| Background: Postoperative recurrence is the main cause of treatment failure in early stage lung adenocarcinoma.To prevent recurrence,postoperative adjuvant therapy is often recommended,but the efficacy is not ideal,because 30-50% of patients ultimately died of recurrent disease within 5 years after operation.Due to the lack of breakthrough in therapeutic approaches,biomarkers that can predict recurrence risk is urgently needed.Giving more aggressive treatment to patients with high risk of recurrence may effectively reduce the recurrence rate.However,TNM staging,which is currently used in clinical practice,has significant limitations in predicting the prognosis of early stage lung adenocarcinoma and cannot accurately distinguish the risk of recurrence in patients at the same pathological stage.Developing biomarkers that can accurately predict the prognosis and act as a supplement or even replacement of TNM stage system to guide the postoperative treatment strategy is vital to reduce the recurrence rate and improve the prognosis of early stage lung adenocarcinoma patients.Methods: Two discovery cohorts were constructed by the lung cancer database of Daping Hospital and the TCGA database.Patients enrolled in discovery cohorts were divided into recurrence and non-recurrence groups according to the recurrence status after surgery.Differential expressed genes(DEGs)and miRNAs were obtained by transcriptome array and RNA sequence.Then we preformed gene set enrichment analysis.Overlapping genes in the two differential expressed genes and miRNAs list were selected as candidate factors for the further validation and mechanism study.The validation sets were constructed by the lung cancer database of Daping Hospital and TCGA database.Firstly,the expression level of candidate genes and miRNAs were obtained by immunohistochemistry(IHC)and PCR assay.Then the correlation between the expression level and recurrence rate was analyzed by the chi-square test,Cox regression model analysis and Kaplan-Meier survival analysis were preformed to study the correlation between candidate factors and prognosis of early stage lung adenocarcinoma.Secondly,we conducted a series of cell models in which the candidate factors were over-expressed or knock-down.Then the proliferation,metastasis and platinum resistance of these cell models were explored by CCK8,tranwell,apoptosis assay in vitro and vivo.Finally,by using immunoprecipitation in combination with mass spectrometry(IP-MS),co-immunoprecipitation(Co-IP),mRNA sequencing and luciferase activity assay,we further explore the molecular mechanism of these candidate factors to regulate recurrence in early stage lung adenocarcinoma.Results: 1.we obtained 734 and 229 differential expressed genes that matched the criteria(ANOVA P<0.05 and |fold change|>1.5)form Daping and TCGA discovery cohorts respectively.The results of GSEA showed that the MITOTIC-SPINDLE and GLYCOLYSIS gene sets were significantly enriched and upregulated in the TCGA and Daping Hospital recurrence groups respectively.Overlapping genes(SFTPB,SFTPD,HLA-DQB1,ITGB8,ANLN,and LRRN1)and miRNAs(miRNA-485-5p,miRNA-376c-3p,miRNA-493-3p,miRNA-134-5p,miRNA-495-3p)in the two DEG lists were selected as candidate factors for further study.2.Cox regression model analysis and Kaplan-Meier survival analysis showed that HLA-DQB1,SFTPB and miRNA-134-5p were associated with early stage lung adenocarcinoma recurrence rate and RFS,OS both in Daping Hospital and TCGA validation sets.3.The results of IHC assay indicated that tumors with higher HLA-DQB1 levels showed greater infiltration of CD4-and CD8-positive T lymphocytes in Daping validation set.Similar,patients with high HLA-DQB1 levels also had a high lymphocyte infiltration rate and greater levels of CD4,CD8,GZMK and GZMH(P<0.0001)in TCGA validation set.4.The results of CCK8 and transwell assay indicated that SFTPB was negatively correlated with the proliferation and migration ability of lung adenocarcinoma cells,these results were confirmed by in vivo animal experiments.GSEA analysis showed that genes involved in AKT down regulated were significantly enriched and upregulated in patients with high SFTPB expression.Then we investigated the relationship between SFTPB and AKT in TCGA database and our tumor specimens,cell models.The results suggest that AKT phosphorylation is significantly negatively correlated with SFTPB in early stage lung adenocarcinoma.IP-MS and Co-IP experiments confirmed that phosphoglycerate kinase 1(PGK1)binds to SFTPB in lung adenocarcinoma cancer cells.TCGA data show that PGK1 is positively correlated with AKT phosphorylation and negatively correlated with SFTPB,the cell experiments further confirm that over expressed SFTPB significantly reduced PGK1 protein levels.5.CCK8 and transwell assay indicated that miR-134-5p was positively correlated with the migration ability and contributes to the development of chemoresistance of lung adenocarcinoma cells.By mRNA sequencing analysis and consulting the miRNA target prediction databases microRNA.org and TargetScan.org,we found that disabled-2(DAB2)is a downstream target gene of miR-134-5p.Further study also confirmed that DAB2 was negatively correlated with the expression of miR-134-5p in clinical specimens and cell lines.Luciferase activity assay showed that DAB2 is a target of miR-134-5p and miR-134-5p inhibits DAB2 expression through directly binding to the 3’-UTR of DAB2 in LUAD cells.the TCGA dataset analysis results show that stage I LUAD patients in the high DAB2 group had longer median recurrence-free survival times and lower recurrence rate than those in the low DAB2 group.In addition,in vitro experiments showed that DAB2 was negatively correlated with the migration ability and chemoresistance of lung adenocarcinoma cells.Overexpression or silencing DAB2 in lung adenocarcinoma cells affects E-cadherin expression.Finally,the results of co-transfection assay showed that DAB2 could reverse the role of miR-134-5p in promoting metastasis and drug resistance of lung adenocarcinoma cells.Conclusion: 1.Seven genes(SFTPB,SFTPD,SFTA1 P,HLA-DQB1,ITGB8,ANLN,LRRN1)and five miRNAs(miRNA-485-5p,miRNA-376c-3p,miRNA-493-3p,miRNA-134-5p,miRNA-495-3p)were differential expressed between the recurrence and non-recurrence groups both in Daping and TCGA study cohorts;2.The MITOTIC-SPINDLE and GLYCOLYSIS gene sets were significantly enriched and upregulated in the TCGA and Daping Hospital recurrence groups respectively.3.Early stage lung adenocarcinoma patients with high expression of HLA-DQB1 in tumor cells(not in stroma cells)experienced lower postoperative recurrence risk and longer RFS and OS,the mechanism is associated with the anti-tumor immune activity;4.SFTPB can negatively regulate the proliferation and migration of lung adenocarcinoma cells,patients with high expression SFTPB had lower recurrence rate and longer RFS and OS.5.The mechanism of SFTPB regulating postoperative recurrence in early stage lung adenocarcinoma may attribute to the combination effect of SFTPB and PGK1.SFTPB may combined with PGK1 in lung adenocarcinoma cells and inhibit AKT/mTOR signaling pathway by promoting PGK1 ubiquity degradation,then reduce tumor cell proliferation and migration,and ultimately inhibit postoperative recurrence and prolong RFS and OS;6.MiR-134-5p can promote metastasis and platinum resistance of lung adenocarcinoma cells,which resulting in higher postoperative recurrence and shorter RFS in patients with high expression of miR-134-5p in stage I lung adenocarcinoma patients.7.DAB2 is a downstream target gene of miR-134-5p in stage I lung adenocarcinoma,DAB2 may mediate the pro-metastasis and platinum resistance effect of miR-134-5p by regulating EMT. |
