The Role Of XRCC2 In Pathogenesis And Progression Of Human Hepatocellular Carcinoma (HCC) And Its Molecular Mechanism

Part Ⅰ Study on the differential expression of DNA damage repair-related molecules between HCC tissues and its corresponding adjacent liver tissuesAims:To explore the differential expression of DNA damage and repair-related molecules between HCC tissues and its corresponding adjacent liver tissues.Methods:1. To collect the tumor tissues and adjacent liver tissues of 40～50 years male HCC patients who had the similar differentiated degrees, tumor sizes and HBV infection history. Primary HCC was confirmed by postoperatively pathological diagnosis. Excised HCC and liver tissues were cutted into small pieces and were rapidly put into the freezing tubes, and then added RNAwait into the tubes and were stored in -80℃ fridge.2. The HCC tissues and adjacent liver tissues were put into cryogenic tank with dry ice and were delivered to Shanghai Bohao Biotechnology Corporation. The Agilent version 16.0 was applied to screen the gene expression profiles involved in the DNA repair pathways between HCC tissues and its adjacent liver tissues.Results:1. Three HCC patients who matched our inclusion criteria were collected.2. The gene chip results indicated that a total of 33 DNA repair molecules expressed differentially. We screened out 8 DNA repair molecules, in which the fold change value was over 10 times for the further studies. Of them, XRCC2 was not reported yet in HCC and the XRCC2 expression in the HCC tissues was up-regulated by 17.5 fold change compared to its adjacent liver tissues.Part Ⅱ Study on the correlation between XRCC2 expression and clinicopathologic features and long-term outcomes of HCC patients undergone liver resectionAim:To explore the relationship between XRCC2 expression and clinicopathologic features and long-term outcomes of HCC patients undergone liver resection.Methods:1. We selected 149 pairs of HCC tissues and the corresponding adjacent liver tissues from HCC specimen library in our hospital. All the HCC patients were performed liver resection in our department from Jan.1st,2009 to Jan.1st,2011. RT-PCR, western blot and immunohistochemistry were used to test the XRCC2 expression in HCC tissues and its adjacent liver tissues.2. The relationship between XRCC2 expression and the malignant biological features of HCC were analyzed. We investigated the overall survival and recurrence to study the effect of XRCC2 expression on long-term outcomes of HCC patients who undergone liver resection.Results:1. Among these 149 patients, the XRCC2 expression in 111 patients was significantly higher in HCC tissues compared to the adjacent liver tissues. IHC and western blot analysis indicated that the protein XRCC2 expression in HCC tissues was significantly higher than their corresponding adjacent liver tissues.2. According to the mRNA XRCC2 expression in HCC tissues compared to their corresponding adjacent liver tissues, we divided these 149 cases into two groups:high expression group (n=111) and low expression group (n=38). We further found that the expression level of XRCC2 was correlated with HBV-DNA copies, severity of liver cirrhosis, differentiation degree, tumor size and tumor capsule.3. The 5-year overall survival and recurrence free survival rates of HCC patients in higher group were significantly poorer than those in lower group. The 5-year overall survival and recurrence rate were 28.7%,54.2% and 17.8%,37.6%, respectively (p<0.01). The cox regression model indicated that XRCC2 expression level was the independent risk factor for overall survival and disease-free survival.Part Ⅲ The effect of XRCC2 on the proliferation of Bel-7402 and SMMC-7721 hepatoma cellsAims:To study the effect of XRCC2 on the proliferation of Bel-7402 and SMMC-7721 hepatoma cells.Methods:1. RT-PCR and western blot were applied to examine the expression level of XRCC2 in 10 HCC cell strains and normal hepatic cell line, and we chosen the HCC cell lines which had high-level and low-level XRCC2 expression for the further studies.2. shRNA lentivirus was used to knock down the XRCC2 expression of Bel-7402 cell line. cDNA lentivirus was employed to up-regulate the XRCC2 expression of SMMC-7721 cell line. These two kinds of lentivirus have the puromycin resistance genes. On day 2 after transfection,5μg/mL puromycin was used to screen out the positive cells. Finally, RT-PCR and Western blot were applied to verify the XRCC2 expression in the above HCC cell strains.3. Cell proliferation assay, colony formation assay and EdU dye assay were used to investigate cell proliferation ability of these 4 HCC cell strains in vitro.4. Xenograft tumor growth of the above 4 HCC cell strains were compared in 4-week nude mice.5×106 cells were injected into the flank of each mouse,10 mice per group. The tumor growth of each group was observed and the tumor sizes were recorded every 3 days.Results:1. By using RT-PCR and Western blot, we detected the XRCC2 expression in 10 HCC cell