ADCY9 functions as a novel cancer suppressor gene in lung adenocarcinoma

Abstract

Background: The process of lucubrating into lung adenocarcinoma (LUAD) is of profound clinical and practical significance in improving the prognosis of LUAD patients. Multiple biomarkers are reportedly involved in the proliferation or metastasis of adenocarcinoma. However, whether the ADCY9 gene influences the development of LUAD remains unknown. Therefore, we aimed to elucidate the relationship between the expression of ADCY9 and the proliferation and migration of LUAD.

Methods: The ADCY9 gene was filtered via a survival analysis of LUAD acquired from the Gene Expression Omnibus (GEO). Then, we conducted a validation analysis and ADCY9-microRNA, microRNA-lncRNA, and ADCY9-lncRNA targeting relationship analysis through the data obtained from The Cancer Genome Atlas (TCGA) dataset. The survival curve, correlation, and prognostic analysis were implemented through bioinformatics methods. Both protein and mRNA expression levels of LUAD cell lines and LUAD patient samples (80 pairs) were detected using western blot assays and quantitative real-time polymerase chain reaction (qRT-PCR). An immunohistochemistry assay was performed to display the correlation between the expression level of the ADCY9 gene and prognosis in LUAD patients (2012-2013; n=115). Overexpression of cell lines SPCA1 and A549 were used for a series of cell function assays.

Results: Compared with the expression level in adjacent normal tissues, ADCY9 expression was downregulated in LUAD tissues. Based on the result of the survival curve analysis, high expression of ADCY9 may lead to a better prognosis and may be seen as an independent predictor for LUAD patients. High expression of ADCY9-related microRNA hsa-miR-7-5p may lead to a worse prognosis, and high expression of hsa-miR-7-5p-related lncRNAs may lead to the opposite effects. Overexpression of ADCY9 restrained the proliferation, invasion, and migration abilities of SPCA1, A549 cells.

Conclusions: Results indicate that the ADCY9 gene acts as a tumor suppressor to restrain the proliferation, migration, and invasion in LUAD and can lead to a better survival or prognosis in LUAD patients.

Keywords: ADCY9 gene; lung adenocarcinoma (LUAD); tumor migration and invasion.

Figure 1

A Kaplan-Meier curve for overall survival in LUAD from GEO database (A) and TCGA database (B). Survival curve of ADCY9 in RCCC, LUSC, BCA and brain LGG (D). Mutations of ADCY9 in different human cancer types from the cBioportal web tools and pan-cancer expression levels of ADCY9 in tumors between adjacent normal tissue from the TIMER database (C,E). **, P<0.01; ***, P<0.001. LUAD, lung adenocarcinoma; GEO, Gene Expression Omnibus; TCGA, The Cancer Genome Atlas Genomes; RCCC, renal clear cell carcinoma; LUSC, lung squamous cell carcinoma; BCA, bladder cancer; LGG, lower grade glioma; CNA, copy number variation; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes.

Figure 2

Representative staining images of high ADCY9 expression slice and low ADCY9 expression slice of LUAD tissues via IHC assay (×4 and ×20) based on LUAD tissue microarray (A) (LUAD specimens n=115) and overall survival curve of low/high ADCY9 expression group was analyzed by R (P=0.033). ADCY9 expression analysis and survival curve of LUAD patients based on data of clinicopathology in terms of age, gender and smoking history (B-D). LUAD, lung adenocarcinoma; IHC, immunohistochemistry; GEO, Gene Expression Omnibus.

Figure 3

Functional enrichment analysis of ADCY9 and its related genes (A). Univariate Cox regression and multivariate Cox regression analysis predicting the overall survival in LUAD patients from GEO and TCGA database (B,C). ROC curve analysis to evaluate diagnostic accuracy of ADCY9 from TCGA database (D). LUAD, lung adenocarcinoma; GEO, Gene Expression Omnibus; TCGA, The Cancer Genome Atlas Genomes; ROC, receiver operating characteristic; AUC, area under the ROC curve.

