The Mechanism Research Of Thioredoxin Regulating HIF-2α In Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. In recent years, an improvement that is believed to be associated with improved prognosis and survival for patients with HCC. However, recurrence and metastasis are still problems for HCC patients after operation. It was reported that tumor recurrence and metastasis complicates70%of cases at5years, combining true recurrence or metastasis, which usually arises within the first2years after resection. Hypoxia is a very common phenonmenon in solid tumours or metastases, especially in HCC and hypoxia-inducible factors (HIFs) play important role in it. HIF-2a is one important member of HIFs, which has less studies and debatable functions in diverse neoplasms. Thioredoxin (TXN) is an antioxidant protein of disulfide reduction, which produced effects in maintain redox staus in intracellular enviroments. TXN has also been proved to play a role in HCC metastasis, but the exact mechanism is not clear. Recent studies found that TXN can regulate the expression of HIF-la. However, it was unknown that whether TXN can regulate HIF-2a. In this study, the analysis was peformed to evaluate the relationships between TXN and HIF-2a in human HCC tissues, as well as their clinical significance in HCC. Then based on different metastatic HCC cell lines, the regulatory functions and mechanism of TXN to HIF-2a were further studied. This study was performed to elucidate the metastatic mechanism of HCC in the perspective of funtions of TXN and HIF-2a, and to provide some effective experimental evidence to explore the new targets for clinic diagnosis and treatment of HCC. This research is divided into three parts below:Part One The expression and clinical significance of TXN and HIF-2a in Hepatocellular CarcinomaObjective:To detect the expression of TXN and HIF-2a in HCC, and explore the relationships and functions of TXN and HIF-2a in progression and metastasis of HCC.Methods:The paraffin sections of HCC were collected from two medical centers (Tainjin Medical University Cancer Hospital and Shanghai Fudan University Zhongshan Hospital), in which all the patients had received hepatoectomy and diagnosed as HCC by postoperative pathology, with the completed follow-up data after operations. TMA were made by HCC paraffins in Shanghai Fudan University. The immunohistochemical staining was performed to dected the expression of TXN and HIF-2a in HCC tissue. According to the results of immunohistochemical staining, the patients were divided into high expression and low expression group. The statistical analysis was perform to correlate.the relationships among the expression of TXN, HIF-2a and clinicopatholical parameters. The survival analysis were performed by Kaplan-Meier methods. Fresh HCC tissue samples were colleted from Tianjin Medical University Cancer Hospital. Western blot were perform to detect the expression of TXN and HIF-2a in fresh HCC sample and different metastatic potential HCC cell line.Results:The immunohistochemical staining results showed that among all336HCC cases, the expression of TXN was negative in85cases (25.3%), low expression in75cases (22.3%), moderate expression in108cases (32.1%), high expression in68ases (20.2%). The expression of HIF-2a was negative in55cases (16.4%), low expression in123cases (36.6%), moderate expression in108cases (32.1%), high expression in50cases (14.9%). The expression of TXN was significantly correlated with HIF-2a in HCC (p=0.031). According to the clinicopathological patameters, TXN expression was statistically associated with preoperative AFP level and vascular invasion, while HIF-2a was statistically associated with history of hepatitis B, tumor size and micro-vascular invasion (MVI). Kaplan-Meier analysis showed high expression of TXN and HIF-2a indicated poor prognosis in HCC patients. Furthermore, TXN expression was significantly higher in HCC patients with extrahepatic metastasis than without metastasis (p=0.036). Western Blot results showed that the expression of TXN and HIF-2a indicated a general decreasing from the tumor tissue, paraneoplastic tissue to distal hepatic tissue. In different fresh tumor tissues, TXN high expression was ofen associated with vessel thrombosis, HIF-2a high expression was prevalent with large tumor size. The expression of TXN and HIF-2a were higher in HCC patients with lung metastasis than those without lung metastasis. In different metastatic potential HCC cell lines, TXN and HIF-2a had higher expression in highly metastatic cell line MHCC97H.Conclusion:The expression of TXN was obviously correlated with HIF-2a in HCC. High expression of TXN and HIF-2a indicated poor prognosis in HCC patients. TXN and HIF-2a both play important role in metastasis of HCC.Part Two TXN promote cell proliferation and prevent senescence through regulating HIF-2a in HCCObjective:In this section, the regulatory function of TXN to HIF-2α was detected by building knock-down TXN in high metastatic HCC cell line MHCC97H. And the biological functions were also evlauted after TXN was knockdown in MHCC97H.Methods:The low expression TXN cell line was established with transfecting lipofectamine2000and siRNA-TXN into highly metastatic HCC cell lines MHCC97H. The HCC cells were collected48h and72h after transfection. Western Blot was performed to detect the expression of TXN and HIF-2a. We assessed the cell proliferation, cell senescence, and DNA damage response by using MTT assays, colony formation assays, flow cytometry, beta-galactosidase staining, western blotting, and immunohistochemistry.Results:In high metastatic potential MHCC97H, the expression of TXN and HIF-2a significantly decreased after transfection, comparing with negative control group and no siRNA group. TXN knockdown reduced cell proliferation in MHCC97H under hypoxic and normoxic.3D matrigel colony formation was also reduced under both hypoxia and normoxia after TXN knockdown. TXN knockdown induced the increasing expression of beta-galactosidase under hypoxia, but not obvious change under normoxia. In normoxic conditions, there was a moderate expression of r-H2AX in MHCC97H with no expression under hypoxia. TXN knockdown reduced the expression of r-H2AX obviously under hypoxia, but no obvious change under normoxia. Conclusion:TXN knockown can induce a decreasing exrpssion of HIF-2a in HCC. TXN knockdown can reduce cell proliferation and3D colony formation in MHCC97H under hypoxia and normoxia. TXN knockdown can induce cell senescense and DNA damage response under hypoxia in MHCC97H.Part Three The mechanism research about TXN regulating HIF-2α in HCCPurpose:In this section, the change of TXN, HIF-2a and target proteins were dected to search for possible pathway in HCC.Methods:The low expression TXN cell line was established with transfecting lipofectamine2000and siRNA-TXN into highly metastatic HCC cell lines MHCC97H. The HCC cells were collected48h and72h after transfection. Western Blot was performed to detect the expression of TXN, HIF-2a and Oct-4.Results:In high metastatic potential cell line MHCC97H, the expression of HIF-2a and Oct-4decreased after TXN knockdown, comparing with negative control group and no siRNA group.Conclusion:Oct-4is one of the target genes of HIF-2a. TXN knockdown can induce the dreacsing expression of HIF-2a and Oct-4. TXN-HIF-2α-Oct-4is an important pathway in progression and metastasis of HCC.