The Experimental Study About Chk-1 And PARP-1 SiRNA Incombination With Radiotherapy To Treat Malignant Tumors

In recent years, studies have shown that the specific gene blockers can inhibit the function of the key regulatory gene which play important in tumor cell DNA damage and repair process, can inhibit DNA repair, and enhance the effectiveness of their radiotherapy. Ionizing radiation can induce signal transduction pathway of DNA damage , activate checkpoint G1 / S and G2 / M, delay cell cycle progression, so take enough time for DNA damage repair. Currently Chk-1 and Chk2 are respectively believed that they are the key regulatory gene of the check points and play an important at G2 / M and G1 / S phase regulating downstream signal transduction pathway. The use of Chk-1 inhibitor UCN-01 could block Chk-1 function, eliminate the cell cycle arrest induced by drugs and radiation, increase cytotoxicity. But the existence of its side effects need further search and the corresponding experimental study to confirm, which limite their application in clinical tumor therapy.Poly(ADP-ribose)polymerase -1 (PARP-1) play an important role in DNA repair, cell death, proliferation and differentiation.The study shows that the blockers of PARP-1 can inhibit PARP-1-mediated DNA repair mechanism, improve radiotherapy and chemotherapy on tumor cell DNA damage by inhibiting PARP-1's activity, thus may have a potential tumor therapeutic value. But there are many defects in blocker technology itself, which significantly limit their clinical application on tumor treatment.Small interference RNA( siRNA) technology can better solve the problem brought about by the application of blockers of the above questions. It may be an effective therapeutic approaches that take Chk-1 and PARP-1 as target and interfere cell damage repair mechanism, improve the treatment of tumor sensitivity. This may provides a new way for radiotherapy for cancer gene therapy.ObjectiveThe RNAi expression vector of Chk-1, PARP-1 were designed,synthesised and identificated. They were studyed on the impact of tumor radiation therapy through the application in vitro and in vivo.They would be a new ways for treatment of cancer research.MethodsAccording to the log in GenBank Chk-1 No. NM₀07691, PARP-1 No. NM₀07415 and shRNA design principles, oligonucleotide used to construct RNAi vector wered design and synthesised, and the recombinant vector RNAi wered identificated through the application of restriction enzyme and sequencing messurement.They were transfected mouse Lewis lung cancer cell lines through Lipofectimine ? 2000 and observed transfection under fluorescence microscope after 48h,thus the Lewis lung cancer cells were re-collected respectively.The effective iRNA vector were chosen by RT-PCR technology.The effective siRNA expression vector were transfected to Lewis lung cancer cell lines. The radiation sensitizing effect of RNAi to Lewis lung cancer cell line were studyed. The inhibited rate of transfected cells were observed by MTT assay , cell apoptosis were observed by flow cytometry, expression of Chk-1 and PARP-1 were analysised by immunohistochemical.The animal models of tumor-bearing mice were built when C57BL / 6 inbred mice were inoculated 5×105 Lewis lung cancer cells into right hind limb subcutaneous. The tumor-bearing mice were irradiated 2Gy body by using deep X-ray treatment machine . RNAi (2 -5mg/kg body weight) were injected by the tail vein after irradiation 4h. Tumor size were observed, tumor-bearing mice survival and mortality were statisted, the expression of Chk-1 and PARP-1 were detected by RT-PCR and immunohistochemistry.ResultsThe correct recombinant PARP-1 RNAi vector by the restriction endonuclease digestion were sequencing analysised, the sequence alignment analysis were taken between sequence of measurements with the theoretical sequence .Results showed that the sequence is fully consistent with the theory. PARP-1 RNAi vector pGPU6/GFP/Neo-PARP-1-1308 were effectively screened when they transfected into Lewis lung cancer cell lines by Lipofectimine ? 2000.The absorbance values of siRNA transfection group and 2Gy irradiation was significant (p <0.05)than the control group, and that of 2Gy irradiation group + siRNA group was significant ( p <0.05) than 2Gy irradiation group and siRNA group respectively. there was no significant difference (P> 0.05) between missense sequence group and the normal control group. The same result took place between 2Gy irradiation group + missense sequence group and 2Gy irradiation group. The inhibited rate of Lewis lung cancer cells in 2Gy irradiation + siRNA group is the highest, 60.5 percent. RT-PCR test results also showed that Chk-1, PARP-1 mRNA expression level is lower in transfected cells .There was significant difference (p <0.05) on Lewis lung cancer cells apoptosis among siRNA transfection, 2Gy irradiation with the normal control group, , and siRNA transfection can increase the Lewis lung cancer cell apoptosis with 2Gy irradiation(p<0.05). siRNA pGPU6/GFP/Neo-PARP-1-1308, pGPU6/GFP/Neo-Chk-1-536 make tumor smaller than sham irradiation group and the negative control group; 2Gy irradiation significantly reduced tumor volume small, slower growing than sham control group and negative control group.United siRNA treatment can decrease tumor volume than irradiation alone. United siRNA pGPU6/GFP/Neo-PARP-1-1308 and pGPU6/GFP/Neo-Chk-1-536 together radiotherapy significantly reduced tumor volume than radiotherapy alone ,but was not obviously reduced with the single united radiotherapy separately. That showed that the siRNA pGPU6/GFP/Neo-PARP-1-1308, pGPU6/GFP/Neo-Chk-1-536 have synergistic effect with radiation on the tumor therapy. but there was no synergy between siRNA pGPU6/GFP/Neo-Chk-1-536 and pGPU6/GFP/Neo-PARP-1-1308.siRNA pGPU6/GFP/Neo-Chk-1-536, pGPU6/GFP/Neo-PARP-1- 1308 could decrease tumor-bearing mice mortality than sham irradiation group, Tumor-bearing mice mortality decreased in 2Gy irradiation group than that in sham control group and negative control group,so is in united siRNA treatment than irradiation alone group.SiRNA pGPU6/GFP / Neo-Chk-1 -536 and pGPU6/GFP/Neo-PARP-1-1308 United radiotherapy significantly decreased mortality than radiotherapy alone and was not obviously reduced with the single united radiotherapy separately.That Showed that the siRNA pGPU6/GFP/Neo-Chk-1- 536, pGPU6/GFP/Neo-PARP-1-1308 have therapeutic effects on the tumor,and has synergistic effect with radiation therapy.Chk-1 expression of tumor cells was down-regulated with siRNA pGPU6/GFP/Neo-Chk-1-536 than sham irradiation group. Chk-1 mRNA expression decreased with the application of siRNA treatment after 2Gy irradiation than irradiation alone group, but decline was not obvious than non- irradiated group . PARP-1 expression of tumor cells was down-regulated with pGPU6/GFP/Neo- PARP-1-1308; PARP-1 mRNA expression decreased with the application of siRNA treatment after 2Gy irradiation than irradiation alone group, but the decline was not obvious than non-irradiated group. That indicated that siRNA has a synergy with irradiation .ConclusionThe RNAi vector of Chk-1, PARP-1 were successfully constructed and screened. The RNAi of Chk-1 and PARP-1 can enhance inhibition of Lewis lung cancer cells and the cell apoptosis induced radiotherapy. They has therapeutic effects on the tumor and has synergistic effect with radiation therapy.