Experimental Study In Influence Of RNAi On Ku70 Gene Upon Radiosensitivity Of Hela Cells

Ku70 is a key molecule in DNA double-strand break (DNA double-strand breaks, DBS) repair pathway and in DNA-dependent protein kinase (DNA-PK) complex. Ku70 could promote the non-homologous end joining (non-homologous end-joining, NHEJ) following DNA broken. Study of the effect of gamma ray exposure on the surviving ratio of Hela cells and the expression of Ku70 gene will provide important biological basis for the radiotherapy in human cervical cancer. In the present study, RNA interference (RNAi) technique was used to knock down Ku70 gene, in order to investigate the changes in radiosensitivity of Hela cells when the protein expression of Ku70 was lowed expression. The results will provide new experimental basis for the gene therapy of cervical cancer.1. Effect of gamma ray exposure on Hela cell survivingThe colony forming unit technique was used to study the effect of gamma ray exposure on the surviving ratio of Hela cells, in order to investigate the Hela cell radiosensitivity. Hela cells were irradiated by gamma ray with the doses of 0, 1.0, 2.0, 4.0 or 6.0 Gy respectively. The number of colony was counted and the surviving ratio was analyzed at 24 h after exposure. The results showed the there was no marked changes for surviving ratio of Heal cells at 24 h after 1.0~4.0Gy gamma ray irradiation compared with 0 Gy group (P>0.05 respectively). These results suggest that there was much higher radioresistance of the Hela cells to gamma ray exposure.2. Effect of gamma ray exposure on Ku70 expression in Hela cellsHela cells were irradiated by gamma ray with the doses of 0, 1.0, 2.0, 4.0 or 6.0 Gy respectively. The quantitative real time PCR (QRT-PCR) technique was employed for the measurement of Ku70 mRNA level and the western-blot analysis was used for the measurement of Ku70 protein expression. In the dose-effect experiments, the results showed that the Ku70 mRNA level was markedly increased and the Ku70 protein expression was significantly increased (P<0.05~P<0.01) at 24 hours after exposures with the doses of 1.0~6.0Gy respectively. In the time course experiments, the results showed that Ku70 mRNA level was markedly increased and Ku70 protein expression was significantly increased (P<0.05~P <0.001) from 8 h to 72 h after 4.0Gy exposures respectively.3. Construction of the RNAi vector on Ku70 and analysis of interference efficiencyBased on siRNA design tool (http://www. ambion.com), three of the siRNA for Ku70 gene were designed. One was located in the 5 `non-coding region and the other two were located in the coding region. After synthesizing fragments of three double-stranded siRNA, three of them were transfected into HeLa cells. The protein expression of Ku70 was detected by western bolt. The results showed that the expression of Ku70 protein was much lower at 72 h after transfection, especially for the third fragment, which was inserted into pSilencer-4.1-CMV-hygro vector. The recombinant vector was constructed which was named pSilencer-4.1+Ku70. The sequence of the recombinant plasmid was correct. It was also found that the expressions of Ku70 mRNA and Ku70 protein were decreased markedly after stable transfecttion of pSilencer-4.1+Ku70 plasmid into Heal cells.4. Influence of RNAi on Ku70 gene upon radiosensitivity of Hela cellsHela cells thansfected with pSilencer-4.1-Ku70 plasmid and with pSilencer-4.1 plasmid were exposed to gamma ray irradiation with the dose of 4.0 Gy respectively. At 24 h after irradiation, the colony number of Heal cells was counted. The results showed that there were no marked changes for the number of colony in 4.0Gy group compared with 0Gy and pSilencer-4.1 -Ku70+0Gy groups. These results indicated that Heal cells were much higher radioresistant to gamma ray exposure. However, it was very interesting to find that there was significantly reduced for the colony number in pSilencer -4.1-Ku70+4.0Gy group compared with pSilencer-4.1 + 4Gy group (P<0.001). These results suggest that the radiosensitivity of Hela cells was increased after Ku70 gene was knocked down by RNA interference.The results mentioned above will provide new experimental basis for the gene therapy of cervical cancer. There will be very import implications and potential available for future uses. Ku70 gene might be a new tagart for the radiotherapy of cervical cancer.