Study On Stereotactic Body Radiation Therapy And Down-regulation Of AMID Combined With Ionizing Radiation In Non-small Cell Lung Cancer

ObjectiveThe purposes of this study were to compare the treatment effect of stereotactic body radiation therapy(SBRT)and surgery for non-small cell lung cancer(NSCLC),and to analyze the risk factors affecting the prognosis of NSCLC treated by radiotherapy.We further investigated the relationship between apoptosis inducing factor homologous mitochondrion associated inducer of death 2(AMID)and the prognosis of patients with NSCLC by the cancer genome atlas(TCGA)lung cancer database and clinicopathological data.And explored the effect and possible mechanisms of down regulation of AMID combined with ionizing radiation on the biological function of NSCLC cells.Methods1.This retrospective analysis included 688 patients with T1-2NOMO NSCLC who underwent SBRT or surgery at Cancer Hospital of the University of Chinese Academy of Sciences from January 2012 to January 2016.An initial propensity score matching(PSM)analysis was performed to compare patients in the two arms based on age,gender,tumor diameter,Karnofsky Performance Status,CCI,and respiratory function.The effects of the two treatments were compared by Kaplan-Meier method.Logistic and Cox regression method were carried out for univariate and multivariate analysis,respectively.2.TCGA database was used to analyze the relationship between expression status of AMID in NSCLC and prognosis.Immunohistochemical method was used to detected the protein levels of AMID in NSCLC and para-carcinoma tissues,and the relationship with progression-free survival(PFS)was analyzed.3.The in vitro experiments were conducted to investigate the effects of down-regulation of AMID combined with ionizing radiation on the biological function of NSCLC.Lentiviral vector of shRNA targeting AMID gene was constructed and the knock-down of AMID was confirmed by RT-PCR and Western blot.Cell apoptosis were detected by flow cytometry assay and protein levels of the Bcl-2 family were analyzed by Western blot.The cell counting kit-8 assay,5-Ethynyl-2'-deoxyuridine assay and clone formation assay were applied to discover the effect of AMID on the proliferation of NSCLC cells;The correlation between down-regulation of AMID and the expression of p-p53,BRCA1 and y-H2AX was detected.The protein levels of Ki-67 in cellular wax blocks were detected by immunocytochemistry method.Wound healing migration and Transwell assays were taken to uncover the migration and invassiveness of PC-9 cells,the protein levels of Claudin-1 and E/N-Cadherin were analyzed by Western blot.Cell cycle test was used to detect the changes in the proportion of lung cancer cell cycle,Western blot was used to explore the expression levels of p-Chk1/2 proteins.Results1.Among the 688 NSCLC patients,579(85%)received surgery treatment,and 109(15%)received SBRT.All patients successfully completed corresponding treatment and were followed up.After PSM,the 5-year overall survival rates(OS)of SBRT and lobectomy group were 79.9%and 91.6%,respectively(P-value=0.097).In the radiation group,only one patient developed grade 3 radiation pneumonitis during the follow-up,no grade 4 or higher toxicity was observed.In addition,multivariate analysis showed that pathological type and maximum diameter of tumor were independent factors affecting the prognosis.2.The AMID expression from 65 NSCLC tissues and 12 adjacent tissues showed that AMID protein exhibited a lower expression in NSCLC compared to para-cancer tissues(P-value=0.037).Univariate analysis showed that the level of AMID expression was significantly correlated with PFS of NSCLC,patients with lower expression of AMID had a worse prognosis,the 3-year PFS rates of the high-expression group and the low-expression group were 51%and 30%,respectively(P-value=0.043).3.The RT-PCR and Western blot analysis revealed that the infection of lentivirus targeting AMID gene was able to inhibited the expression of AMID significantly(P-value<0.01).The flow cytometry assay showed that knock-down of AMID in the NSCLC cells could reduced the rates of radiation-induced cell apoptosis.Moreover,Western blot results revealed interactions between down-regulation of AMID and proteins of the Bcl-2 families.The CCK-8 assay,5-Ethynyl-2'-deoxyuridine assay in addition to clone formation assay all exhibited that the knock-down of AMID could significantly up-regulated the proliferation of NSCLC cells;These results suggested a role of AMID in management of cell proliferation.Compared with the control group,the expression of Ki-67 in lentivirus infection group was significantly increased.The knock-down of AMID revealed a significant raise in cell migration and invasiveness;Western blot analysis showed that the down of AMID in PC-9 cells decreased the protein levels of Claudin-1 and E-Cadherin,increased the protein levels of N-Cadherin,conversely.The down-regulation of AMID could significantly reduce the G2/M stage block of mitosis in NSCLC cells compared to the control group.Conclusions1.For patients with early stage NSCLC,SBRT is a safe and effective treatment with similar efficacy as surgery and mild toxicity;Pathological type and maximum tumor diameter are independent risk factors affecting the prognosis of radiotherapy patients with NSCLC.2.The expression of AMID protein is generally low in NSCLC,and there was a significant correlation between the expression level of AMID and the PFS,the lower expression of AMID suggested a poorer prognosis.3.AMID was able to conduct the biological function in NSCLC cells through differing molecular mechanisms.The knock-down of AMID was ascertained to correlated with the proliferation of NSCLC cells significantly,which may serve as a therapeutic target for the treatment of lung cancer.