The Expression Of Common Fragile Gene WWOX And Its Role In Human Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC) is one of the most common malignancies in China. The 5-year survival rate was very low because of the high recurrent and metastasis ratio after radical resection. Therefore, it makes sense to explore the mechanism of the tumorigenesis and progression and the new therapy of HCC. Risk factors for HCC include chronic hepatitis B and C virus infections, alcohol intake, non-alcoholic steatohepatitis, and dietary aflatoxins. Some factors can result in breakage of chromosome fragile site, which affect gene encoding, lead to inactivation of the gene and promote the formation and development of tumor. The WW-domain con-taining oxidoreductase(WWOX) gene was identified by Bednarek in 2000. WWOX spans 1.1 Mb within the FRA16 D fragile site at 16q23. Low, undetectable expression or aberrant transcripts of WWOX have been reported in different types of cancer and several tumor cell lines of different origin. The frequent deletion of WWOX protein expression could be associated with certain clinical or pathological parameters. Recent studies have also shown that WWOX may act as a tumor suppressor gene and a proapoptotic protein. Normal expression of WWOX may play an important role in suppressing tumor. The aim of this study is to detect the expression of WWOX in HCC, and to evaluate its clinical significance and study effects of overexpression of WWOX gene in vivo and in vitro on the biological behavior of human hepatocellular carcinoma cell, and to provide theory basis on the development, progress and therapy of HCC.Part 1 The clinicopathological significance of common fragile gene WWOX expression in human hepatocellular carcinomaObjective:The aim of this study is to detect the expression of WWOX in HCC and evaluate its clinical significance.Methods:Expression of WWOX was detected by semi-quantitative RT-PCR and western blot in Hepatocellular carcinoma cell lines and HCC tissues. Immunofluorescence was used to detect the WWOX and p-WOX subcellular localization of Hep G2 cells. Immunohistochemistry was employed to investigate the expression of WWOX, p-WOX, FHIT, Ki-67, Survivin, P53, Bcl-2, Bax and Cyclin D1 protein in HCC tissues, and to study the relationship among these proteins expression with the clinical pathologic characteristics and prognoses of HCC.Results: Low expression of WWOX was detected in SK-HEP-1 and MHCC97-H cells. Immunofluorescence was used to demonstrate that WWOX protein was located in both cytoplasm and nucleus, and p-WOX protein was located in nucleus in Hep G2 cell. The expression level of WWOX, especially WWOX protein expression, in HCC tissues was lower than that in normal liver tissues(P<0.05). Immunohistochemical staining revealed that 88 out of 172(51.2%) HCCs expressed decreased levels WWOX protein. And the expression level of WWOX was significant lower in the tumors with cirrhosis than that without cirrhosis(P=0.002). The p-WOX expression is associated with histological grading and differentiated degree of HCC, the expression of p-WOX protein increased with the decrease of tumor differentiation level and increase of histological grading(P=0.003, P=0.003). WWOX expression was positively correlation with the expression level of Bax, and was negative correlation with the expression level of Cyclin D1(P=0.001, P=0.009); The expression of pWOX was positively associated with the expression level of Survivin, Bax and Bcl-2(P<0.001, P=0.011 and P<0.001). Age, preoperative levels of AFP, differential degree, TNM stage, vascular invasion and the expression of KI-67 were independently related to patients disease-free survival by COX multivariate survival analysis(Hazard radio(HR)=2.382, P<0.001; HR=1.562, P=0.034; HR=1.448, P=0.023; HR=2.041, P=0.001; HR=2.217, P=0.003; HR=2.111, P<0.001, respectively). Tumor size, vascular invasion, recurrence, WWOX expression, p-WOX expression and Survivin expression were independently related to patients overall survival by COX multivariate survival analysis(Hazard radio(HR) =2.640, P<0.001; HR=2.649, P= 0.033; HR=6.232, P<0.001; HR=0.449, P=0.004; HR=2.763, P=0.001, HR=1.719, P=0.050, respectively).Conclusion:Decreased expression of WWOX probably promote the occurrence and development of HCC. WWOX and p-WOX proteins may play an important role in the regulation of the cell cycle and cell apoptosis of hepatocellular carcinoma cell. Combined detection of WWOX, p-WOX, FHIT, Ki-67, Survivin, Bcl-2 and Bax protein helps us to predict the recurrence and overall survival of HCC patients.Part 2 Construction and identification of lentiviral vector plasmid containing WWOXObjective:To provides an experiment foundation for further function researches of WWOX gene and the tumor therapy with targeting WWOX gene by constructing of Lentiviral vector carrying WWOX gene.Methods: WWOX gene was amplified by PCR and cloned into the expression plasmid of lentiviral