Study Of RNAi Targeting Survivin Gene Combined With Radiotherapy On Lung Adenocarcinoma

Lung cancer is highest incidence of cancer in world, and accounts for the first one of the proportion of cancer deaths, the occurrence, the development, and the invasion,the metastasis of lung cancer are a multi-gene participating, multi-step developing process. The general treatment of lung cancer includes operation, radiotherapy and chemotherapy, but has limitations, in recent years, the rise of molecular targeting therapy of lung cancer are expected to combine with traditional regimen and solve thorough the therapy of lung cancer treatment. The so-called molecular targeting therapy is utilizing molecular targeting technology to block specific molecular target and its biological function, thereby reverses the biological behavior of malignant tumor cells on the molecular level, to reach purposesof tumor growth inhibition. Survivin gene is the inhibitor of apoptosis protein family member, it can inhibit apoptosis, involve in cell cycle regulation and promote angiogenesis function, is expected to become a target for cancer therapy; RNAi is a new development techniques of specific functional gene expression inhibition, using RANi technique to silence Survivin gene expression, thus to inhibit the role of Survivin in tumors can induce tumor cell apoptosis. Ionizing radiation can be directly used to treat tumors, but the long application has radiation tolerance phenomenon, and the combination of silencing Survivin gene and ionizing radiation can significantly increase the efficacy of radiation therapy. In this study, using the features of the three and combining them, to investigate the inhition effects on A549 cells and their graft tumor in vitro and in vivo after Survivin gene silencing, and to explore the mechanism. This experiment can provides a new treatment programs and experimental evidence for molecular target for clinical treatment of lung cancer. 1 Effects on A549 cell apoptosis and Survivin protein expression of ionizing radiationIonizing radiation can induce tumor cell apoptosis, its mechanism is very complicated, involves many genes. In this study, the apoptosis percentage of lung adenocarcinoma A549 cells 0 ~ 72 h after 0, 0.5, 1, 2.5 and 5 Gy X-ray irradiation were measured by flow cytometry, at same time, Survivin protein expression regularity in cells were detected by flow cytometry. The results showed that the apoptosis percentage of lung adenocarcinoma A549 cells 0 ~ 72 h after the same dose rradiation increased, and reached a peak at 12 or 24 h, then back down, but still were higher than the control; the percentage of apoptosis increased with dose increase same period of time after different doses irradiation, and reached the maximum 24 h after 5 Gy irradiation; at the same time, Surivin protein expression also had similar regulatity with apoptosis.Although Survivin is the apoptosis inhibition protein, but as the expression increased after radiation, apoptosis was not inhibited but actually increased, this suggested that there mignt be other factors effect them.2 Construction of shRNA vectors targeting Survivin and it's efficiency evaluationIn order to study lung adenocarcinoma A549 cell biological behavior change after Survivin was inhibited, the research used RNAi technique to specifically block Survivin gene expression. Two pairs of silencing sequence targeted Survivi gene were designed mainly with biological means, and linkaged to pGenesil-2 vector, to construct shRNA expression plasmid targeted Survivin, pGenesil-2-Survivin was identificated by restriction enzyme and sequencing, then transfected the expression vector into A549 cells with liposome, transfection efficiency was evalued with eukaryotic expression vector pGenesil-1 carried green fluorescence protein by flow cytometry and fluorescence microscope, and after shRNA expression plasmid was trasfected into A549 cells, Survivin mRNA and protein expressions were measured with RT-PCR and Western blot, and to evaluate interferencing efficiency. The results showed that 389 bp and 4206 bp fragments could be seen after pGenesil-2-Survivin plasmid digested by KpnⅠand EcoRⅠrestriction enzyme, this indicated that pGenesil-2-Survivin was right, the sequencing results were consistent with design, expression vector was entirely correct. cells with a green fluorescent were more than cells of normal group under fluorescence microscope, the flow cytometry results showed that green fluorescent protein expression rate was (56.22±8.16)%, transfection efficiency could reach 51.64%, the transfection efficiency could meet the experimental needs. After cells were transfected with the pGenesil2-Survivin, the Survivin mRNA and protein expression in cells were significantly inhibited, indicating Survivin gene is effectively silenced.3 Effect on A549 cells biological behavior of shRNA vector targeted SurvivinAfter the shRNA targeting Survivin expression vector pGenesil-2-Survivin were transfected into A549 cells, to measure cell proliferation change by plotting cell survival curve and MTT assay; and to measure cell