FAM83A Regulates The Proliferation,Migration And Invasion Of Lung Adenocarcinoma Cells

BackgroundThe morbidity and mortality of primary lung cancer ranks first among all kinds of malignant tumors,especially in developing countries such as China.The burden of prevention of lung cancer is increasing because of the economic development,environmental damage and aging population increase.Non-small cell lung cancer(NSCLC)accounts for the majority of lung cancer,and is the priority for prevention of lung cancer.NSCLC is mostly in advanced stage and has no chance to receive surgery resection;the main therapy of advanced NSCLC is platinum-based combination chemotherapy.Although this combination chemotherapy including the third generation chemotherapy drugs can bring benefits to patients,the toxicity is obvious and the therapeutic effect has reached the bottleneck and make little breakthrough.Targeted therapy such as epidermal growth factor receptor tyrosine kinase inhibitors(EGFR TKIs)can significantly improve the quality of life and overall survival of patients with advanced non-small cell lung cancer,especially lung adenocarcinoma,compared with traditional chemotherapy.EGFR TKIs resistance is inevitable,appearing after 1 year or so,even if the new generation of targeted drugs.Finding new therapeutic targets and exploring new drug resistance mechanisms are very important for the prevention and treatment of lung cancer.FAM83A,a member of the FAM83 protein family,contains the highly conserved 1699 domain(DUF1699)structurally and regulates MAPK and PI3K/AKT pathways downstream of the EGFR signaling pathway functionally to control cell proliferation and transformation.At present,a number of literatures had described that FAM83 A can promote the proliferation and invasion of cancer cells and EGFR TKI resistance in breast cancer,pancreatic cancer and other malignant tumors.These studies had also found that high expression of FAM83 A was associated with poor prognosis.However,It is confused whether FAM83 A is a oncogene of lung cancer and lead to EGFR TKI resistance.The public database analysis showed that FAM83 A was overexpressed in lung cancer tissues.So we speculated that FAM83 A play an important role in the pathogenesis of NSCLC,especially lung adenocarcinoma.Therefore,it is feasible to carry out studies on FAM83 A in lung adenocarcinoma for finding new potential therapeutic targets.Objective1.To observe FAM83 A expression in lung adenocarcinoma and its significance in clinicopathologic characteristics and prognosis of the disease.2.To investigate the effect of FAM83 A on the proliferation,migration and invasion of lung adenocarcinoma cells.3.To explore the effect of FAM83 A on the epithelial mesenchymal transition(EMT)of lung adenocarcinoma cells.MethodsA total of 86 paraffin-embedded lung adenocarcinoma and adjacent lung tissue samples were used to construct tissue chips,and the expression of FAM83 A in the chip samples was detected by immunohistochemistry;FAM83A expression in five fresh lung adenocarcinoma tissues and adjacent tissues was detected by Western blotting;Chi-square test was used to analyze the relationship between the expression of FAM83 A and histopathological characteristics of lung adenocarcinoma,and Cox regression analysis and Kaplan-Meier survival analysis was used to analyze the relationship between the expression of FAM83 A and the prognosis of the disease;Cell culture,passage and cryopreservation technique was used to gain and store the cells for experiments;The expression of FAM83 A protein in the cell line was detected by Western blotting;Lentivirus infected lung adenocarcinoma cells,and RNA interference technique inhibited the FAM83 A expression;Real-time fluorescence quantitative PCR was used to detect knock down effect of FAM83A;CCK8 cell proliferation assay,wound healing assay as well as Transwell cell invasion assay was used to evaluate the proliferation,invasion and metastasis of lung adenocarcinoma cell;Western blotting was used to detect the expression of molecular markers and transcription factors associated with EMT.Results1.Bioinformatics analysis revealed that the transcription of FAM83 A in lung adenocarcinoma was up-regulated in Oncomine database.2.Western blotting results showed that the protein expression of FAM83 A in fresh lung adenocarcinoma tissues was significantly higher than that in adjacent lung tissues,P = 0.036.3.The results of immunohistochemistry in lung adenocarcinoma tissues microarray showed that FAM83 A was mainly located in the cytoplasm,and the immunohistochemical staining index of FAM83 A in lung adenocarcinoma was significantly higher than that in adjacent lung tissues,P < 0.001.Subgroup analysis showed that FAM83 A expression was higher in patients with stage III-IV than that with stage I-II,P = 0.004.4.Correlation analysis showed that FAM83 A expression was significantly correlated with clinical stage(P = 0.008)and lymph node grade(P = 0.008).Univariate Cox regression analysis showed that FAM83 A high expression increase the risk of death of lung adenocarcinoma patients compared with FAM83 A low expression,(HR: 1.765,95% CI: 1.065-2.926,P= 0.028),multivariate Cox regression analysis showed that FAM83 A expression is an independent predictor of overall survival of lung adenocarcinoma patients(HR: 1.899,95% CI: 1.085-3.323,P = 0.025).Kaplan Meier survival analysis showed the lung adenocarcinoma patients with high FAM83 A expression(n = 37)had shorter overall survival(P = 0.024)than those with low expression(n = 49).5.Western blotting results showed that A549 and H1795 cell lines had higher FAM83 A expression,while H1395 and Calu cell lines had lower FAM83 A expression.After the knockdown of FAM83 A in A549 and H1795 cell lines by RNA interference,the m RNA of FAM83 A was significantly decreased compared with the control group by real-time fluorescence quantitative PCR,P < 0.001.6.The proliferation activity of A549 cells with knockdown FAM83 A was not significantly decreased at all time points(0,12,24,48,72 h)compared with that of control group,P > 0.05.The proliferation activity of H1795 cells with knockdown FAM83 A was significantly decreased at all time points(0,12,24,48,72 h)compared with that of control group,P < 0.001.7.The healing area of wound healing assay conducted in A549 cells with knockdown FAM83 A was significantly reduced compared with that conducted in control group,P < 0.01.The healing area of wound healing assay conducted in H1795 cells with knockdown FAM83 A was significantly reduced compared with that conducted in control group,P < 0.01.8.The cell count of A549 cells with knockdown FAM83 A on the basal membrane in Transwell cell invasion assay was significantly decreased compared with that of control group,P < 0.01.The cell count of H1795 cells with knockdown FAM83 A on the basal membrane in Transwell cell invasion assay was significantly decreased compared with that of control group,P < 0.01.9.The expression of E-cadherin was enhanced,the expression of the Vimentin and the EMT-related transcription factor Snail was decreased on A549 cells with knockdown FAM83 A compared with that on control group,P < 0.001.The expression of E-cadherin was enhanced,the expression of the Vimentin and the EMT-related transcription factor Snail was decreased on H1795 cells with knockdown FAM83 A compared with that on control group,P < 0.001.Conclusions1.FAM83 A is overexpressed in lung adenocarcinoma.2.High expression of FAM83 A predicted advanced clinical stage and prognosis of lung adenocarcinoma,and FAM83 A could be used as a potential diagnostic and prognostic biomarker of disease.3.FAM83 A regulates the proliferation,invasion and metastasis of lung adenocarcinoma cells.4.FAM83 A promoted epithelial mesenchymal transformation of lung adenocarcinoma cells.FAM83 A may regulate the epithelial mesenchymal transformation of lung adenocarcinoma cells by the Snail pathway.