Figure 4

Analysis of the ADCY9-related gene in LUAD from the GEO database. Expression heat map (A), correlation analysis of the ADCY9-related gene in LUAD (B) and circus plot (C). Analyzing KEGG pathways by GSEA (FDR <0.05 and P value <0.05 were considered statistically significant), multi-analysis of KEGG pathways via GSEA and the gg plot package in R (D). Volcano plot of LUAD: green, downregulated; red, upregulated (E). LUAD, lung adenocarcinoma; GEO, Gene Expression Omnibus; KEGG, Kyoto Encyclopedia of Genes and Genomes; GSEA, gene set enrichment analysis; FDR, false discovery rate.

Figure 5

PPI network (A) and mcode (B-D) of ADCY9-related genes. PPI, protein-protein interaction.

Figure 6

Analysis of the ceRNA mechanism between the ADCY9 gene and gene-related miRNAs and miRNA-related lncRNAs. PPI network of ADCY9-related miRNAs (A). Correlation analysis of ADCY9 gene and its related RNA hsa-miR-7-5p (B). The difference in the expression level of miRNA hsa-miR-7-5p in normal tissues and tumor tissues (P<0.01) (C). The survival curve was calculated using R software between hsa-miR-7-5p high/low-expressed group (P<0.05) (D). Correlation analysis showing the correlation of ADCY9 gene and its related miRNA hsa-miR-7-5p between the miRNA hsa-miR-7-5p associated lncRNAs (LINC00663, MIR29B2CHG, MIR497HG) (E). Expression of miRNA hsa-miR-7-5p-related lncRNAs LINC00663 (P<0.05), MIR29B2CHG (P<0.001), and MIR497HG (P<0.00001) in normal tissues and tumor tissues (F). The overall survival rate between high-expressed miRNA hsa-miR-7-5p related lncRNAs and the low-expressed group (P<0.001) (G). Network of ceRNA mechanism between ADCY9 gene, hsa-miR-7-5p, and associated lncRNAs (H).

Figure 7

Relationship between the ADCY9 gene and the infiltrating immune cells performed by correlation analysis (A). Correlation analysis between ADCY9 gene expression and immune-related biomarkers (B). Correlation between expression of ADCY9 gene and the infiltrating degree of neutrophils, dendritic cells, and T cells from the TIMER database (B). Variance in gene copy number of ADCY9 resulted in distinctions in immune cells infiltrating. TPM, transcripts per million.

Figure 8

ADCY9 is lower expressed in LUAD cell lines and tissues. ADCY9 expression level in terms of tissue type, age, gender and stage from TCGA and GEO database. (A,B). ADCY9 protein is higher expressed in normal tissue than LUAD (C) and validated by western blot assay (D,E). Expression level of ADCY9 in BEAS-2B, SPCA1, A549, and HCC827 cell lines shown by qRT-PCR (F). The expression of ADCY9 in 80 pairs of LUAD tissues and adjacent normal tissues. *, P<0.05; **, P<0.01; ****, P<0.0001. LUAD, lung adenocarcinoma; GEO, Gene Expression Omnibus; TCGA, The Cancer Genome Atlas Genomes; WB, western blot; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 9

ADCY9 overexpression suppresses cell proliferation, migration, and invasion as shown by flow cytometry and proliferation assays of ADCY9 and ADCY9 overexpression cell lines. Differentiated expression of ADCY9 gene between SPCA1, A549 and SPCA1, A549 overexpression cell lines shown by qRT-PCR and western blot (A,B). CCK-8 proliferation curve of SPCA1, A549 cells (C,D). Flow cytometry assays of cell cycle distribution (G,H). Flow cytometry of cellular apoptosis (I,J). Clone formation assay of SPCA1, A549 and SPCA1, A549 for the ADCY9 overexpression cell lines (E,F). Cell migration of SPCA1, A549 and SPCA1, A549 overexpression cell lines (×20) (K,L). Cell invasion of SPCA1 and SPCA1 overexpression cell lines for 48 h (×20) (M,N). **, P<0.01; ***, P<0.001; ****, P<0.0001. OD, optical density; qRT-PCR, quantitative real-time polymerase chain reaction.