APEX1/Ref-1 Promotes Malignant Progression Of Non-Small Cell Lung Cancer Through Wnt/β-Catenin Signaling Pathway

Background and purpose :lung cancer was the most common cause of cancer-related mortality globally,Non-small-cell lung cancer(NSCLC)accountted for comprising 85% of all cases.Apurinic endonuclease 1/redox factor-1(APEX1/ Ref-1)is a dual-function molecule in DNA repair and redox regulation of transcription factors.Its importance in the occurrence and progression of cancer has also aroused a lot of research and attention.However,the regulatory mechanism of APEX1 in NSCLC is not fully understood.Small molecule RNA was one of the crucial post-transcriptional regulatory mechanisms of APEX1.So far,the human genome has encoded more than two thousand micrornas,which regulate about one third of the genes in the genome.However,the clinical significance of APEX1 in NSCLC,its upstream and downstream regulatory mechanisms,and its role in tumor progression remain unclear.Objective:To explore the mechanism of hsa-miR-338-3p/APEX1 in NSCLC occurrence and development and its biological influence on human NSCLC cells,and to provide a novel theoretical premise and experimental basis for early diagnosis and lung cancer therapy.Methods:The expression profile of APEX1 and its diagnostic and prognostic value were analyzed by bioinformatics.The upstream regulatory small RNA was screened with high throughput screening,and the relationship between APEX1 and the expression of downstream genes in Wnt/β-catenin signaling was analyzed.Immunohistochemistry was performed to detect the protein level in paraffin specimens of lung cancer and the protein expression in the tumor body of subcutaneous transplantation in nude mice.Western blotting was applied to detect APEX1 expression and other molecules connected with cell proliferation,invasion and apoptosis(KI-67,PCNA,MMP-1,Caspase-3)in clinical surgical specimens and nude mice.And the role of APEX1 on Wnt pathway in various cell models;Fluorescence quantitative PCR was used to investigate the regulation of hsa-miR-338-3p and APEX1 on oncogenes in Wnt/β-catenin signaling.The intracellular localization and alteration of APEX1 and Wnt/β-catenin pathway molecules were investigated by immunofluorescence method.Transwell experiment was conducted to determine APEX1 influence on invasion or migration of NSCLC cells.CCK-8 and Edu experiments were used to study APEX1 influence on NSCLC cell proliferation.APEX1 influence on apoptosis of NSCLC was studied by flow cytometry.8×GTIIC luciferase reporter gene system and RT-q PCR were performed to confirm the direct binding relationship of hsa-miR-338-3p and APEX1.A nude mouse model of xenograft tumor derived from cell lines was constructed to verify APEX1 biological function in NSCLC.Results:Bioinformatics analysis revealed that APEX1 m RNA and protein were up-regulated in NSCLC.Subsequently,we collected 8 pairs of postoperative samples from NSCLC and adjacent tissues,and RT-q PCR and Western blot experiments confirmed the high expression of APEX1 molecule in NSCLC.Further,immunohistochemical method was applied to test the expression of APEX1,PCNA,MMP-1 and Caspase-3 in NSCLC tissues and adjacent tissues.The clinical significance of APEX1 in NSCLC and its relationship with clinicopathologic features of lung cancer were further analyzed The correlation of A total of 83 pairs of paraffin tissue samples of NSCLC were included,and it was found that APEX1over-expression was related to smoking history(p=0.037),T(p=0.017),N(p=0.033),and M stage(p < 0.001).Overexpression of APEX1 promotes cell proliferation,invasion and metastasis in NSCLC and inhibits their apoptosis,while knockdown of APEX1 has the opposite biological effect.We used Target Scan,miRWalk and star Base databases to download the presumed upstream miRNAs bound with APEX1,and finally determined the target miRNA as hsa-miR-338-3p.miRNA and m RNA data of NSCLC and clinical data of cases were obtained from TCGA,and the data were merged and standardized by using R language.GSEA analysis suggested that APEX1 regulated Wnt/β-catenin signaling related target genes cyclin D1,c-Myc,etc.Western blotting,RT-q PCR and immunofluorescence tests showed that hsa-miR-338-3p and APEX1 regulated the Wnt pathway,and the former inhibited the entry of β-catenin into the nucleus.Dual luciferase reporter gene assay and RT-q PCR suggested the binding of hsa-miR-338-3p and APEX1,and APEX1 could be post-transcriptionally regulated by hsa-miR-338-3p,forming a negative feedback relationship with hsa-miR-338-3p,and jointly involved in the progression of lung cancer.Overexpression of APEX1 can promote the growth of subcutaneous transplanted tumors.And immunohistochemical analysis of transplanted tumors showed that after overexpression of APEX1,PCNA and Ki-67 expression level increased,while Caspase-3 expression level decreased,which once again confirmed the function and mechanism of APEX1 at the molecular level.Conclusion:Our study proves that APEX1 and hsa-miR-338-3p directly combine,and APEX1 could be post-transcriptionally regulated by hsa-miR-338-3p,forming a negative feedback relationship with hsa-miR-338-3p,and jointly participate in lung cancer progression.Meanwhile,the hsa-miR-338-3p-APEX1 axis can regulate the Wnt signaling and promote the malignant progression of lung cancer.Our results are expected to provide a novel reference for early diagnosis and treatment of NSCLC.