCC patients undergoing liver resection (p<0.05). PRDX6 level in the serum of HCC patients is significantly higher than that in healthy subjects (p<0.05).2. Stable over-expression of PRDX6 inhibited colony formation and xenograft growth. PRDX6 knock-down decreased the proliferative activity of BEL-7402. PRDX6 over-expression increased S phase and decreased G2 phase HepG2 cell number, and PRDX6 knock-down had opposite effects in HepG2 cells. PRDX6 over-expression increased BEL-7402 cell apoptosis, but PRDX6 knock-down had no effects. When treated with H2O2, PRDX6 inhibited apoptosis. When treated with TNF-a/CHX, PRDX6 promoted apoptosis. Inhibition of iPLA2 activity of PRDX6 decreased the apoptosis induced by TNF-a/CHX. When treated with TNF-a/CHX, PRDX6 knock-down changed the expression of BAX, Caspase3 and Caspase8.Conclusion1. PRDX6 is over-expressed in peri-tumoral tissues of HCC, and its level is high in the serum of HCC patients. PRDX6 can be used as a potential tumor marker for the diagnosis and prognosis for HCC.2. PRDX6 can inhibit the carcinogenesis of HCC. PRDX6 can inhibit H2O2-induced cancer cell apoptosis. However, PRDX6 can also promote TNF-a- induced apoptosis, probably through its iPLA2 activity.