The Effect And Mechanism Of C9orf72 In Lung Adenocarcinoma

BackgroundAutophagy is a mechanism by which cells sequester substances to be decomposed and deliver them to lysosomes for degradation in response to physiological and pathological changes.Autophagy dysfunction causes tumors,nervous system degeneration,immune dysfunction and other diseases.C9orf72 is involved in the initiation of autophagy,membrane transportation,and affects lysosomal and immune function.We found that the expression of C9orf72 in lung adenocarcinomas in the TCGA database was significantly different between tumors and adjacent normal tissues,and patients with high levels of C9orf72 expression improved survival in patients than lower expression.Whether different levels of C9orf72 will affect the development of lung adenocarcinoma has not been reported.AimOverexpression and silencing of C9orf72 were carried out in lung adenocarcinoma cell lines to observe phenotypic changes such as autophagy,proliferation,migration and apoptosis in different culture conditions.C9orf72 overexpressed A549 cells were planted in nude mice to observe tumor growth in vivo.Bioinformatics analyses were carried out in both TCGA lung adenocarcinoma database and RNA-seq of overexpressed C9orf72 cells to explore the functions and mechanisms of C9orf72.Protein changes in autophagy pathways were carried out to explore the effect of C9orf72 on lung adenocarcinoma.Methods1.C9orf72 overexpressed and silenced A549 and H1299 cells were constructed.Stably transfected cells with GFP-RFP-LC3 fluorescence were used to observe the status of autophagy.Under normal culture and/or serum starvation conditions,the changes in autophagy fluorescence were observed in C9orf72 silenced A549 cells.Under normal culture and/or serum starvation conditions,experiment of cell proliferation(CCK8,colony formation assay),cell migration(transwell,scratch experiments),and apoptosis were observed after overexpression or silence of C9orf72.2.Nude mice were used for subcutaneous tumorigenesis experiments to observe the growth of tumors by transmited A549 cells overexpressing C9orf72.3.Samples of lung adenocarcinoma in TCGA database were divided into two groups according to the expression of C9orf72.The bioinformatics analysis of C9orf72 single gene in the lung adenocarcinoma data of TCGA database was performed to explore the differences of expression and survival.Functional enrichment,immune cell infiltration and gene correlation were analyzed.RNA-seq of A549 cells overexpressed C9orf72 was used to analyze the related functional changes of C9orf72.PCR and immunofluorescence co-localization analyses were used to verify the results.4.Protein changes of autophagy pathway in C9orf72 overexpressed and silenced A549 cells by western blot under different culture conditions.Results1.Silenced C9orf72 by small interfering RNA caused changes of autophagy fluorescence.Under normal culture conditions,the proliferation ability(colony formation assay,CCK8 experiments)was decreased in C9orf72 overexpressed cells,while promoted proliferation in C9orf72 silenced cells.Under serum starvation condition,C9orf72 overexpression enhanced proliferation ability(CCK8 experiments)while C9orf72 silencing inhibited proliferation.The cell migration(wound healing test,transwell)ability was decreased with the overexpression of C9orf72,while increased with C9orf72 silencing.Under normal culture conditions,the apoptosis of A549 cells were enhanced compared with the control group after overexpression of C9orf72,but there was no s ignificant difference in H1299.After 48 h of serum starvation,the apoptosis of the two cell lines was decreased compared with the control group.2.The A549 cells overexpressing C9orf72 were cultured normally and then subjected to the tumorigenesis experiment in nude mice.The results showed that the tumor growth in the C9orf72 overexpression group was slower than that in the control group,and the tumor mass was smaller than that in the control group.3.The bioinformatics analysis of TCGA database showed that the survival of the C9orf72 high expression group was improved.The odds ratio of smoking patients with low C9orf72 expression was 30.6 t imes higher than that of non-smoking patients(OR= 30.6).Immune infiltration analysis showed that C9orf72 was associated with neutrophils,monocytes,macrophages,M2 macrophages,and CD4+ T lymphocytes.Gene correlation analysis showed that C9orf72 was associated with CD80,CD86,ICOS,CTSB,SLC38A6,APAF1,CD276,PD-L1,and ALOX5.Sequencing data of C9orf72 overexpression in A549 cells under normal culture conditions overlapped with TCGA grouped data enrichment analysis for multiple pathways.Under normal culture conditions,the expressions of CD80,PD-L1,and SLC38A6 in A549 cells overexpressing C9orf72 were significantly increased,and CTSB was significantly decreased.Under serum starvation conditions,the differences in PD-L1 and SLC38A6 were more significant;immunofluorescence co-localization analysis of lung adenocarcinoma tissue showed that C9orf72 co-localized with ICOS and CD206.4.Under normal culture conditions,the overexpression of LC3,P62 and m TOR in C9orf72 was lower than that in the control group.After silencing C9orf72,LC3,P62 and m TOR were all increased compared with the control group.Under the condition of serum starvation,there was no significant difference in LC3 and P62 in the overexpression group.After silencing C9orf72,both LC3 and P62 were increased compared with the control group.When autophagy was induced by rapamycin,LC3 in the C9orf72 overexpression group was higher than that in the control group,and P62 was lower than that in the control group.Under normal culture and serum starvation conditions,the M6 PR in the C9orf72 overexpression group was lower than that in the control group,and the difference was not statistically significant in the rapamycin group.ConclusionHigh expression level of C9orf72 increases the level of basal autophagy in lung adenocarcinoma,and C9orf72 inhibits tumor in a condition-dependent manner.C9orf72 increases the expressions of PD-L1,is positively correlated with CD206 and ICOS,and may be involved in immune remodeling.The effect of C9orf72 on immune function maybe achieved by regulating the composition and function of lysosomes during autophagy.Fully understanding the effects of C9orf72 on a utophagy and pathways under different conditions may provide new diagnostic ideas for the treatment of lung adenocarcinoma.