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. 2017 Feb 6:17:18.
doi: 10.1186/s12935-017-0394-x. eCollection 2017.

Human basonuclin 2 up-regulates a cascade set of interferon-stimulated genes with anti-cancerous properties in a lung cancer model

Egon Urgard  1   2 , Anu Reigo  3 , Eva Reinmaa  4 , Ana Rebane  2 , Andres Metspalu  1   3
Affiliations

Human basonuclin 2 up-regulates a cascade set of interferon-stimulated genes with anti-cancerous properties in a lung cancer model

Egon Urgard et al. Cancer Cell Int. .

Abstract

Background: Human basonuclin 2 (BNC2) acts as a tumor suppressor in multiple cancers in an as yet unidentified manner. The role and expression of the BNC2 gene in lung cancer has not yet been investigated.

Methods: BNC2 expression was studied in the A549 and BEAS-2B cell lines, as well as in lung cancer tissue. Illumina array analysis and a viability assay were used to study the effects of transient transfection of BNC2 in A549 cells. Ingenuity pathway analysis and g:Profiler were applied to identify affected pathways and networks. RT-qPCR was used to validate the array results.

Results: We showed the reduced mRNA expression of BNC2 in non-small cell lung cancer tissue and lung cancer cell line A549 compared to non-cancerous lung tissue and BEAS-2B cells, respectively. Further array analysis demonstrated that the transfection of BNC2 into A549 cells resulted in the increased expression of 139 genes and the down-regulation of 13 genes. Pathway analysis revealed that half of the up-regulated genes were from the interferon/signal transducer and activator of transcription signaling pathways. The differential expression of selected sets of genes, including interferon-stimulated and tumor suppressor genes of the XAF1 and OAS families, was confirmed by RT-qPCR. In addition, we showed that the over-expression of BNC2 inhibited the proliferation of A549 cells.

Conclusion: Our data suggest that human BNC2 is an activator of a subset of IFN-regulated genes and might thereby act as a tumor suppressor.

Keywords: BNC2; Lung cancer; OAS family; Type I IFN; XAF1.

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Figures

Fig. 1
Fig. 1
The expression of BNC2 is reduced in the lung carcinoma cell line A549 and lung cancer tissue. a Relative BNC2 mRNA expression in A549 and BEAS-2B cell lines. The mean expression of BNC2 in BEAS-2B cells was normalized to 1. Data represent the mean ± SEM from four transfections. Student’s t-test, ***p value < 0.001. b Expression of BNC2 in 8 pairs of squamous cell carcinoma (SCC) and adjacent non tumor tissues (NL). Wilcoxon matched pair test, *p value <0.05
Fig. 2
Fig. 2
The effect of BNC2 transfection in A549 cells. a Relative BNC2 mRNA expression was measured 48 h after the transfection. Human A549 cells were transfected with either BNC2 expression vector or the control (empty vector). Data represent the mean ± SEM of four transfections. Student’s t-test, **p value <0.01. b The proliferation rate of A549 cells was measured 48 h after transfection. A549 cells were transfected with either BNC2 expression vector or the control (empty vector) followed by Luminescent Cell Viability Assay. Student’s t-test, **p value <0.01. c Heatmap of the top 30 of the most differentially expressed genes from array data chosen based on fold changes. Red represents the lower and yellow the higher expression of each gene in all six samples. Data represents the quantile normalized expression values across all of the samples in heatmap. The column-side dendrogram represents the hierarchical clustering of control and BNC2-transfected samples using the complete linkage method with Euclidean distance measures. The samples were collected from two sets of transfections performed in triplicate both times. d Comparison of microarray and RT-qPCR results. Data are normalized to the control-transfected cells and are shown as a log2-transformed mRNA fold change. The RT-qPCR results represent four independent transfections with the error bar indicating SEM
Fig. 3
Fig. 3
The functional network of the interferon signaling pathway by IPA. Genes that were significantly up-regulated in BNC2-transfected A549 cells are shown in red. The intensity of red corresponds to an increase in fold change
Fig. 4
Fig. 4
The over-expression of BNC2 induces the expression of ISGs associated with the repression of cancer development. Human A549 cells were transfected with either BNC2 or the control. Data represent the mean ± SEM of four transfections
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