vector to generate the lentiviral expression vector, WWOX/GV208. The correct WWOX gene was confirmed by PCR identification and sequencing. Recombinant lentiviruses carrying WWOX gene were produced by 293 T cells following the co-transfection of WWOX/GV208 and packaging plasmids p Helper1.0 and p Helper2.0. Finally, the virus titer was measured.Results: PCR analysis and DNA sequencing confirmed that the WWOX sequences were successfully inserted into the lentiviral vectors.The titer of concentrated virus was 2.00E+8 TU/m L.Conclusion:The lentiviral vector plasmid carrying WWOX has been successfully constructed,which will provide a foundation for the further study on function of WWOX gene in HCC and gene therapy.Part 3 Inhibition of hepatocellular carcinoma cell growth in vitro and in vivo: effect of restoration of WWOX expressionObjective:To study effects of overexpression of WWOX gene in vivo and in vitro on the biological behavior of human hepatocellular carcinoma cell, and to provide theory basis on the development, progress and therapy of HCC.Methods:(1) MHCC97-H, SK-HEP-1 and Hep G2 cell were infected by lentiviral vector plasmid carrying WWOX(LV-WWOX) and negative vector(LV-GFP) respectively. The proliferative capability of these cells were estimated by MTT method and clone formation test, and the cell cycle distribution was detected by flow cytometry. WWOX, p-WOX, Ki-67, Cyclin D1, Cyclin E, Bax, Bcl-2, EGFR, Procaspase-3 and Procaspase-9 were detected by Real-time PCR, Western Blot and IHC.(2) To investigate the effect of WWOX overexpression on STS induced apoptosis in MHCC97-H cells by morphological observation, MTT method and flow cytometry. Western Blot was employed to examine the level of Procaspase-3 and Procaspase-9.(3) In vivo, MHCC97-H and SK-HEP-1cells, include LV-WWOX group and LV-GFP group, were implanted subcutaneously into nude mice respectively to observe tumor growth. Collect the tumor, calculate the anti-tumor rate. The expression of WWOX, pWOX, Ki-67, Cyclin D1, Bax and Bcl-2 protein in the transplanted tumor tissues were detected by IHC. TUNEL method measured apoptotic tumor cells, and calculated the apoptotic index.Results:(1) The real-time PCR and western blot results showed that the expression levels of WWOX m RNA and protein were significantly increased in MHCC97-H, SK-HEP-1 and Hep G2 cells infected with LV-WWOX. MTT result showed that increased expression of WWOX can inhibit the cell proliferation of MHCC97-H and SK-HEP-1. Cloning formation assay show the inhibition rate of LV-WWOX group is higher than the LV-GFP group and uninfected group of SK-HEP-1 cell.However, WWOX overexpression showed no anti-tumor activity on Hep G2 by inhibiting growth of tumor cells. The result of flow cytometry showed that WWOX overexpression arrested MHCC97-H and SK-HEP-1cell cycle at G1 period, while there was no change in Hep G2. Real-time PCR, Western Blot and immunohistochemistry were applied to detected the expression level of some proteins in MHCC97-H, SK-HEP-1 and Hep G2 which were infected with LV-WWOX. The result showed that p-WOX expression was significantly increased in nuclear, Ki-67 proliferation index decreased, cell cycle related proteins cyclin D1 and Cyclin E decreased, apoptosis associated protein Bax increased and there no significant changes in Bcl-2 in MHCC97-H(LVWWOX) and SK-HEP-1(LV-WWOX). Procaspase-3 and Procaspase-9 proteins expression also significantly reduced. But there were no changs in Hep G2(LVWWOX).(2) The inhibition rate of LV-WWOX group was higher than the LV-GFP group by MTT assay after MHCC97-H treated with STS. The result of flow cytometry showed that the overall apoptosis ratio and the late apoptosis ratio of LV-WWOX group were higher than the LV-GFP group. Compared with LV-GFP group, Procaspase-3 and Procaspase-9 proteins expression significantly reduced in LV-WWOX group.(3) The tumor growth inhibitory rate of MHCC97-H(LV-WWOX) group was 52.63%, and SK-HEP-1(LV-WWOX) group was 90.00%. The protein level of WWOX, pWOX and Bax expression in LV-WWOX group were significantly higher than those of LV-GFP group. And The protein level of Ki-67、Cyclin D1、Bcl-2 expression in LV-WWOX group were significantly lower than those of LV-GFP group. It was found that the apoptosis index of LV-WWOX group was higher than LV-GFP group by TUNEL method.Conclusion:(1) Overexpression of WWOX gene in MHCC97-H and SK-HEP-1 by infected lentivirus vector can inhibit the cell proliferation and the growth of transplant tumor.(2) Increased expression of WWOX can inhibit the cell proliferation of MHCC97-H and SK-HEP-1 by down-regulating the expression of Cyclin D1 and Cyclin E.(3) Increased expression of WWOX can promote the cell proliferation of MHCC97-H and SK-HEP-1. WWOX protein is involved in the regulation of cell apoptosis through protein phosphorylation and nuclear translocation.(4) Overexpression of WWOX can facilitate the apoptosis of hepatoma carcinoma cell under STS action.