Identification And Exploration Of Serine Peptidase Inhibitor Kazal Type â…  (SPINK1) As A Potential Biomarker Correlated With The Progression Of Non-small Cell Lung Cancer

ObjectiveLung cancer is one of the most common malignancies worldwide and is the leading cause of cancer-related death.Approximately 85%of lung cancer patients represent as non-small cell lung cancer（NSCLC）.Advanced diagnosis and early metastasis restrict the five-year survival rate of patients with lung cancer less than 18%.Therefore,it is particularly important to identify new biomarkers related to the oncogenesis and progression of NSCLC for the early diagnosis and clinical treatment.Methods1.mRNA expression profiles of four datasets（GSE19804,GSE18842,GSE27262,GSE43458）were downloaded from the GEO（Gene Expression Omnibus）database,and the differentially expressed genes（DEGs）in NSCLC tissues were analyzed by GEO2R.Venn Diagram demonstrated the intersections of DEGs obtained from the four datasets.Functional annotation of differentially expressed genes was conducted by FUNRICH,and protein-protein interaction（PPI）analysis of differentially expressed genes was conducted by using STRING database.2.Among the 144 DEGs,mRNA expression levels of SPINK1 was significantly increased in NSCLC tissues.Furthermore,mRNA expression levels of SPINK1 were further verified in TCGA（The Cancer Genome Atlas）,GTEx（The genotype-tissue Expression）and IST（In Silico Transcriptomics）online databases.In vitro,qRT-PCR and western blot were used to detect the mRNA and protein expression levels of candidate gene SPINK1 in human normal bronchial epithelial cell line HBE,human lung adenocarcinoma epithelial cell line A549,H1299,human lung squamous cell carcinoma epithelial cell line SK-MES-1,and human lung large cell carcinoma cell line H460.Peripheral blood serum of 20 normal adults,38 patients with NSCLC and pleural effusion of 11 patients with NSCLC were collected,and the expression levels of SPINK1 in body fluids of normal control and NSCLC patients were detected by enzyme-linked immunosorbent assay（ELISA）.3.CCK8 and continuous culture cell counting assays were used to detect the effect of candidate gene SPINK1 on proliferation of non-small cell lung cancer cell lines.The influence of SPINK1 on autophagy was detected by western blot assay.The effects of candidate gene SPINK1 on cell cycle distribution and apoptosis were detected by flow cytometry and western blot.The role of candidate gene SPINK1 in cell migration was detected by scratch healing and transwell assays.Western blot assay was used to screen the relevant signal pathways by which SPINK1 exerted biological functions.Results1.A total of 23 common up-regulated genes and 121 common down-regulated genes were identified by GEO2R analysis from the four datasets:GSE19804,GSE18842,GSE27262,GSE43458,with p value<0.05 and|LogFC|>1.5 as the screening criteria.Functional annotation of the above 144 differentially expressed genes（DEGs）indicated that these DEGs were mainly expressed in the plasma membrane,cytoplasm,extracellular and nucleus of cells,acting as cell adhesion molecules,metallopeptidases and receptors to involved in a variety of biological process such as cell communication,signal transduction,growth supporting and metabolism process.In addition,these genes are abnormally expression in breast cancer,liver cancer,kidney cancer,head and neck tumors.Furthermore,these 144 DEGs were subjected to STRING database for a protein-protein interaction analysis and results suggested that IL-6,SPP1,VWF,CDH5,CAV1 and PECAM1 may play a central regulatory role in the progression of NSCLC2.SPINK1 was significantly increased in non-small cell lung cancer tissues,and that was further verified in TCGA,GTEx and IST online databases.In vitro,mRNA and protein expression levels of SPINK1 in human NSCLC cell lines were both significantly higher than that of HBE.For the collected clinical samples,the average concentration of SPINK1 in peripheral blood serum of normal adults was 644.5pg/mL,the average concentration in peripheral blood serum of NSCLC patients was1092.9pg/mL,and the average concentration of SPINK1 in pleural effusion of NSCLC patients was 1928.8pg/mL.3.Silence of SPINK1 significantly inhibited the proliferation of NSCLC cell lines,while exogenous addition of rhSPINK1（5ng/mL）could partially rescue the vitality impaired by endogenous knockdown of SPINK1.rhSPINK1（1ng/mL）was exogenously added to stimulate H460cell proliferation.Knockdown of SPINK1 resulted in cell cycle arrest,with increased proportion of G₁-phase cells and decreased proportion of S phase cells.In addition,silencing SPINK1 induces excessive autophagy and cell apoptosis.Mechanically,SPINK1 accelerated the progression of NSCLC mainly through MEK/ERK and PI3K/Akt signaling pathways.ConclusionIn this study,144 DEGs in NSCLC tissues were screened by an integrated bioinformatic analysis.Among these genes,SPINK1 was significantly up-regulated in NSCLC tissues and cell lines and accelerated the development of NSCLC mainly through MEK/ERK and PI3K/AKT signaling pathways.