Targeting DNA Repair Genes Efficiently Radiosensitizes Human Tumor Cells

BackgroundRadiotherapy, one of the leading approaches in cancer therapy, contributes to more than50%of cancer treatment. However, the intrinsic radiation resistance and radiatherapeutic-induced tumor cell repopulation are major obstacles for cancer radiotherapy which greatly limit its clinical and potential value. Therefore, enhancing the radiosensitivity of tumor cells and inhibiting tumor cell repopulation can effectively improve the clinical radiotherapeutic outcome.The main mechanism of Ionizing radiation (IR) killing tumor cells is mainly inducing DNA double-strand breaks (DSBs), leading to loss of proliferative capacity. However, the tumor cells have a strong DNA DSBs repair capacity which directly results in tumor radiation therapy resistance. In mammalian cells, there are two pathways to repair DNA DSBs:homologous recombination (HR) repair and non-homologous end joining (NHEJ) repair. Regulation of DNA DSBs repair genes can enhance the tumor cell sensitivity to radiotherapy. XRCC2and XRCC4genes are essential factors in HR and NHEJ repair pathway, respectively. Targeting XRCC2and XRCC4might sensitize human tumor cells to IR-induced killing.Tumor cell repopulation is an important factor for radiation treatment failure and recurrence. It has been demonstrated that IR-induced dead cells can activate caspase3 gene and its downstream pathway caspase3-arachidonic acid-prostaglandin E2(PGE2) to stimulate the accelerated proliferation. Therefore, inhibition of radiation therapy induced caspase3can enhance the radiosensitivity of human tumor cells.MiRNAs are evolutionally conserved endogenous non-coding RNAs with19to23nucleotides that negatively regulate gene expression by binding to the3'-untransltaed region (UTR) of target genes via mRNA degradation or (and) translation inhibition. As artificial miRNA (amiRNA) has been reported to mitigate shRNA-mediated cytotoxicity, it has been widely used in the specifically silencing expression of target genes. AmiR-based RNA interference has become the basic tools for cancer target therapy and is expected to provide a new strategy for cancer therapeutic and clinical research.Partâ… RNAi-Mediated Targeting of Noncoding and Coding Sequences in DNA Repair Gene Messages Efficiently Radiosensitizes Human Tumor CellsObjectives:Aritificial miRNAs targeting DNA repair genes XRCC2and XRCC4in HR repair and NHEJ repair were constructed to investigate the silencing effect on XRCC2and XRCC4genes expression in human tumor cells and cell radiosensitivity after X-ray exposure. We were interested in testing the hypothesis that combining amiRNAs to target the3'-UTR of XRCC2and XRCC4gene and siRNA to target the coding sequence of the same gene could maximally knock down the genes and sensitize human tumor cells to IR-induced killing. The mechanism of gene inhibition by theses mall RNAs was also investigated. Then the possibility that whether XRCC2and XRCC4could be used for the therapeutic radiosensitizer and the efficiency that RNA interference (RNAi) technology by combining amiRNA and small interference RNA (siRNA) could enhance genes knockdown were further analyzed.Materials and Methods:The specific double-strand oligonucleotides of XRCC2and XRCC4genes were designed and synthesized based on the BLOCK-iTTM Pol II miR RNAi expression system and generated by annealing equal amounts of single-strand oligonucleotide, which was cloned into pLenti6/V5-DEST vector to form pLenti6/V5-GW/EmGFP-miRNA expression plasmids by Gateway technology. Luciferase assay was performed to identify the specific binding of amiRNAs with potential-targeted sequences of3'-UTR. The amiRNA expression plasmids including amiR-Vector, amiR-XRCC2and amiR-XRCC4were combinedd with ViraPowerTM Lentivirual Packaging Mix and transfected into the293FT cells to generate lentiviral particles.The virus containing supernatant was collected and transduced into human glioblastoma cell U87MG and lung cancer cell A549. The cells were harvested after postinfection for Western blot analysis to evaluate