phage change by flow cytometry; to detect cell apoptosis by flow cytometry after PI / AnexinⅤstaining and TUNEL; to measure the change of radio sensitivity; to measure the relative gene mRNA and protein expresson by RT-PCR and Western blot. The results showed that A549 cell proliferation were inhibited in blank vector group, transfected pGenesil-2-Surivin group, 5 Gy irradiation group and transfected pGenesil-2-Surivin+5 Gy irradiation group at 0, 2, 4, 6 and 8 d, in turns, the cells in blank vector group were inhibited small, then in 5 Gy irradiation group were inhibited more, at last, in transfected pGenesil-2-Survivin plasmid and in transfected pGenesil-2-Survivin plasmid +5 Gy group were inhibited most, particularly cells inhibition in the two combined group was strongest; MTT results showed that cell proliferation in blank plasmid was basically same with normal cell proliferation from 0 h to 72 h, but cell proliferation was inhibited in transfected pGenesil-2-Survivin group and 5 Gy irradiation group, cells proliferation was inhibted most in the two combined group. Flow cytometry results showed that cells cycle phage in transfected blank vector and 5 Gy irradiation group change little, while cells percentage of G0/G1 in transfected pGenesil- 2-Survivin group significantly increased, S phase shortened, in particular, cells percentage of G0/G1in transfected pGenesil-2-Survivin+5 Gy irradiation group increased more. Flow cytometry results with PI / AnnexinⅤstaining showed that cell apoptosis percentage did not change in transfected blank vector, and increased in 5 Gy irradiation group, and increased obviously in transfected pGenesil-2-Survivin shRNA, and was more in transfected pGenesil-2-Survivin+5 Gy group. Colony forming assay results showed that the cells in transfected blank vector survival fraction changed little, while cell survival fraction in transfected pGenesil-2-Survivin decreased obviously, the D0 values of normal cells, transfected blank vector and transfected pGenesil-2-Survivin were 3.26 Gy, 3.31 Gy and 2.11 Gy, respectively, this indicated cell radiosensitivity in transfected blank vector blank changed little, but cell radiosensitivity in transfected pGenesil-2-Survivin group increased. RT-PCR, immunocytochemistry and Western blot results showed that Survivin mRNA and protein expression in the cells transfected blank vector were affected little, and Survivin mRNA and protein expression in the cells of 5 Gy irradiation group increased, but Survivin mRNA and protein expression in the cells transfected pGenesil-2-Survivin and transfected pGenesil-2-Survivin+5 Gy irradiation were inhibited significantly. Western blot results showed that caspase-3 protein in transfected blank vector group, transfected pGenesil-2-Survivin plasmid group, 5 Gy irradiation group and combination of two increased, and in combination group the caspase-3 expression was maximum. These results suggested that successfully constructed RNAi expression vector targeting Survivin gene, after pGenesil-2-Survivin transfection into A549 cells, Survivin mRNA and protein in cells were inhibited obviously, and cell proliferation was suppressed, G0/G1 cycle arrest was induced, apoptosis of cell increased and cell radiosensitivity was enhanced.4 The inhibitory effect on A 549 cell xenografts in nude mice of targeting Survivin shRNATo establish the lung adenocarcinoma A 549 cells xenograft tumor in nude mice model, pGenesil-2-Survivin expression vector was injected into the tumor with liposome, then the tumor was irradiated by 5 Gy, to observe tumor growth inhibition, and to explore mechanism, results showed that the graft tu mor model successfully established, after A549 cells were injected into nude mice 4 d, the mass 5 mm in diameter could be seen. The tumor growth injected by saline, pGenesil-2-Survivin shRNA, irradiated by 5 Gy, and injected by pGenesil-2-Survivin shRNA +5 Gy irradiation was significantly different, tumor growth injected saline was very rapidly, The tumor growth injected pGenesil-2-Survivin shRNA, irradiated by 5 Gy, and injected by pGenesil-2-Survivin shRNA+ 5 Gy irradiation slowed down, tumor growth irradiated by 5 Gy was inhibited relatively weakly, while tumor growth injected expression plasmid inhibition was very strong, and combination of pGenesil-2-Survivin and 5 Gy was most strong. To detect mRNA and protein expression by RT-PCR, Western blot and immunohistochemistry, results showed that 5 Gy X-rays irradiation could induce Survivin mRNA and protein increase, while the injection of pGenesil-2-Survivin plasmid and injection added by 5 Gy irradiation could inhibit Survivin mRNA and protein expression, these results suggested that after injection of pGenesil-2-Surivin, Survivin expression was inhibited, then induced tumor cell apoptosis, this might be main reason for tumor growth inhibition. At present, there is no reports about application of RNAi technology combined with radiotherapy for lung cancer at home and abroad, the experimental results, the conclusion might provid the necessary experimental data for gene therapy for adenocarcinoma of the lung.