Expression Characteristics Of IL-24 In Patient With Non-Small Cell Lung Cancer And Its Relationship With Autophagy And Metastasis

Background: Currently,lung cancer is the leading cause of cancer deaths worldwide,and the non-small cell lung cancer(non-small cell lung cancer,NSCLC)accounts for about 80% of all lung cancer.A large number of studies have shown that IL-24 can inhibit tumor angiogenesis,tumor cell-growth,promote tumor cell apoptosis,and has a good prospect in tumor therapy.However,the effection of IL-24 on tumor inhibiting is selective.The expression characteristics of IL-24 in NSCLC patients,and its relationship with NSCLC autophagy,invasion and metastasis were not seen the related literature at home and abroad.In this study,the clinical specimens of NSCLC patient and NSCLC cell lines A549 were used to investigate the effection of IL-24 on NSCLC invasion and metastasis and its preliminary mechanism on the organization,cell and molecular level,in order to search for new drug targets for treatment of NSCLC.Part 1 Study on the expression characteristics of IL-24 in NSCLC patients and its relations with autophagy and invasion and metastasis-ralated proteinObjective: To determine the content of IL-24 in NSCLC patients serum and resected tissue and analyze its correlation with clinical pathological factors,simultaneously,to research the expression level of autophagy and the invasion and metastasis-ralated protein(LC3,Beclin-1,MMP-9 and VEGF)in resected tissure of NSCLC patients,and to analyze the correlation between the expression level of LC3,Beclin-1,MMP-9 and VEGF as well as clinical pathological factors respectively,furtherly to analyze the relevance of expression level of IL-24 and these proteins.Methods:(1)The Elisa detection method was used to detect the content of IL-24 in healthy people and patient serum.Statistical tool was used to analyze its difference,and its relationship with clinical pathological factors.(2)The real-time PCR was used to detect IL-24 m RNA expression level in normal and tumor tissue of NSCLC patients.The NSCLC cells were isolated from the tumor tissues by microdissection,and IL-24 m RNA expression in NSCLC cells was assayed by Q-PCR.Then,we analyze respectively the relationship between the expression level of IL-24 m RNA on organization and cell in patients and clinical pathological factors.(3)We detect IL-24,LC3,Beclin-1,MMP-9 and VEGF protein expression level respectively in normal and tumor tissue by Western blot method,and analyze separately the correlation between expression level of these proteins in tumor tissues and the clinical pathological factors.And the correlation between IL-24 and autophagy-associated protein LC3,Beclin-1,or the invasion and metastasis-ralated protein MMP-9,VEGF was also analyzed.(4)The tissue microarray and immunohistochemical method were used to stain and score for IL-24 and LC3 immunoreactive score separately.Further,we analyzed respectively the relationship between IL-24 or LC3 immunoreactive score and the patient clinical stage,and the correlation between IL-24 and LC3 immunoreactive score was also analyzed.Results:(1)The IL-24 content in patient’s serum is higher than that of healthy people,and is positively correlated with clinical TNM staging(IA-B→IIIA-B)significantly.(2)The IL-24 m RNA expression in tumor cells is negatively correlated with T stage(T0→T3).(3)The IL-24,LC3 and Beclin-1 in tumor tissue were significantly higher than that in normal tissue,and the IL-24 expression level in squamous carcinoma was significantly higher than that in adenocarcinoma.The IL-24 protein expression was positively related to tumor differentiation(poor to well differentiation)and negatively to N staging(N0 to N1-3).The expression of MMP-9 and VEGF were positively correlated with TNM staging(IA-B→IIIA-B)and the expression of MMP-9 also positively correlated with T stage(T0→T3).The expression of IL-24 was positively correlated whith Beclin-1 and LC3 respectively,and LC3-II/LC3-I was negatively correlated with MMP-9.The expression of IL-24 and LC3 in NSCLC were related with patient’s survival rat.Conclusion: The content of IL-24 in patient serum,IL-24 m RNA in tumor cells and IL-24 protein expression level in tumor tissue may be associated with NSCLC tumor progress.IL-24 could inhibit NSCLC invasion and metastasis,which may be associated with higher cell autophagy level.Part 2 IL-24-induced autophagy and its effection on the invasion and migration of NSCLC cell lines A549Objective: To validate the relationship bettween expession of IL-24 and autophagy,invasion and metastasis-ralated protein on cellular level,and preliminarily investigate the mechanism of IL-24 inhibition of invasion and metastasis of NSCLC.Methods: MTT method was used to evaluate the growth curve of NSCLC cell lines A549 transfected with the recombinant adenovirus-IL-24.IL-24,LC3,MMP-9 and VEGF expression were respectively detected in A549 cells transfected with the recombinant adenovirus-IL-24 or LC3-GFP by Western blot.The characteristics of invasion and metastasis of A549 cells transfected with the recombinant adenovirus-IL-24 or LC3-GFP were investigated respectively by the scratches and transwell experiments.Results:(1)The proliferation of A549 cells was restrained after transfection of excess Ad-IL-24.(2)The positive correlation between IL-24 and LC3 was observed after over expression IL-24 and LC3 in serum cultured A549 cells transfected with the Ad-IL-24 or LC3-GFP respectively,which was also found by free serum cultured A549 cells.(3)Over expression of IL-24 can reduce the expression of VEGF and MMP–9 in A549 cells.The scratches healing degree was smaller for over expression of IL-24 and LC3 group respectively than that of control group,and the cell number through the basement membrane also decreased significantlyConclusion: IL-24 could inhibit invasion and metastasis of NSCLC cell lines A549,which mechanisms may be that mutual induction of IL-24 and LC3,at the same time,decreases the expression of MMP-9 and VEGF.