Enhanced Radiosensitivity Of HepG-2 Cancer Cells By RNA Interference Targeting DNA-PKcs Gene

Radiotherapy constitutes a major part of the treatment of cancer patients. Of all cancer patients, 70% receives radiotherapy. All cancer cells can be killed theoretically with enough doses, which are hampered by the limited endurance of normal tissue.DNA is the main target of irradiation and DSB is believed to be one of the most severe types of DNA damage, and if left unrepaired is lethal to the cell. The most important DSB repair in mammalian cells is NHEJ and HRR. NHEJ is the predominant type of DSB repair in mammalian cells, as opposed to lower eucaryotes, but HRR has recently been implicated in critical cell signaling and regulatory functions that are essential for cell viability. The NHEJ reaction relies on Ku (a heterodimer of Ku70 and Ku86), DNA-PKcs, Artemis, Xrcc4, and DNA ligase IV (Lig4). First, Ku binds to the ends of a DSB and recruits the DNA-PKcs/Artemis complex. This complex would then trim the ends to make them ligatable. Additional nucleases and/or polymerases may also be involved in this process. Finally, the Lig4/Xrcc4 complex is recruited for ligation.The correlation between radiosensitivity and DSB repair property was confirmed by enhancement of radiosensitivity in cells defect in DSB repair genes and highly expression of DSB repair proteins in radio-resistant cells. As a result, inhibition expression of DSB repair proteins became an important radiosensitization strategy. A number of experiments proved the radiosensitization by inhibition expression of DNA-PKcs as well as other DSB repair proteins.RNAi is the process of using specific sequences of dsRNA to knock down the expression levels of complementary genes. SiRNAs are loaded into RISC and cleave the target mRNA. RNAi has been widely applied in the research of gene function and gene therapy as an efficient tool for specific gene silencing.This research aimed to reveal the radiosensitization property of inhibited DNA-PKcs gene by RNAi in HepG-2 cell line. Two confirmed siRNA, targeting 352 bases downstream from the start codon and kinase domain of the mRNA sequence of DNA-PKcs gene separately, and a scrambled sequence (control) were synthesized. The siRNA-encoding complementary single-stranded oligonucleotides hybridize to give BaraH I and HindIII-compatible overhang. After annealing and 5'phosphorylation by T4 polynucleotide kinase, oligonucleotides were ligated into plasmid pSilencer2.1-U6, which then was transformed into DH5 a . The obtained plasmids were identified by PCR and sequencing and were named pSilencer-1, pSilencer -2 and pSilence -C. To reduce theamounts of carbohydrate and increase transfection efficiency, Qiagen plasmid purification kit was selected. The pcDNA3.0-GFP vector, which encodes GFP, was used to assess transfection efficiency. The most optimal efficiency was obtained when cell confluence was 90% and the ratio of DNA: lipofectamine 2000 was 0.8ug: 2ul. The experiment included four groups: pSilencer-1, pSilencer-2, pSilencer-C and untransfected group. After transfection, each group was cultured in DMEM medium added with 200ug/ml hygromycin B. About two weeks later, cell clones were obtained. Three clones of each group were selected and transfered to fresh 96 well culture dish and then to culture dishes. Each clone including pSilencer vector sequence was confirmed by PCR.The protein expression of intracellular was determined by western blotting. The protein level of pSilencer -C group was similar to untransfected group. The level dropped to 15%, 25% and 34% in pSilencer-1, and to 54%, 36% and 39% in pSilencer-2, compared to pSilencer -C group. The protein level in pSilencer-2 is significantly lower than that of pSilencer -C (p<0.01) and significantly higher than that of pSilencer-1 (p<0.05).The mRNA levels of DNA-PKcs were measured by RT-PCR. The mRNA level of pSilencer -C group was similar to untransfected group. The level dropped to 18%, 24% and 38% in pSilencer-1, and to 48%, 37% and 41% in pSilencer-2, compared to pSilencer -C group. The mRNA level in pSilencer-2 is significantly lower than that of pSilencer -C (p<0.01) and significantly higher than that of pSilencer-1 (p<0.05).The clones that protein level dropped to 17% in pSilencer-1, to 36% in pSilencer-2, along with the clone that had 97% protein level in pSilencer -C, were selected. MTT results indicated that the survival fractions of pSilencer-1 and pSilencer-2 group were significantly lower than that of pSilencer-C (p<0.05) after irradiated with 4Gy> 8Gy^ lOGy. Radiosensitization was ascertained by clonogenic survival assays. Cell survival was plotted as a function of dose and fitted using the linear quadratic model SF =exp(- aD-P D2). In 2Gy, 4Gy and 6Gy group, the survival fractions of pSilencer-2 is significantly lower than that of pSilencer-C (p<0.001) and significantly higher than that of pSilencer-1 (p<0.001), whereas no significant survival difference was seen in both 8Gy and lOGy.Conclusions1 Inhibiting gene expression by RNAi radiosensitized HepG-2 cell line by prohibiting DSB repair, which suggested DNA-PKcs could be a candidate target for tumor gene therapy.