Cell Signaling Pathway in 12-O-Tetradecanoylphorbol-13-acetate-Induced LCN2 Gene Transcription in Esophageal Squamous Cell Carcinoma

LCN2 is involved in various cellular functions, including transport of small hydrophobic molecules, protection of MMP9 from proteolytic degradation, and regulating innate immunity. LCN2 is elevated in multiple human cancers, frequently being associated with tumor size, stage, and invasiveness. Our previous studies have shown that LCN2 expression could be induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in esophageal squamous cell carcinoma (ESCC) by the binding of five nucleoproteins (MISP, KLF10, KLF15, PPP1R18, and RXRβ) at a novel TPA-responsive element (TRE), at −152~−60 bp of the 5′ flanking region of the LCN2 promoter. However, much is unknown about whether these proteins can respond to TPA stimulation to regulate LCN2 transactivation and which cell signaling pathways mediate this process. In this study, expression plasmids encoding these five nucleoproteins were stably transfected into EC109 cells. Then, stable transfectant was characterized by a Dual-Luciferase Reporter Assay System. RT-PCR, real-time PCR, western blotting, specific kinase inhibitor treatment, and bioinformatics analyses were applied in this study. We found that MISP, KLF10, KLF15, PPP1R18, and RXRβ proteins could strongly respond to TPA stimulation and activate LCN2 transcriptional expression. MEK, ERK, JNK, and P38 kinases were involved in the LCN2 transactivation. Furthermore, the MEK-ERK signal pathway plays a major role in this biological process but does not involve PKCα signaling

The k = 12 clique from the downregulated miRNA-miRNA network and its co-regulated subpathways.

<p>Green nodes represent downregulated miRNAs, while upregulated miRNA is colored red. The size of the miRNA nodes corresponds to the node degree. <i>P</i>-value strength is represented by edge line width, with darker edges representing more significant interactions.</p

Graphic representation of three miRNA-subpathway networks.

<p>(<b>A</b>) Downregulated miRNA-subpathway network. (<b>B</b>) Upregulated miRNA-subpathway network. (<b>C</b>) Total miRNA-subpathway network. Nodes colored in green are downregulated miRNA, and red nodes are upregulated miRNAs. Blue nodes represent the subpathways. The size of the miRNA nodes correspond to the node degree (the number of subpathways that miRNA connected). <i>P</i>-value strength is represented by edge line width, with wider edges representing more significant interactions. Hsa-miR-320b and hsa-miR-1248 had the biggest degree are shaded in yellow.</p

Power law of node degree distribution for the miRNA-subpathway networks.

<p>(<b>A</b>) Degree distribution of the downregulated miRNA-subpathway network. (<b>B</b>) Degree distribution of the upregulated miRNA-subpathway network. (<b>C</b>) Degree distribution of the total miRNA-subpathway network.</p

Schematic presentation of the analysis workflow to construct the miRNA-subpathway, miRNA-miRNA and subpathway-subpathway sub-networks.

<p>miRNA target genes were subjected to subpathway enrichment analysis to generate a miRNA-subpathway network. Then two sub-networks were constructed by the hypergeometric test.</p

ESCC patient clusters and survival analysis.

<p>(<b>A</b>) The cluster of miR-31 and miR-338-3p in 89 ESCC patients. The prefix 0 represents deceased ESCC patients, while the prefix 1 represents living ESCC patients. (<b>B</b>) Survival of grouped ESCC patients is analyzed by Kaplan-Meier analysis and the log-rank test.</p

Network parameters of miRNA-subpathway and subpathway-subpathway networks.

<p>Network parameters of miRNA-subpathway and subpathway-subpathway networks.</p

The k = 6 clique from total miRNA-miRNA and its co-regulated subpathways.

<p>Red nodes represent upregulated miRNAs, while blue nodes are downregulated miRNAs. The size of the miRNA nodes corresponds to the node degree. <i>P</i>-value strength is represented by edge line width, with wider edges representing more significant interactions.</p

Summary of differentially-expressed ESCC miRNAs applied in this study.

<p>Summary of differentially-expressed ESCC miRNAs applied in this study.</p