Purpose  Retinoblastoma is the most common intraocular malignancy of childhood. The cancer-associated gene, hPAX6, is a key regulator of the embryological development of the retina. Our study is to assess whether the hPAX6 gene is involved in the development of retinoblastoma and to discuss the possible ways how the effects occur.
Methods  Two human retinoblastoma cell lines (Y79 and SO-Rb50) were transfected with either hPAX6-GFP lentiviral vector (hPAX6 overexpression group, OE group) or GFP lentiviral vector (negative control group, NC group). The overexpression of hPAX6 gene in retinoblastoma cells was confirmed by Q-PCR and western blot analysis. After 7 days post-infection, cell growth curve assays were evaluated by CCK8 (colorimetric cell counting kit-8). The cell cycle of Y79 and SO-Rb50 cells was analyzed by fluorescence-activated cell sorting (FACS). TUNEL assay was performed followed by a FACS analysis to confirm the cell apoptosis induced by hPAX6 gene overexpression. Western blot analysis was performed to detect the levels of cell cycle and apoptosis related proteins P53, P21, P27, cdc2, PCNA and cleaved caspase-3 in the OE groups, NC groups and untransfected control groups (CON groups).
Results  After 3 days’ post-infection, exogenous hPAX6 was detected by fluorescent microscopy. Q-PCR analysis demonstrated that the hPAX6 mRNA levels in Y79 and SO-RB50 cells transfected with hPAX6-GFP lentiviral vector were markedly increased (667.768±12.157 and 10.443±1.016, respectively) compared to those in the NC groups (1.001±0.060 and 1.001±0.066, respectively) (ANOVA, P＜0.001). The expression of PAX6 protein also increased significantly in the OE groups. After 7 days’ post-infection, a significantly promotion of cell growth was found in the OE groups of two human retinoblastoma cell lines. In Y79 and SO-Rb50 cells, the percentages of total cells in the G2/M phase were significantly higher in the OE groups (22.85±0.32% and 12.06±0.74%, respectively) than those in the NC groups (13.35±0.47% and 8.65±0.67%, respectively) (ANOVA, P＜0.01). Meanwhile, the percentages of total cells in the G0/G1 phase were significantly lower in the OE groups (47.64±0.74% and 60.24±0.30%, respectively) than those in the NC groups (57.11±0.34% and 70.14±0.48%, respectively) (ANOVA, P＜0.001). In addition, the percentage of total SO-Rb50 cells in the S phase was significantly higher in the OE group (27.69±0.50%) than that in the NC group (21.20±1.06%) (ANOVA, P＜0.001). On the contrary, the percentage of total Y79 cells in the S phase did not vary significantly between the OE group (29.52±0.97%) and the NC group (29.53±0.25%) (ANOVA, P=0.928). The levels of the P53 and the P21 protein were down-regulated in two retinoblastoma cell lines transfected with hPAX6-GFP lentiviral vector in comparison to the NC groups. The level of the P27 protein was slightly down-regulated and the level of the cdc2 protein was up-regulated. Alteration in the level of the PCNA protein was not detectable in Y79 and SO-Rb50 cells. The percentages of TUNEL positive apoptotic cells to total cells were significantly lower in the OE groups (0.040±0.026% and 0.000±0.000%, respectively) than those in the NC groups (0.597±0.032% and 0.403±0.035%, respectively) in Y79 and SO-Rb50 cells (ANOVA, P＜0.001). Correspondingly, western blot analysis indicated that the level of apoptosis-related protein, cleaved caspase-3, was down-regulated in the cells transfected with hPAX6-GFP lentiviral vector.
Conclusions  The overexpression of hPAX6 gene in human retinoblastoma cells resulted in an increased cell growth and an inhibited apoptosis in vitro, paralleled by a down-regulation of the P53 and the P21 protein and an up-regulation of the cdc2 protein. It may induce a P53 mediated caspase-dependent cell apoptosis. Our present study suggests the potential value of hPAX6 as a therapeutic target in the treatment of human retinoblastoma.