the protein level of XRCC2and XRCC4. Both U87MG and A549cells stably expressing amiRNAs were established by blasticidin treatment. These satble cells were then irradiated at different doses by X-ray machine. Then, y-H2AX assay was performed to examine the DNA DSB repair efficiency by immumofluorescence cell staining technique. Cellular sensitivity to radiation was determined by clonogenic surviving assay. The siRNAs against XRCC2and XRCC4were synthesized and transfected into U87MG and A549with stably expressing amiR-XRCC2and (or) amiR-XRCC4. The protein level of XRCC2and XRCC4was examined after transfection at72h by Western blot to evaluate the inhibition effect and clonogenic surviving assay was performed to reveal the effect of combining amiRNA expression plasmid and siRNA transfection on tumor cell radiosensitivity. The amiRNA mimics against XRCC2and XRCC4were synthesized and co-transfected with siRNAs against XRCC2and XRCC4into U87MG and A549cells for72h, and then the mRNA and protein levels of XRCC2and XRCC4were determined by quantitative real-time PCR (RT-PCR) and Western blot. The effect of combined RNA interference (RNAi) with siRNA and amiRNA on sensitizing human tumor cells to radiation was determined by clonogenic assay.Results:In this study, the mRNA and protein levels of XRCC2and XRCC4were higher in human glioblastoma and lung cancer cells than that in human non-tumor cells. The amiRNA against XRCC2or XRCC4could specifically bind to the potential targeted sequences of3'-UTR, suggesting the amiRNA could work as a miRNA to inhibit the targeted gene expression. The amiRNA against XRCC2or XRCC4 efficiently inhibited the targeted gene expression and significantly sensitized human tumor cells to IR-induced killing, respectively. This radiosensitization effect was further enhanced by combining amiR-XRCC2and amiR-XRCC4. The stably amiRNA expressed cells showed similar proliferation and clonogenic rate, indicating that the amiRNA expression had less toxicity. There was no apparent difference in the y-H2AX-positive ratios among the amiRNA stably expressed cells immediately after IR at30min. however, the y-H2AX-positive ratios clearly increased in the irradiated cells expressing amiR-XRCC2or amiR-XRCC4when compared with the irradiated cells transfected with the amiR-Vector alone. The y-H2AX-positive ratios left even more in the irradiated cells expressing both amiR-XRCC2and amiR-XRCC4than in the irradiated cells expressing amiR-XRCC2or amiR-XRCC4alone. The specific siRNA against XRCC2or XRCC4maxinally inhibited the targeted gene at72h after transfection and inhibited more XRCC2or XRCC4expresssion in U87MG and A549cells stably expressing the amiRNA against XRCC2or XRCC4. In addition, the siRNA against XRCC2or XRCC4more efficinently radiosensitized U87MG and A549cells expressing the amiR-XRCC2or amiR-XRCC4than the cells containing amiR-Vector. Mixing the amiRNA mimic with siRNA against XRCC2or XRCC4more efficiently decreased their expression level and sensitized the cells to IR-induced killing than using amiRNA or siRNA against XRCC2or XRCC4alone. The protein level of XRCC2and XRCC4between the cells treated with amiRNA mimic and the cells treated with siRNA did not shown any apparent difference (-70%of control level), but the mRNA level of the targeted genes was lower in the cells treated with siRNA (-30%of control level) than in the cells treated with amiRNA mimic (-65%of the control level). When compared with siRNA alone, combining the amiRNA and siRNA did not further decrease the mRNA stability (-30%) although the combined approach did further decrease the protein level.Conclusion:Combining an amiRNA engineered to target3'-UTR of XRCC2or XRCC4along with a siRNA to target the gene coding region and non-coding region can improve silencing efficiencies to achieve more robust radiosensitization than a single approach alone. Mechanistically, the combinatorial knockdown decreased targeted gene expression through both a reduction in mRNA stability and a blockade to mRNA translation. Together, our findings establish a general method of gene