Ku80 Correlates With Resistance To Neoadjvant Chemotherapy In Patients With Stage ⅢA Lung Adenocarcinoma, And Reduces Cisplatin/pemetrexed-induced Apoptosis In A549 Cells

BackgroundLung cancer is one of the top leading causes of death among all malignant cancers worldwide, and only 15.9% of patients with lung cancer are alive more than 5 years after diagnosis, while lung adenocarcinoma becoming most common histological subtype of total lung tumor. Due to deficiency of health examination,when patients are diagnosed as lung cancer,most of them have been at the middle-late stage,according to the tumor-node-metastasis (TNM) classification for non-small cell lung cancer (NSCLC) of UICC in 2009. For the stage (N2) of NSCLC patients, the prognosis of only curative resection remains unsatisfactory, because it is difficult for surgeons to achieve complete resection. According to NCCN guideline,after 2 cycles of chemotherapy, only tumor regresses apparently and ipsilateral mediastinal metastatic lymph nodes disappear, curative surgery could be effective. Therefore, the routine preoperative neoadjuvant radiotherapy or chemotherapy has been widely accepted. Platinum combines pemetrexed as the first-line neoadjuvant chemotherapeutic agents for lung adenocarcinoma. However, when reevaluating the tumor status through contrast Computed Tomography (CT) after 2 cycles of neoadjuvant chemotherapy, we find that only a few patients can obtain ideal efficiency.Due to the genetic variability and heterogeneity of lung adenocarcinoma, the effect of cisplatin combined with pemetrexed has obviously individual variation. Therefore, it is urgent to find an indicator to predict the resistance to neoadjuvant chemotherapy to improve the effects of treatment. With the development of cancer research, gene therapy has become the first line treatment of mid-late stage non-small cell lung cancer instead of traditional chemotherapy.DNA damage and repair mechanism takes a significant part in the carcinogenesis and development of lung adenocarcinoma. Among all types of DNA damage, DNA double-strand breaks (DSBs) is the most severe and dangerous type, and unrepaired DSBs will lead to tumorigenesis and genomic instability. Homologous recombination(HR) and nonhomologous end joining (NHEJ) constitute the major mechanism of DSBs repair. For the most mammalian cells, the NHEJ is the most method to repair DSBs. It has been reported that at least 4 proteins join in the NHEJ: the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku70/86, X-ray cross complementing 4(XRCC4) and DNA ligase IV. The heterodimeric Ku protein acted as detector for DSBs and composed of Ku70 and Ku80. And Ku protein could bind to DNA terminal to activate DNA-PK. Gu et al reported that inhibited the DNA-PK or Ku80 expression would result in deficiency of DSBs repair process. Ma et al reported that high expression of Ku80 was correlated with resistance to apoptosis caused by cisplatin and inhibited the Ku80 expression would enhance the sensitivity of lung adenocarcinoma cells to chemotherapy or radiotherapy. However, most of studies are based on resection specimen after the operation. It is unknown whether Ku80 is associated with chemosensitivity for cisplatin combined with pemetrexed before the neoadjuvant chemotherapy initiation in lung adenocarcinoma.In this study, we detected the mRNA and protein level of Ku80 of tumor samples obtained by fiberoptic bronchoscopy examination from 110 patients with stage Ⅲ A lung adenocarcinoma and explored the relationship among Ku80 expression level,clinical features and response to the neoadjuvant chemotherapy based on cisplatin combined with pemetrexed. In vitro experiments, to knockdown and upregulate Ku80 expression, A549 cells were transfected with lentiviral vector including specific shRNA and full-length cDNA of Ku80. Then we determined the expression level of Ku80 correlated with the biological function of A549 cells and the apoptosis-resistance caused by cisplatin combined with pemetrexed to explore a new strategy for gene therapy in lung adenocarcinoma.Part ⅠThe expression level of Ku80 in lung adenocarcinoma tissues and correlation with clinicopathological significanceObjective1. To detect the expression of Ku80 in lung adenocarcinoma tissue samples.2. To explore the relationship of Ku80 expression with clinical,pathological features and the effect of neoadjuvant chemotherapy.Methods1. Lung cancer patients consulted in the Department of Thoracic Surgery, Provincial Hospital Affiliated to Shandong University from September 2013 to September 2016 were examined by bronchoscopy. 110 patients with pathological diagnosis as lung adenocarcinoma with clinical classification stage IIIA were recruited for this study.No patient received chemotherapy, radiotherapy or gene-targeted treatment. All patients received 2 cycles of same first-line neoadjuvant chemotherapy based on cisplatin combined with pemetrexed. Then we reevaluated the response to chemotherapy by chest CT scan.2. All tumor samples obtained from bronchoscopy biopsy. Then we detected the Ku80 mRNA and protein expression level through qRT-PCR and immunohistochemistry.SPSS 19.0 was used for data analysis. The relationship between Ku80 mRNA and protein expression level and clinicopathological parameters was calculated by the chi-square test.Results110 patients of clinical classification were stage ⅢA and pathology diagnosed by bronchoscopy examination. After 2 cycles of neoadjuvant chemotherapy, 38 patients(34.5%) obtained obvious response to therapy and other 72 patients (65.5%) did not obtain the apparent effect of chemotherapy. Ku80 mRNA expression level of 38 patients in the response group is significantly lower than 72 patients in the nonresponse group (3.612±2.392,7.981 ±2.684; p<0.05). Ku80 protein expression level of response group is obviously lower than nonresponse group (2.079±1.617,5.597±2.114; p<0.05). Both mRNA and protein expression of Ku80 were related to tumor size,lymph node metastasis and