| Background and objectiveNowadays,lung cancer is the leading cause of cancer-related death in both men and women.In China,the incidence of lung cancer is about 781,000 per year,and the number of deaths is about 626,000.Lung cancer can be divided into two types:non-small cell lung cancer and small cell lung cancer.Non-small cell lung cancer accounts for 85%of lung cancer,including three subtypes:adenocarcinoma,squamous cell carcinoma and large cell carcinoma.Lung adenocarcinoma can account for more than 50%of all lung cancer patients,and it is the main subtype of lung cancer now.In the past few decades,great progress has been made in targeted treatment,immunotherapy and development of new anticancer drugs for lung adenocarcinoma.However,lung adenocarcinoma is still one of the most common and fatal malignant tumors.Because of its early stage without obvious symptoms,it is often diagnosed later,and the patients with advanced lung adenocarcinoma have higher drug resistance and metastasis rate,which is the main cause of disease deterioration and treatment failure.Therefore,the 5-year survival rate of patients with lung adenocarcinoma is lower than 18%.So it is urgent to further study the new molecular mechanism of the occurrence and development of lung adenocarcinoma in order to find new factors of potential prognosis and therapeutic targets of lung adenocarcinoma.In recent years,with the improvement of genome sequencing,the popularization of biochip and the open use of large-scale data,a large amount of bioinformatics has been applied to clinical research,so that a large amount of disease-related data can be obtained.More and more researchers have begun to use bioinformatics to analyze the differentially expressed genes in lung cancer and adjacent tissues,which will be meaningful for the diagnosis and treatment of lung cancer,and find more potential targets for more effective treatment strategies.In the study,two sets of genes,GSE116959 and GSE19188 were selected from the public gene expression database.Then the differentially expressed genes were obtained using R language,and the ASPM gene was selected from the core genes as the follow-up research target.ASPM mainly plays a role in the regulation of mitotic spindle and the coordination of mitotic process.Its abnormal expression not only can promote the occurrence of liver cancer,bladder cancer,ovarian cancer,but also accelerate tumor cell self-renewal.Lung adenocarcinoma as the main subtype of lung cancer.At present,ASPM is rarely studied in lung adenocarcinoma tissues,and the role of this gene in the development of lung adenocarcinoma is unclear.This study then analyzed the relationship between the expression level of ASPM and the clinicopathological characteristics,prognosis of patients with lung adenocarcinoma based on TCGA database and immunohistochemistry.Meanwhile the effects of ASPM on the proliferation and invasion of lung adenocarcinoma and the possible mechanism of promoting the development of lung adenocarcinoma were studied in vitro experiments,so as to provide a new potential target for the treatment of lung adenocarcinoma.Methods1.The eligible data sets GSE116959 and GSE19188 were selected from the GEO database.GSE 116959 includes 57 lung adenocarcinoma tissues and 11 normal tissues.GSE 19188 includes91 non-small cell lung cancer tissues and 65 normal tissues.2.R software was used to process the original data.Differential expression genes were screened based on the criteria of |log2(FC)|? 1 and P<0.05,and integrated by the robustrankogg software package.Then FunRich software was used for function and pathway enrichment,including molecular function(MF),biological pathway(BP),and biological pathway(BPA).3.The PPI network of differentially expressed genes was obtained by string database and Cytoscape software,and the core genes were identified using the MCODE plug-in in Cytoscape.4.The expression data of ASPM mRNA in lung adenocarcinoma and related pathological parameters were downloaded through TCGA database.55 specimens of lung adenocarcinoma and 30 adjacent tissues were collected from postoperative patients at the same time,and the expression level of ASPM protein in lung adenocarcinoma was measured by immunohistochemistry.Then the relationship between the mRNA and protein expression level of ASPM and clinicopathological characteristics and its prognosis were analyzed.5.Lung adenocarcinoma cell lines A549 and h1650 were selected for subsequent in vitro experiments.The expression of ASPM was knockdown by plasmid transfection and confirmed by Western blot.CCK-8 and colony formation assay were used to detect the proliferation capacity of lung adenocarcinoma cells.Transwell and wound healing assay were used to detect the invasion capacity of lung adenocarcinoma cells.Flow cytometry was used to detect the effect of ASPM knockdown on the cell cycle of lung adenocarcinoma cells.6.GSEA was used to predict the gene pathways that ASPM may participate in the development of lung adenocarcinoma.Then the Western blot was used to verify the effect of ASPM knockdown on some proteins of signal pathways in order to explore the biology of ASPM in lung adenocarcinoma.Results1.GSE116959 and GSE19188 datasets were integrated and analyzed,and 287 up-regulated and 551 down-regulated differentially expressed genes were obtained.Follow-up analysis was performed through the string database and Cytoscape software to obtain important gene module.The six core genes KIAA0101,PBK,UBE2C,ASPM,TOP2A,and CEP55 with higher degree are related to OS in patients with lung adenocarcinoma and may be the potential prognostic indicators.Pathway analysis results showed that they are closely related to the cell cycle.2.ASPM mRNA was highly expressed in lung adenocarcinoma tissues(P<0.001)and significantly associated with lymph node metastasis,distant metastasis,survival status,and TNM stage(P=0.046,P=0.031,P<0.001,P=0.029).COX regression analysis showed that ASPM mRNA expression level(HR=1.754,P=0.002),T stage(HR=2.010,P=0.002),and N stage(HR=2.001,P<0.001)were independent prognostic factors for OS of lung adenocarcinoma.3.ASPM protein was highly expressed in lung adenocarcinoma tissues(P<0.001)and was significantly associated with lymph node metastasis(P=0.011).COX regression analysis showed that ASPM protein expression level(HR=5.227,P<0.001),lymph node metastasis(HR=7.160,P<0.001),gender(HR=2.851,P=0.004)were independent prognostic factors of OS.Meanwhile,ASPM protein expression level(HR=2.373,P=0.022)and lymph node metastasis(HR=1.959,P=0.030)were independent prognostic factors for patients with RFS.4.The results of in vitro experiments showed that the low expression of ASPM significantly inhibited lung adenocarcinoma cells proliferation and invasion,and the cell cycle was blocked in the G0/G1 phase.5.GSEA analysis showed that the high expression group of ASPM mRNA was significantly enriched in the Wnt signaling pathway.Western blot experiments confirmed that knocking down ASPM expression can decrease the levels of ?-catenin,c-myc,and survivin on the Wnt signaling pathway.ConclusionKIAA0101,PBK,UBE2C,ASPM,TOP2A,and CEP55 may play a crucial role in the development of non-small cell lung cancer.High expression levels of ASPM mRNA and protein are independent prognostic factors for OS in lung adenocarcinoma.In vitro experiments,knockdown of ASPM expression can inhibit lung adenocarcinoma cells proliferation and invasion capacity,and the cell cycle is blocked in the G0/G1 phase.ASPM may participate in the development of lung adenocarcinoma through the Wnt signaling pathway.ASPM is expected to become a new therapeutic target for lung adenocarcinoma,providing a new idea for targeted treatment of lung adenocarcinoma. |
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