silencing that is more efficient and particularly suited for suppressing genes that are difficult to downregulate by amiRNA-or siRNA-based methods alone. Part IICombining Heavy Ion Radiation and Artificial miRNA to Target Homologous Recombination Repair Gene Efficiently Kills Human Tumor CellsObjectives:Heavy ion radiotherapy is defined as the best and most effective approach to chieve good cancer controllability because of the advantages of its physics and biological effects compared with conventional radiograph radiotherapy. Heavy ions can lead to dense ionization along their trajectories, cause clustered DNA damage and alter cellular ultrastructure. Moreover, much clustered and shorter DNA fragments could interfere with the efficiency of NHEJ, but bot HRR. Clinical trials with carbon ions provided excellent outcome, especially for radiotherapy-resistant tumors, which resulted in an increased interest in heavy ion radiotherapy especially combinated with molecularly targeted approaches or chemical agents. Previously, we have shown that our designed amiRNAs could efficiently target DNA DSBs repair gens of XRCC2or XRCC4and sensitize human tumor cells to X-ray. Based on these data, we were interested in testing the hypothesis that combining heavy ions and amiRNAs to target HRR but not NHEJ should efficiently kill human tumor cells.Materials and Methods:Human glioblastoma cell line U87MG and lung cancer cell line A549and their counterparts, over-expressing amiRNA to target XRCC2, XRCC4or both, were used in this study. In addition, these cell lines were subcutaneously injected into nude mice to form xenografts. Both cells and xenografts were exposed to low-linear energy transfer (LET) and high-LET radiation, respectively. The low-LET radiation was performed with X-ray machine at320KV,10mA and the dose rate was 2Gy/min for cells but1Gy/ml for animals. The high-LET radiation was performed with Fe ions,1GeV/amu, at the dose rate of1Gy/min. Survival sensitivities were examined using a clonogenic assay after these cells were exposed to X-ray or heavy ion. In addition, the mice were sacrificed one week after the final irradiation. The tumors were removed and weighed, and the expression of XRCC2and XRCC4was also measured by immunohistochemical staining.Results:Targeting either XRCC2or XRCC4sensitized U87MG or A549cells to low-LET radiation. Targeting XRCC2in U87MG or A549cells dramatically sensitized the cells to high-LET radiation, but targeting XRCC4in these cells did not change the sensitivities of the cells to high-LET radiation when compared with that of their counterpart cells expressed with empty vector alone. In addition, although targeting both XRCC2and XRCC4significantly sensitized the tumor cells to low-LET radiation versus targeting XRCC2or XRCC4alone, it did not further sensitize the tumor cells to high-LET radiation versus targeting XRCC2alone. High-LET radiation significantly reduced the tumor size versus low-LET radiation in all groups. After exposure to X-ray, the sizes of the tumor from the cells expressed with amiRNA, targeting either XRCC2or XRCC4, were smaller than that from the tumor cells transfected with vector alone. After exposure to iron, however, the size of the tumors from the cells expressed with amiRNA targeting only XRCC2but not XRCC4were smaller than that from the tumor cells transfected with vector alone. Similar to the in vitro data, although targeting both XRCC2and XRCC4significantly sensitized the tumors to low-LET radiation versus targeting XRCC2or XRCC4alone, targeting both XRCC2and XRCC4did not further sensitize the tumors to high-LET radiation versus targeting XRCC2alone.Conclusion:Although targeting either XRCC4(NHEJ factor) or XRCC2(HRR factor) sensitized the human tumor cells to X-ray, in vitro and the xenograft animal model, only targeting XRCC2but not XRCC4sensitized the human tumor cells to heavy ions in vitro and in the xenograft animal model. Combining heavy ions with molecularly targeting the HR repair pathway, but not the NHEJ repair pathway, could significantly improve the efficiency of tumor cell killing.