strains and normal hepatic cell line. We found that the XRCC2 expression in HCC cells was significantly higher compared to the normal hepatic cell line. We selected Bel-7402 and SMMC-7721 which were the relatively high and low XRCC2 expression for the further studies.2. Fluorescence microscope was used to detect the transfection efficiency and we found all the cells were transfected successfully. RT-PCR and Western blot results showed that the inhibition ratio was about 75% in Bel-7402-sh-XRCC2 cells compared to Bel-7402-vector cells. Moreover, the XRCC2 expression was up-regulated nearly 32 times in SMMC-7721-XRCC2 cells compared to SMMC-7721-vector cells.3. CCK-8 assay suggested that, the OD45o value of the Bel-7402-sh-XRCC2 cells on day 3 were significantly fewer than vector-transfected cells (0.45±0.098 vs 1.16±0.21,p<0.01); the OD450 value of the SMMC-7721-XRCC2 cells on day 3 were significantly more than vector-transfected cells (1.56±0.18 vs 0.72±0.11,p<0.01). The plate colony assay indicated that, the colony number of the Bel-7402-sh-XRCC2 cells on day 12 were significantly less than those of the vector-transfected cells (145±16 vs 344±24,p<0.01); the colony number of the SMMC-7721-XRCC2 cells on day 12 were significantly more than those of the vector-transfected cells (582±32 vs 410±21, p<0.05). Soft agar colony formation assay indicated that, the colony number of the Bel-7402-sh-XRCC2 cells on day 18 were significantly fewer than those of the vector-transfected cells (49±6 vs 117±9, p<0.01); the colony number of the SMMC-7721-XRCC2 cells on day 12 were significantly more than those of the vector-transfected cells(128±10 vs 81±7,p<0.01). Edu assay showed that the Edu positive cells were down-regulated more than 5 times in Bel-7402-sh-XRCC2 cells compared to Bel-7402-vector cells.4. Tumor growth assay indicated that, Bel-7402-sh-XRCC2 subcutaneous tumors grew slower and were smaller at each time points measured since day 8 after injection compared to Bel-7402-vector group (p<0.01). When the tumors were excised from the mice on day 32 after injection, the mean volumes (0.26±0.05cm3 vs 0.86±0.19cm3) and weights (0.44±0.08g vs 0.11±0.01g) of the tumors derived from the Bel-7402-sh-XRCC2 cells were significantly smaller than those derived from the corresponding vector-transfected cells (p<0.01). SMMC-7721-XRCC2 subcutaneous tumors grew faster and were bigger at each time points measured since day 12 after injection compared to SMMC-7721-vec group (p<0.01). The xenograft tumor volumes (1.82±0.19cm3 vs 1.14±0.28cm3) and weights (1.32±0.12g vs 0.54±0.09g) of the tumors derived from the SMMC-7721-XRCC2 cells were significantly bigger than those derived from the corresponding vector-transfected cells (p<0.01).Part Ⅳ The potential molecular mechanism of XRCC2 in regulating the proliferation of Bel-7402 and SMMC-7721 cellsAims:To explore the potential molecular mechanism of XRCC2 in regulating the proliferation of Bel-7402 and SMMC-7721 cells.Methods:1. Cell cycle distributions of all the four HCC cell strains were investigated using flow cytometry analysis.2. The apoptosis rates of Bel-7402-vector cell and Bel-7402-sh-XRCC2 cell were examined using flow cytometry analysis.3. The expression of molecules involved in the regulation of cell cycle and apoptosis were analyzed by RT-PCR and western blot.4. The expression of cell cycle related molecules were analyzed by immunohistochemical staining. Cell apoptosis levels in xenograft tumor tissues were detected by TUNEL assay.Results:1. Flow cytometry analysis indicated that, the percent of Bel-7402-vector cells in S phase were 10.46%±2.09%; the corresponding Bel-7402-sh-XRCC2 cells were 41.08%±3.17%. There was a significant difference in the percentages of S phase between the two kinds of cells (p<0.01); the percent of SMMC-7721-vector cells in S phase were 28.87%±2.37%; the corresponding SMMC-7721-XRCC2 cells were 15.65%±2.23%. There was a significant difference in the percentages of S phase between these two kinds of cells (p<0.01)2. Flow cytometry results showed that, the apoptosis rates in Bel-7402-vector cells were 1.36%±0.54%, whereas, the apoptosis rates in Bel-7402-sh-XRCC2 cells were 9.8%±1.1%. There was a significant difference in cell apoptosis rate between these two cell strains (p<0.01). Down-regulating the XRCC2 expression in Bel-7402 cells increases the cell apoptosis compared to its vector-transfected cell strains.3. RT-PCR and western blot analyses showed that the expression levels of Rad 51, p53 p21 and p15 significantly increased after sh-XRCC2 was transfected into the Bel-7402 cells, whereas the expression levels of MDM4, PARP-1,cyclinD1, CDK2 and CDK4 significantly decreased; the expression level of Bax, Caspase-3 and c-myc significantly decreased, whereas the expression of Bcl-2 increased significantly. After up-regulating the XRCC2 expression in SMMC-7721 cells, the expression of MDM4, PARP-1, CDK4 and cyclinD1 significantly increased, whereas the expression of the above apoptotic factors