Function of low ADARB1 expression in lung adenocarcinoma

Abstract

Adenosine deaminase RNA-specific B1 (ADARB1), an adenosine-to-inosine (A-to-I) RNA-editing enzyme, has been found to play an essential role in the development of cancer. However, the specific function of ADARB1 in lung cancer, especially in lung adenocarcinoma (LUAD), is still not fully understood and requires further study. In our study, integrative bioinformatics were used to analyze the detailed function of ADARB1 in LUAD. By conducting bioinformatics analyses of several public databases, such as Gene Expression Profiling Interactive Analysis (GEPIA), GE-mini, and Oncomine, we found significantly decreased ADARB1 expression in LUAD cells and tissues. Moreover, RT-PCR and Western blot showed lower ADARB1 expression in H358 and A549 LUAD cells compared to human bronchial epithelial Beas-2B cells. Wound Healing Assay indicated that knockdown ADARB1 could promote LUAD cell metastasis. By using the Kaplan-Meier Plotter tool, we found that downregulation of ADARB1 was related to shorter first progression (FP), overall survival time (OS) and post-progression survival time (PPS). The relevant clinical data acquired from the Wanderer database indicated that the expression and methylation values of ADARB1 were significantly associated with the clinical characteristics of LUAD. Using DNA methylation inhibitor, we found DNMT inhibitor 5-aza-2-deoxycytidine (5-azaD) could promote the expression of ADARB1 and reverse the inhibition effect of ADARB1 in migration. In addition, functional enrichment analysis of ADARB1-associated coexpression genes was further conducted. Our investigation demonstrated that low levels of ADARB1 were specifically found in LUAD, and this gene might be a potential target in the diagnostic and prognostic evaluation of LUAD patients.

Fig 1. The Oncomine database indicated the downregulated ADARB1 in LUAD tissues.

The expression of ADARB1 in five datasets (Hou Lung, Landi Lung, Okayama Lung, Selamat Lung and Su Lung) acquired from the Oncomine database.

Fig 2. Analysis of ADARB1 expression levels in LUAD tissues and cell lines.

(A) One dataset GSE2514, downloaded from the GEO database, described the mRNA expression of ADARB1 in LUAD in relation to normal lung tissues. (B) ADARB1 mRNA levels were significantly decreased in 44 LUAD cell lines, compared to 2 normal cell lines. (C-E) The mRNA expression of ADARB1 was evaluated from the database UALCAN, GEPIA and GE-mini, respectively. (F-G) Expression of ADARB1 was analyzed by qPCR and western blot.

Fig 3. ADARB1 could suppress LUAD cell metastasis.

(A) The CCLE database showed lower levels of ADARB1 in metastatic LUAD cells. Western blot (B) and RT-PCR (C) analyzed the expression of ADARB1 in 95C and 95D cells. The protein levels of ADARB1 after transfected with siADARB1 in Beas-2B (D) and 95C (F) cells. The mRNA levels of ADARB1 after transfected with siADARB1 in Beas-2B (E) and 95C (G) cells. The results of wound healing assay after transfected with siADARB1 in Beas-2B (H) and 95C (I) cells.

Fig 4. Analysis of ADARB1 expression on the prognosis of LUAD patients.

(A-C) The relationship between ADARB1 expression and FS, OS or PPS, described by Kaplan-Meier Plotter, respectively.

Fig 5. Methylation values of ADARB1 in LUAD patients.

(A) Global ADARB1 methylation in LUAD samples compared with that in normal samples was analyzed by the DiseaseMeth version 2.0 database. (B) The data acquired from the Wanderer database indicated the highest methylation value of cg19810954 in ADARB1. (C) Negative correlations between cg19810954 methylation and the ADARB1 expression level.

Fig 6. Modulation of DNA methylation inhibited ADARB1-reduced LUAD cell metastasis.

Beas-2B (A, B) and 95C cells (C, D) treated with siADARB1 and/or DNMT inhibitors 5-azaD subjected to Western blot and RT-PCR assays. The wound healing assays were performed after treating Beas-2B (E) and 95C (F) cells with siADARB1 and/or DNMT inhibitors 5-azaD.

Fig 7. Functional enrichment analysis of ADARB1-associated co-DEGs in LUAD samples.

(A) The coexpression genes of ADARB1 are shown as a volcano plot. (B) The PPI network of ADARB1-associated co-DEGs as completed by the STRING and Cytoscape software. (C) GO analysis of ADARB1 associated co-DEGs.