Effects On The Proliferation And The Expression Of VEGF In Glioblastoma Cells After Silencing The Oncogene C-Myc Through ShRNA

Objective:This study was to construct the shRNA plasmid targeting the oncogene c-Myc and slience the expression of c-Myc gene in human glioblastoma cell lines IN500Δcells using RNA interference technique. Then, this study was also to approach the effects of c-Myc oncogene on glioma cells'proliferation and apoptosis both in vivo and in vitro. Meanwhile, this study detected the influence on the expression of VEGF in IN500Δcells after silencing the c-Myc oncogene and discussed the position of c-Myc gene in glioma and its role on the regulation of angiogenesis. Finally, significant experimental data was provided for the gene therapy and anti-angiogenesis therapy to gliomas.Methods:1 Construction of shRNA plasmids and cell transfection: Construct the shRNA interference plasmid neamed pCMYC targeting c-Myc gene and also construct the pHK plasmid as negative control. Transfect the pCMYC and pHK plasmids into the IN500Δcells respectively through liposome mediating and establish the IN500Δ-pCMYC and IN500Δ-pHK cell lines following the green fluorescent protein marking the plasmid after G418 screening.2 Detection of the shRNA's silencing effects on the targeting gene: Seen the non-transfection IN500Δcells as blank control, detect the expressive change of c-Myc and VEGF both on mRNA and protein level by RT-PCR and immunocytochemistry method respectively among different cell groups.3 The influence of pCMYC plasmid on the cell cycle and cell apoptosis in IN500Δcells: Detect the change of cell cycle and cell apoptosis among IN500Δ, IN500Δ-pHK and IN500Δ-pCMYC cell groups through flow cytometry.4 The influence of pCMYC plasmid on cell proliferation in vivo through bearing cancer nude mice experiment: Inoculate the IN500Δ, IN500Δ-pHK and IN500Δ-pCMYC cells into nude mice subcutaneously. Observe the tumor formation rate and the tumor growing information and draw the tumor volume growth curve and tumor weight bar graph.5 The influence of pCMYC plasmid on the expression of c-Myc and VEGF on protein level in IN500Δcells in vivo: Decte the expression of c-Myc and VEGF on protein level in the three different xenograft tumors by immunohistochemistry.6 The influence of pCMYC plasmid on the cell apoptosis of IN500Δcells in vivo: Detect the cell apoptosis information in the three different xenograft tumors by TUNEL in situ cell apoptosis detecting method. 7 Statistical analysis: Using SPSS15.0 statistical software, analyse the experimental data scientifically. The comparison of interclass measurement data applied the one-way ANOVA method and the comparison of two classes applied LSD and SNK-T test. The comparison of enumeration data applied non-parameter test. P<0.05 suggested the difference had statistical significance. Correlation analysis applied Pearson correlation and Spearman rank correlation. 0.40.7 suggested high correlation.Results:1 The pCMYC plasmid targeting c-Myc gene was successfully constructed and the cell lines with stable expression of the shRNA plasmid were also successfully established.2 The influence of pCMYC plasmid on the expression of c-Myc and VEGF on mRNA level in IN500Δcells: After RT-PCR, the electrophoresis results showed that the ratios of the average optical density value of the c-Myc mRNA's electrophoresis strip and that of GAPDH mRNA in IN500Δ, IN500Δ-pHK and IN500Δ-pCMYC cell groups were 1.185±0.145, 1.098±0.128 and 0.273±0.028 respectively. The according ratios of VEGF were 1.116±0.072, 1.208±0.133 and 0.443±0.048. One-way ANOVA and LSD, SNK-T tests showed that the mRNA's expression of c-Myc and VEGF in IN500Δ-pCMYC cells was less than that of IN500Δand IN500Δ-pHK cells. The difference had statistical significance (P<0.01). The difference of the mRNA's expression of c-Myc and VEGF between IN500Δcells and IN500Δ-pHK cells was not significant (P>0.05). Pearson correlation analysis suggested that the mRNA expression of c-Myc was highly correlated to that of VEGF (r=0.938, P<0.01).3 The influence of pCMYC plasmid on the expression of c-Myc and VEGF on protein level in IN500Δcells: The results of immunocytochemical stain showed that the expression of c-Myc and VEGF in both IN500Δcells and IN500Δ-pHK cells were high. The average positive rates of c-Myc and VEGF in the IN500Δcells were 90.02%±3.36% and 84.39%±5.84% and there were 86.79%±2.22% and 23.52%±3.44% in the IN500Δ-pHK cells accordingly. Kruskal-Wallis H test and Mann-Whiney U test showed that there was no significant difference in the expression of c-Myc and VEGF between IN500Δcells and IN500Δ-pHK cells (P>0.05). In IN500Δ-pCMYC cells, the expression level of c-Myc and VEGF protein was low and the positive cells were few. The average positive rates of c-Myc and VEGF were 24.52%±4.39% and 23.52%±3.44%. Compared with both IN500Δcells and IN500Δ-pHK cells, the difference had statistical significance (P<0.05). Spearman correlation for ranks analysis suggested that among the three cell groups, the expression of c-Myc was correlated with that of VEGF. The coefficients of correlation were 0.886, 0.829 and 0.771 respectively and three coefficients all had statistical significance (P<0.05).4 The influence of pCMYC plasmid on the cell cycle in IN500Δcells: The cell cycle information was detected by flow cytometry and the results were as follows. For IN500Δcells, the cells in phase G1 were account for 36.9%±4.7% and that in phase S were account for 43.2%±6.1%. For IN500Δ-pHK cells, the cells in phase G1 were account for 34.4%±5.3% and that in phase S were account for 50.1%±7.6%. For IN500Δ-pCMYC cells, the cells in phase G1 were account for 72.9%±7.5% and that in phase S were account for 9.8%±3.3%. Kruskal-Wallis H test and Mann-Whiney U test showed that the percentage which phase G1 cells and phase S cells occupied respectively between IN500Δcells and IN500Δ-pHK cells had no significant difference (P>0.05). The percentage which phase G1 cells and phase S cells occupied respectively between IN500Δcells and IN500Δ-pCMYC cells had significant difference (P<0.01) and that between IN500Δ-pHK and IN500Δ-pCMYC cells also had significant difference (P<0.01). 