remained unchanged.4. Immunohistochemical staining showed that the expression of the proteins in the above results was the same in xenograft tumors. TUNEL assay showed that the apoptosis rates in tumor tissue obtained from Bel-7402-vector cell strains and Bel-7402-sh-XRCC2 cell strains were 2.6%±1.0% and 11.8%±2.8%, respectively. The cell apoptosis rates was significantly increased in Bel-7402-sh-XRCC2 cell strains compared to its vector-trasfected cells group (p<0.01).Part V The effect of XRCC2 expression on the chemotherapy of HCCAims:To investigated whether down-regulating the XRCC2 expression could increase the inhibition effect of minocycline on the proliferation of HCC.Methods:1. CCK-8 and plate colony assays were employed to investigate the effect of minocycline on the proliferation of Bel-7402-vec and Bel-7402-sh-XRCC2 cell strains.2. Four-week male nude mice (n=24) were equally divided into two groups. Bel-7402-vec and Bel-7402-sh-XRCC2 cells (5×106) were injected into the flanks of the each mouse. When the maximum volume of xenograft tumor in each group was about 100 mm3, the mice were randomly divided into two groups:the minocycline and control groups. Minocycline was injected intraperitoneally at 20 mg/kg, and saline was injected at the same volume for the control groups, three times a week. The tumors sizes were measured every 4 days and tumor weights were calculated on day 40.Results:1. The results indicated that minocycline significantly inhibited the proliferation of Bel-7402-sh-XRCC2 cells, but not Bel-7402-vector cells. CCK-8 assay suggested that, the OD450 value of the Bel-7402-sh-XRCC2 cells on day 2 were significantly less than its corresponding vector-transfected cells (0.48±0.08 vs 0.93±0.11,p<0.01); the OD450 value of the Bel-7402-sh-XRCC2+mino cells on day 3 were significantly less than Bel-7402-sh-XRCC2 cells (0.39±0.07 vs 0.63±0.14,p<0.01); the OD450 value of the Bel-7402-sh-XRCC2+mino cells on day 3 were significantly less than Bel-7402-vec+mino cells (0.39±0.07 vs 1.07±0.12,p<0.01). The plate colony assay indicated that, the colony number of the Bel-7402-sh-XRCC2 cells on day 14 were significantly fewer than those of the vector-transfected cells (124±16 vs 208±21,p<0.01); the colony number of the Bel-7402-sh-XRCC2+mino cells on day 14 were significantly less than those of the Bel-7402-sh-XRCC2 cells (74±9 vs 124±16, p<0.01); the colony number of the Bel-7402-sh-XRCC2+mino cells on day 14 were significantly less than those of the Bel-7402-vec+mino cells (74±9 vs 189±18,p<0.01).2. On day 40, all the mice were performed euthanasia, and xenograft tumors were excised from the flanks. The results indicated that, the mean volumes (2.44±0.35cm3 vs 2.21±0.29cm3) and weights (0.75±0.083g vs 0.66±0.075g) of the tumors obtained from the Bel-7402-vec cell strains and Bel-7402-vec+mino cell strains remained unchanged; the mean volumes (0.78±0.19cm3 vs 2.44±0.35cm3) and weights (0.24±0.032g vs 0.75±0.083g) of the tumors derived from the Bel-7402-sh-XRCC2 cells were significantly smaller than those derived from their corresponding vector-transfected cells (p<0.01); the mean volumes (0.32±0.065cm3 vs 2.21±0.29cm3) and weights (0.11±0.023g vs 0.66±0.075g) of the tumors derived from the Bel-7402-sh-XRCC2 +mino cells were significantly smaller than those derived from Bel-7402-vec+mino cells (p<0.01); the mean volumes (0.32±0.065cm3 vs 0.78±0.19cm3) and weights (0.11±0.023g vs 0.24±0.032g) of the tumors derived from the Bel-7402-sh-XRCC2+mino cells were significantly smaller than those derived from Bel-7402-sh-XRCC2 (p<0.01).Statistical analysisAll the previously mentioned continuous variable data were expressed as mean±standard deviation represented in the form and all the categorical variable data were expressed as percentage. SPSS 19.0 software was used for statistical analysis. The t-test or univariate analysis of variance (ANOVA) was carried out to analyse the differences among the groups, and categorical variables be used chi-square test more generally, and the Kaplan Meier methods were applied to analyze the survival between the two groups. All the results in the experiments represent the averages in three independent times and the p<0.05 was considered a statistically significant difference.Conclusions1. The XRCC2 expression was higher in tumor tissues compared to the corresponding adjacent liver tissues, and its expression level has a reverse relationship with the long-term outcome after liver resection.2. XRCC2 was highly expressed in HCC cell lines in comparison with normal liver cell line. Down-regulating the XRCC2 expression in HCC cell strains could inhibit the proliferation of HCC cells in vitro and in vivo.3. Down-regulating the XRCC2 expression caused S-phase arrest and cell apoptosis in Bel-7402 HCC cells.4. XRCC2 promotes HCC progression by regulating MDM4/p53/p21 signaling pathway.5. Down-regulating the XRCC2 expression enhanced the inhibitory effect of minocycline on the proliferation of HCC cells in vitro and in vivo.