Tuberc Respir Dis.  2017 Jul;80(3):247-254. 10.4046/trd.2017.80.3.247.

ACN9 Regulates the Inflammatory Responses in Human Bronchial Epithelial Cells

• Jeong JH
• Kim J
• Kim J
• Heo HR
• Jeong JS
• Ryu YJ
• Hong Y
• Han SS
• Hong SH
• Lee SJ
• Kim WJ

• KMID: 2385039
• DOI: http://doi.org/10.4046/trd.2017.80.3.247

Abstract

BACKGROUND
Airway epithelial cells are the first line of defense, against pathogens and environmental pollutants, in the lungs. Cellular stress by cadmium (Cd), resulting in airway inflammation, is assumed to be directly involved in tissue injury, linked to the development of lung cancer, and chronic obstructive pulmonary disease (COPD). We had earlier shown that ACN9 (chromosome 7q21), is a potential candidate gene for COPD, and identified significant interaction with smoking, based on genetic studies. However, the role of ACN9 in the inflammatory response, in the airway cells, has not yet been reported.
METHODS
We first checked the anatomical distribution of ACN9 in lung tissues, using mRNA in situ hybridization, and immunohistochemistry. Gene expression profiling in bronchial epithelial cells (BEAS-2B), was performed, after silencing ACN9. We further tested the roles of ACN9, in the intracellular mechanism, leading to Cd-induced production, of proinflammatory cytokines in BEAS-2B.
RESULTS
ACN9 was localized in lymphoid, and epithelial cells, of human lung tissues. ACN9 silencing, led to differential expression of 216 genes. Pathways of sensory perception to chemical stimuli, and cell surface receptor-linked signal transduction, were significantly enriched. ACN9 silencing, further increased the expression of proinflammatory cytokines, in BEAS-2B after Cd exposure.
CONCLUSION
Our findings suggest, that ACN9 may have a role, in the inflammatory response in the airway.

Keyword

Succinate Dehydrogenase; Cadmium; Pulmonary Disease, Chronic Obstructive; Gene Expression; Inflammation

MeSH Terms

Cadmium
Cytokines
Environmental Pollutants
Epithelial Cells*
Gene Expression
Gene Expression Profiling
Humans*
Immunohistochemistry
In Situ Hybridization
Inflammation
Lung
Lung Neoplasms
Pulmonary Disease, Chronic Obstructive
RNA, Messenger
Signal Transduction
Smoke
Smoking
Succinate Dehydrogenase
Cadmium
Cytokines
Environmental Pollutants
RNA, Messenger
Smoke
Succinate Dehydrogenase

Figure

• 

  Figure 1 ACN9 localization in human lung tissues. (A) In situ hybridization showing ACN9 mRNA localized in human lung tissues. Paraffin sections were rehydrated and incubated with an anti-ACN9 or scramble probe as negative control. Results are normalized to anti-PPIB probe (internal control) used. (B) Protein-level expression of ACN9 in the liver and lung tissues was analyzed using immunohistochemistry. Data shown is representative of at least two independent experiments.

• 

  Figure 2 Heatmaps for representative gene sets dysregulated by cadmium (Cd)-treated ACN9-silenced cells. Effect of Cd and siACN9 on the expression of ACN9 mRNA level. (A) BEAS-2B cells transfected with siACN9 (15 nM) compared to the control scrambled siRNA. (B) BEAS-2B cells (2×105 cells per well for quantitative polymerase chain reaction) were treated with the indicated concentrations of Cd (10 µM) for 24 or 48 hours. For quantitative real time polymerase chain reaction (qRT-PCR), statistical evaluation was performed using Student's t test. Data shown is representative of at least three independent experiments. (C) Comparison of gene expression patterns obtained from cDNA microarray and qRT-PCR with ACN9 knockdown in Cd-treated bronchial epithelial cells. Heat map and bars indicate the magnitude of gene expression changes. (D) Numbers of differentially expressed genes in the ACN9-silenced BEAS-2B cells with/without Cd. (E) Gene ontology analysis showing Cd and ACN9 silencing responsive relationships in terms of biological processes. *p≤0.05, **p≤0.01, and ***p≤0.001 were considered significant. Mock: transfection reagent alone; siNC: small interference RNA of non-targeting control; siACN91-3: small interference RNA for ACN9 at 10 nM, 15 nM, and 25 nM.

• 

  Figure 3 ACN9-silenced cells drive inflammatory responses. BEAS-2B cells were pretreated for 24 hours with small interference RNA for ACN9 (siACN9; 15 nM) after being stimulated with cadmium (Cd) (10 µM). (A) mRNA was extracted from total cell lysates and analyzed by quantitative polymerase chain reaction for proinflammatory cytokines. (B) Supernatants were analyzed by enzyme-linked immunosorbent assay for interleukin 6 (IL-6). (C) Whole-cell lysates were analyzed by Western blotting for proinflammatory cytokine (IL-1β) and p38/mitogen-activated protein kinase (MAPK) signal pathway and normalized by β-actin. (D) The protein expression levels of phosphorylated JNK, ERK, and p38 MAPK were calculated by normalized to total ERK, and IL-1β and IL-1RA were normalized by β-actin. For enzyme-linked immunosorbent assay and quantitative real time polymerase chain reaction, statistical evaluation was conducted, using Student's t test *p≤0.05, **p≤0.01, and ***p≤0.001 were considered significant. Data shown is representative of at least three independent experiments. TNF-α: tumor necrosis factor α; COX2: cyclooxygenase 2; iNOS: inducible nitric oxide synthase; MMP9: matrix metalloproteinase 9; siNC: small interference RNA of non-targeting control.

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