PLXNC1 Enhances Carcinogenesis Through Transcriptional Activation of IL6ST in Gastric Cancer

Abstract

Background: Transcriptional factors (TFs) are responsible for orchestrating gene transcription during cancer progression. However, their roles in gastric cancer (GC) remain unclear. Methods: We analyzed the differential expressions of TFs and, using GC cells and tissues, investigated plexin C1 (PLXNC1) RNA levels, as well as PLXNC1's clinical relevance and functional mechanisms. The molecular function of PLXNC1 was evaluated in vitro and in vivo. Kaplan-Meier curves and the log-rank test were used to analyze overall survival (OS) and disease-free survival (DFS). Results: PLXNC1 was frequently up-regulated in GC and associated with poor prognosis. The expression level of PLXNC1 could serve as an independent biomarker to predict a patient's overall survival. Notably, knockdown of PLXNC1 significantly abolished GC cell proliferation, and migration, and overexpression of PLXNC1 accelerated carcinogenesis in GC. The gene set enrichment analysis (GSEA) indicated that high-expression of PLXNC1 was positively correlated with the activation of epithelial-mesenchymal transition (EMT), TNF-α, and IL-6/STAT3 signaling pathways. PLXNC1 promoted proliferation and migration of GC cells through transcriptional activation of the interleukin 6 signal transducer (IL6ST), which could rescue the malignant behavior of PLXNC1-deficient GC cells. Conclusions: Our study demonstrated that the PLXNC1 plays an oncogenic role in GC patients. The PLXNC1-IL6ST axis represents a novel potential therapeutic target for GC.

Keywords: IL6ST; PLXNC1; carcinogenesis; gastric cancer; transcriptional factor.

Figure 1

Differentially expressed transcription factors predict a patient's overall survival in TCGA-STAD cohort. (A) Three-dimensional scatter plot generated from the differential expression profiles of TCGA-STAD 27 paired gastric tissue. (B) The circled diagram of differentially expressed TF genes in TCGA-STAD cohort. In the figure comprised of five tracks, the first track refers to the average expression level (log2 transfer) of TFs; the second track indicates the fold change of differential expression analysis of TFs in the STAD paired tissue dataset; the third track shows the P-value (–log10 transfer) of log-rank test for each TF; the fourth track represents the hazard ratio value of univariate-cox model (HR value and it's 95% CI (lower and upper) were both highlighted as red, blue, and green ligatures, respectively); the last track displays the correlation coefficients between tumor stage and TF expression level. The darker color indicates a higher quantitative value to distinguish TFs. The sector with light red shows the high-risk TFs, which indicate poorer outcomes for GC patients. (C) Correlation of clinicopathological features with tumor PLXNC1 expression level in TCGA-STAD cohort.

Figure 2

PLXNC1 predicts prognosis in gastric cancer. (A) The differential expression level of PLXNC1 expressed in our 111 paired STAD tissues. (B,C) Kaplan–Meier curves of overall survival and disease-free survival in our internal 111 gastric patients, validated by PLXNC1 mRNA expression levels. (D) The results of multi-variate analyses using the Cox proportional hazard regression model for PLXNC1 mRNA levels and other clinical indices in our internal cohort. (E) The comparison of diagnostic efficacy of CEA and PLXNC1 mRNA levels for predicting the time period of tumor OS. *P < 0.05; **P < 0.01.

Figure 3

PLXNC1 plays oncogenic roles in gastric cancer both in vitro and in vivo. (A) Colony formation assays (up) and Transwell migration assays (down) for HGC-27 and AGS cells transfected with PLXNC1 siRNAs or negative control (NC) siRNA. (B) Colony formation assays (up) and Transwell migration assays (down) for HGC-27 and AGS cells infected with the PLXNC1 knockdown-mixed sgRNAs or control sgRNA lentivirus. (C) Colony formation assays (up) and Transwell migration assays (down) for HGC-27 and AGS cells infected with PLXNC1 overexpression lentivirus or GFP control. (D) Xenograft tumors of Cas9 or PLXNC1 knockdown AGS cells in nude mice. (E,F) The knockdown of PLXNC1 reduces the (E) weight and (F) volume of xenograft tumors (n = 6 mice per group). Values represent the mean ± SEM, (A–C) n = 3 and (D–F) n = 6. *P < 0.05; **P < 0.01.

Figure 4

PLXNC1 activates cancer-associated signatures in gastric cancer. (A) The heatmap of hierarchical clustering shows the enrichment score of cancer hallmark gene sets enriched in the PLXNC1 high/low expressed group based on single sample gene set enrichment analysis (GSEA) for TCGA-STAD cohort. (B) KEGG pathway analysis performed by the DAVID platform for PLXNC1 high-expressed group. The KEGG pathway with P < 0.05 is shown in a bubble plot. (C) GSEA of hallmark gene sets in high-level-group of PLXNC1. All transcripts were ranked by log2 (fold change) between two groups. Each run was performed with 500 permutations. Enrichment results with significant associations with PLXNC1 were shown. (D) The relative candidate cancer hallmark pathway gene mRNA expression infected with PLXNC1 or GFP overexpression lentivirus in AGS gastric cancer cells using qRT-PCR analysis. (D) Values represent the mean ± SEM, n = 3. *P < 0.05; **P < 0.01.

Figure 5

PLXNC1 controls IL6ST expression at the DNA level. (A) The relative mRNA levels of the IL-6/STAT3 pathway genes in AGS cells transfected with PLXNC1-mixed siRNAs and negative control siRNA. (B) IL6ST mRNA levers and protein levels in AGS cells transfected with PLXNC1 siRNAs and negative control. (C) ChIP-qPCR revealed the enrichment of PLXNC1 in IL6ST promoter in AGS cells. (D) The IL6ST promoter activity transfected with PLXNC1-mixed siRNAs and negative control siRNA in AGS cells. (E) Expressional correlation of PLXNC1 and IL6ST in GC tissues. (F) Colony and migration assays of AGS cells transfected with PLXNC1-mixed siRNAs, PLXNC1-mixed siRNAs plus IL6ST overexpression plasmids, or negative control. (A–D,F) Values represent the mean ± SEM, n = 3. *P < 0.05; **P < 0.01.