chemosensitivity. While no obvious correlation with age, gender and smoking status.Conclusion1. The Ku80 mRNA or protein expression level was higher in nonresponse group to chemotherapy compared to response group.2. The overexpression of Ku80 is correlated with poor clinical and pathological parameters of lung adenocarcinoma patientsPart ⅡEffects of knockdown and overexpress Ku80 gene on biological behavior of lung adenocarcinoma cells and the chemosensitivity to cisplatin combined with pemetrexed in vitroObjective1. To detect the role of Ku80 gene to regulate the biological function of A549 cells2. To explore the effects of suppress and upregulate Ku80 expression on chemosensitivity based on cisplatin combined with pemetrexed of A549 cells.Methods1. Knockdown of Ku80 expression of lung adenocarcinoma cells were obtained through transfected with lentiviral vector including shRNA targeted to Ku80. And lung adenocarcinoma cells with Ku80 overexpression were obtained though transfected of lentiviral containing the full length of cDNA. Then, qRT-PCR and Western blot were used to detect the Ku80 expression of transfected cells.2. CCK-8 assay tested proliferation ability of A549 cells before and after transfection,and then, draw a cell growth curve to analysis the difference among different groups.3. CCK-8 assay was used in determining cell toxicity of transfected and untransfected A549 cells cultured in different concentration of cisplatin combined with pemetrexed.4. Transwell assay and cell scratch experiments were used in exploring transfected and untransfected A549 cells migration and invasion ability.5. Flowcytometry was performed to detect the apoptosis rate of transfected and untransfected A549 cells cultured in the same concentration of cisplatin combined with pemetrexed.Results1. We selected three siRNA sequences to interfere in Ku80 expression, and successfully construct lentiviral vector GV115 containing specific shRNA: shRNA86,shRNA87 and shRNA89 by Genechem (Shanghai, China). Then we built lentiviral GV115 contained scramble shRNA as negative control based on the scramble sequence. These lentiviral containing shRNA transfected A549 cells,and the infection rate confirmed over 80%.2. We obtained the lentivirus containing the full length cDNA of Ku80 to upregulate Ku80 expression in A549 cells and the control lentiviral vector from Genechem(Shanghai, China). After transfected with A549 cells, the infection rate confirmed over 90%.3. qRT-PCR results displayed that the Ku80 relative mRNA expression of normal A549 cells was 1, and relative expression of A549 cells transfected with scramble shRNA, shRNA86, shRNA87, shRNA89, empty vector for overexpressin and Ku80 cDNA were 1.083±0.15,0.534±0.17,0.663±0.13,0.916±0.11,1.051 ±0.112,2.231 ±0.194. Ku80 relative mRNA expression obviously decreased in shRNA86 transfected A549 cells compared to negative control (P<0.05). While the relative expression of Ku80 mRNA significantly increased in A549 cells transfected with lentivirus containing full length cDNA of Ku80 compared with the negative control and normal A549 cells, indicating that Ku80 cDNA could effectively upregulate Ku80 expression in A549 cells.4. Western blot results showed that Ku80 relative protein expression of untransfected A549 cells was 1, and the Ku80 relative protein expression of A549 cells transfected with shRNA86, shRNA87, shRNA89, empty vector for overexpression and Ku80 cDNA were 0.892±0.15, 0.033±0.029, 0.072±0.009, 0.530±0.061,1.083±0.182 and 2.102±0.574. Ku80 protein expression apparently decreased in A549 cells transfected with shRNA86, compared with negative control (p<0.05). And Ku80 protein expression was higher in A549 cells transfected with Ku80 cDNA than the negative control vector. Thus, we selected A549 cells transfected with shRNA86 and full length of cDNA as A549kd cells and A549oe cells to further experiments.5. CCK-8 assay showed proliferation ability of shRNA86 transfected A549 cells was decreased obviously compared to shRNA scramble transfected cells and normal A549 cells. The cell proliferation of Ku80-cDNA transfected A549 cells was obviously increased compared to empty vector transfected cells and normal A549 cells. The proliferation of shRNA scramble transfected A549 cells and empty vector transfected cells has no obvious difference with untransfected cells5. Transfected and untransfected A549 cells were cultured in different concentration of cisplatin combined with pemetrexed and CCK-8 analysis was used to calculate the IC50 of cells. Ku80-silencing A549 cells were 2.2-fold sensitive to mixed drugs (IC500.451 vs. 0.972) compared to untransfected A549 cells. While Ku80-overexpression A549 cells were 0.304-fold sensitive to mixed drugs (IC50 3.192 vs. 0.972) than untransfected A549 cells (p<0.05). There was no significant difference among the two negative control and normal A549 cells.6. Scratch experiments displayed that knockdown of Ku80 expression could suppress migration abilities compared with negative control group. And Ku80-overexpression could improve migration capacity of A549 cells compared with the negative control.7. Transwell experiment results confirmed that the ability of migration and invasion of shRNA86-transfected A549 cells were significantly decreased, while these abilities of Ku80 cDNA-transfected A549 cells were obviously increased.8. Date from flow cytometry analysis displayed that A549 cells treated by mixed drugs underwent apoptosis, which was augmented in shRNA86-transfected A549 cells(58.9%). However, Ku80 cDNA-transfected A549 cells apoptosis was reduced compared to negative control and normal A549 cells treated by the same drugs (18.7%,P<0.05).Conclusion1. Knockdown of Ku80 expression suppresses migration and invasion abilities of A549 cells and enhances the chemosensitivity to cisplatin combined with pemetrexed.2. Upregulation of Ku80 expression could promote migration and invasion capacities of A549 cells and enhances the chemoresistance to cisplatin combined with pemetrexed.