5 The influence of pCMYC plasmid on the cell apoptosis in IN500Δcells: The cell apoptosis information was detected by flow cytometry and the results were as follows. The apoptosis rates of IN500Δcells, IN500Δ-pHK cells and IN500Δ-pCMYC cells were 7.13%±3.57%, 10.40%±4.88% and 46.51%±4.99% respectively. Kruskal-Wallis H test and Mann-Whiney U test suggested that the difference of the apoptosis rate between IN500Δcells and IN500Δ-pHK cells had no statistical significance (P>0.05). The apoptosis rate between IN500Δcells and IN500Δ-pCMYC cells had significant difference (P<0.01) and that between IN500Δ-pHK and IN500Δ-pCMYC cells also had significant difference (P<0.01).6 The influence of pCMYC plasmid on the proliferation of IN500Δcells in vivo of nude mice: The results of tumor formation test on nude mice were as follows. The 12 nude mice which were injected with IN500Δcells or IN500Δ-pHK cells subcutaneously all developed tumor and the rate of the tumor formation rate was 100%. The xenograft tumors grew fast and the average volume of the tumors in the two groups were (563.1±40.0)cm~3 and (521.1±32.1)cm~3; the average weight of the tumors were (2.10±0.53)g and (1.87±0.35)g when they were harvested. Among the 6 nude mice which were injected with IN500Δ-pCMYC cells subcutaneously, only 4 nude mice developed tumors and the tumor formation rate was 66.7%. Furthermore, the xenograft tumor grew slowly and the average volume and weight were (224.6±28.3)cm~3 and (0.60±0.12)g. One-way ANOVA and SNK-T tests suggested that the tumor volume and weight between IN500Δcells and IN500Δ-pHK cells neither had significant difference (P>0.05); that between IN500Δcells and IN500Δ-pCMYC cells, IN500Δ-pHK cells and IN500Δ-pCMYC cells both had significant difference (P<0.01). 7 The influence of pCMYC plasmid on the expression of c-Myc and VEGF on protein level in IN500Δcells in vivo: The expression of c-Myc and VEGF protein in xenograft tumors was detected by immunohistochemistry method and the results were as follows. In IN500Δcells tumor and IN500Δ-pHK cells tumor, the c-Myc or VEGF positive cells were so many and they diffused as lamellar. The average positive cell rates of c-Myc and VEGF in IN500Δcells tumor were 81.68%±4.22% and 80.73%±3.12% respectively. The rates in IN500Δ-pHK cells tumor were 77.77%±3.54% and 76.39%±5.74%. In IN500Δ-pCMYC cells tumor, the c-Myc and VEGF positive cells were diffused sporadically. The average positive cell rates of c-Myc and VEGF in IN500Δ-pCMYC cells tumor were 28.74%±1.69% and 22.99%±4.90%. Kruskal-Wallis H test and Mann-Whiney U test suggested that the expression of c-Myc and VEGF protein in IN500Δ-pCMYC cells tumor was lower than that of IN500Δcells tumor and IN500Δ-pHK cells tumor. The difference had statistical significance (P<0.05). The expression of c-Myc and VEGF between IN500Δcells tumor and IN500Δ-pHK cells tumor had no significant difference (P>0.05). Spearman correlation for ranks analysis suggested that among the three different cells transfection tumors, the expression of c-Myc was correlated with that of VEGF respectively. The coefficients of correlation were 0.829, 0.886 and 1.000 respectively and all the three coefficients had statistical significance (P<0.05). 8 The influence of pCMYC plasmid on the cell apoptosis of IN500Δcells in vivo: The cell apoptosis information of the xenograft tumors was detected through TUNEL method and the results were as follows. In IN500Δcells tumor and IN500Δ-pHK cells tumor tissues, the apoptosis cells were diffused sporadically and the average apoptosis index were 23.44%±3.58% and 24.00%±2.15% respectively. In IN500Δ-pCMYC cells tumor tissues, there were many apoptosis cells and the average apoptosis index was 76.50%±2.65%. Kruskal-Wallis H test and Mann-Whiney U test suggested that the difference of apoptosis index between IN500Δcells tumor and IN500Δ-pHK cells tumor had no statistical significance (P>0.05). The apoptosis index of IN500Δ-pCMYC cells tumor was higher than IN500Δcells tumor and IN500Δ-pHK cells tumor and the difference between IN500Δ-pCMYC cells tumor and IN500Δcells tumor, IN500Δ-pCMYC cells tumor and IN500Δ-pHK cells tumor both had statistical significance (P<0.05).Conclusion:1 After transfected into the IN500Δcells, the shRNA plasmid targeting the oncogene c-Myc could effectively silence the expression of c-Myc both on mRNA and protein levels in vivo or in vitro.2 Sliencing the expression of c-Myc oncogene could effectively inhibite the proliferation of IN500Δcells and could induce most of tumor cells to apoptosis both in vivo and in vitro.3 There was existing correlation to some degree between the expression of VEGF and c-Myc in glioblastoma cells. When the expression of c-Myc gene was silenced, the expression of VEGF was also decreased accordingly and c-Myc might regulate tumor's angiogenesis through its regulation of VEGF.4 The oncogene c-Myc could be an effective target in the gene therapy to malignant glioma.