Effect of Ku70 expression on radiosensitivity in renal carcinoma 786-O cells

In order to construct the pBaBb-puro-Ku70 vector containing a Ku70 cDNA and the pSUPERretro-puro-siKu70 vector, a DNA fragment encoding Ku70 was amplified by PCR from human genomic DNA and three pairs of effective RNA interference sequences (siKu70-1, siKu70-2 and siKu70-3) to target Ku70 gene were designed and synthesized. To determine the effect of various siRNAs on Ku70 protein expression, we measured the amount of Ku70 by Western blot analysis. Of the three Ku70 siRNAs, siKu70-1 was the most effective in suppressing the expression of Ku70 (Figure 1A). Therefore, the recombinant plasmid vector containing siKu70-1 was screened out for establishing stably knock-down 786-O cell line.Ku70 expression in stable cell lines was determined by RT-PCR and Western blot analysis. Compared with stable 786-O control cells, the expression of Ku70 mRNA was increased by more than 13-fold in 786-O-Ku70 cells, and reduced by 76% in 786-O-siKu70 cells (Figure 1B). Additionally, 786-O-vector cells and 786-O-scramble cells showed no significant change of Ku70 expression (Figure 1B and1C). The results indicate the stable 786-O cell lines with overexpression or downexpression of Ku70 were successfully generated by retrovirus-mediated method.

To investigate whether the amount of Ku70 could affect the apoptosis of RCC cells, flow cytometry and TUNEL assay were performed to analyze the apoptosis of the stable 786-O cell lines.The flow cytometry analysis indicated that the apoptosis rate of 786-O-Ku70 cells (0.97 ± 0.12%) was significantly reduced in comparison with those of 786-O-vector cells (3.17 ± 0.06%). However, compared with the 786-O-scramble cells (3.33 ± 0.15%), 786-O-siKu70 cells (12.07 ± 0.15%) was significantly increased (Figure 2A and2C).The similar results were found by TUNEL assay. As shown in Figure 2B and2D, the apoptosis rate of cells was lower in 786-O-Ku70 cells (13.43 ± 2.89%) than in 786-O-vector cells (21.03 ± 4.18%), and the apoptosis rate of 786-O-siKu70 cells (45.70 ± 3.99%) was significantly higher than 786-O-scramble cells (25.56 ± 3.70%). All of the above results of flow cytometry analysis and TUNEL assay had statistical significance (P < 0.05). These results showed that Ku70 can suppress the apoptosis of 786-O cells.

In order to determine the correlation between Ku70 expression and radiation response, we exposed the stable 786-O cell lines (786-O-Ku70, 786-O-siKu70, 786-O-vector and 786-O-scramble) to γ radiation, and assessed the cellular radiosensitivity using MTT assay (Figure 3). Compared to the 786-O-vector cells, the survival fractions of 786-O-Ku70 cells were much higher at each point, and at 16Gy, 32Gy and 48Gy, the survival fraction of 786-O-Ku70 cells was increased by 4.9%, 3.8% and 2.3%, respectively. Conversely, the survival fractions of 786-O-siKu70 cells were significantly lower than the 786-O-scramble group at each point, and at 16Gy, 32Gy and 48Gy, the survival fraction of 786-O-siKu70 cells was decreased by 6.0%, 9.7% and 19.6%, respectively. Together, the results indicated that upregulating Ku70 expression can reduce the radiosensitivity of 786-O cells. On the contrary, RNAi-mediated Ku70 knockdown enhances radiosensitivity of 786-O cells.

Human renal carcinoma cell line (786-O) was obtained from Keygen company (Nanjing, China) and cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) at 37°C with 5% CO₂ and humidity-saturated incubator.

To construct a retroviral vector expressing Ku70, a DNA fragment encoding Ku70 was amplified by PCR from human genomic DNA and the cloning primer was designed as Additional file1: Table S1. The PCR product was digested with the restriction enzymes BamH I and Sal I, and inserted into the pBaBb-puro vector. The recombinant vector was designated as pBaBb-puro-Ku70 and the correction of the inserted fragment was verified by enzymatic treatment and DNA sequencing. We also designed and synthesized three pairs of effective RNA interference (RNAi) sequences (Additional file1: Table S2) to target Ku70 gene. Then each sequence was annealed to form double-stranded DNA and connected to pSUPERretro-puro vector digested by Bgl II and Hind III. The recombinant vectors were designated as pSUPERretro-puro-siKu70 and identified by sequencing. At the same time, we also constructed the corresponding control vectors named pBaBb-puro-vector and pSUPERretro-puro-scramble. After the Ku70 siRNA target sequence with the strongest silencing effect was screened, all of the vectors were co-transfected in 293FT cells. Generated virus particles subsequently infected 786-O cells, the positive clones were obtained following puromycin selection. The stable cell lines achieved were correspondingly designated as 786-O-Ku70, 786-O-siKu70, 786-O-vector and 786-O-scramble.

Cultured cells were collected and washed twice with 1 ml of PBS. After cracked with total protein lysis buffer, the cells were cooled on ice for 10 min and collected in a centrifuge tube. 50 μl of protein sample were mixed with 50 μl of SDS loading buffer and incubated for 5 min at 100°C. Then the samples were run on a SDS-PAGE gel, after electrophoresis, the proteins were transferred onto PVDF membrane and detected by immunolabelling with primary and secondary antibodies. In this experiment, we made the β-actin as the internal reference.

Total RNA was extracted using the Trizol reagent according to the instructions. The RNA purity and concentration were determined by the UV spectrophotometer. CDNA was reversibly transcribed from the extracted total RNA using an MMLV reagent kit (TaKaRa, Japan) and the primers were designed as Additional file1: Table S1. PCR was then carried out as follows: denaturing at 95°C for 20 s, 40 cycles of 10 s at 95°C, 20 s at 58°C and 30 s at 72°C.

The stable 786-O cell lines were harvested by centrifugation for 3 min at 1000 rpm and were resuspended in binding buffer. Aliquots containing 1 × 10⁵ cells in 190 μl of buffer were stained with 10 μl of PI solution and with 5 μl of Annexin V-FITC (eBioscience, USA) for 10 min at room temperature. Then Flow cytometric analysis was performed using a flow cytometer (BD, USA) to detect the cell apoptosis.

TUNEL assay was also performed to detect the apoptosis of the stable 786-O cell lines. Briefly, the cells were smeared on slides and fixed with 4% paraformaldehyde at room temperature for 30 min. Then the slides were incubated with 0.1% Triton X-100 for 10 min, rinsed with PBS, incubated with 3% H₂O₂ to block the endogenous peroxidase activity, and rinsed with PBS. After that, the slides were TUNEL stained using a apoptosis detection kit (Keygen, China) in accordance with the manufacturer’s instructions. The TUNEL-stained slides were observed under a fluorescence microscope (Olympus, Japan).

The stable 786-O cells were plated in a 96-well culture plate at an initial density of 2 × 10³ cells/well and then exposed to 0-48 Gy of a ¹³⁷Cr source in a γ-irradiator. Cell viability was determined by the MTT assay immediately after radiation treatment. In brief, 20 μl of MTT solution was added into each well and incubated for 4 hours at 37°C in a humid, 5% CO₂ atmosphere. Then, the media was removed, and cells dissolved in 150 μl of DMSO. Finally, absorbance was recorded at 490 nm using the enzyme-linked immunosorbent assay (ELISA) Reader (Bio-RAD, USA).