HOXA11-As Promotes Lymph Node Metastasis Through Regulation of IFNL and HMGB Family Genes in Pancreatic Cancer

Abstract

Recent studies have shown that long noncoding RNAs (lncRNAs) play pivotal roles in the development and progression of cancer. In the present study, we aimed to identify lncRNAs associated with lymph node metastasis in pancreatic ductal adenocarcinoma (PDAC). We analyzed data from The Cancer Genome Atlas (TCGA) database to screen for genes overexpressed in primary PDAC tumors with lymph node metastasis. Our screen revealed 740 genes potentially associated with lymph node metastasis, among which were multiple lncRNA genes located in the HOXA locus, including HOXA11-AS. Elevated expression of HOXA11-AS was associated with more advanced tumor stages and shorter overall survival in PDAC patients. HOXA11-AS knockdown suppressed proliferation and migration of PDAC cells. RNA-sequencing analysis revealed that HOXA11-AS knockdown upregulated interferon lambda (IFNL) family genes and downregulated high-mobility group box (HMGB) family genes in PDAC cells. Moreover, HMGB3 knockdown suppressed proliferation and migration by PDAC cells. These results suggest that HOXA11-AS contributes to PDAC progression, at least in part, through regulation of IFNL and HMGB family genes and that HOXA11 AS is a potential therapeutic target in PDAC.

Keywords: HMGB3; IFN-λ; PDAC; lncRNA; lymph node metastasis.

Figure 1

Identification of HOXA11-AS upregulation in PDAC with lymph node metastasis. (A) Heatmap showing expression of 740 genes upregulated in PDAC with lymph node metastasis in TCGA dataset. (B) GO analysis of genes upregulated in PDAC with lymph node metastasis. (C) Circular plot showing the chromosome positions of upregulated genes in PDAC with lymph node metastasis. (D) Heatmap showing expression of HOXA cluster genes upregulated in PDAC with lymph node metastasis. (E) Heatmap showing expression of HOXA cluster genes in PDAC cell lines in the CCLE dataset. (F) qRT-PCR analysis of HOXA11-AS in normal pancreatic tissue and PDAC cell lines (n = 3). Error bars represent SDs.

Figure 2

Association between HOXA11-AS expression and clinical and molecular features in primary PDAC. (A) Levels of HOXA11-AS expression in primary PDAC tumors with the indicated N factors (left) or T factors (right) in TCGA dataset. (B) Kaplan–Meier curves showing the effect of HOXA11-AS expression on survival of PDAC patients (n = 176). (C) HOXA11-AS expression in normal pancreatic tissue (n = 5) and primary PDAC tumors (n = 42) in the GSE55643 dataset. (D) Summarized results of GSEA of the indicated gene sets using genes upregulated in PDAC with high HOXA11-AS expression in TCGA dataset. NES, normalized enrichment score; FDR, false discovery rate. (E) Results of GSEA of the indicated gene sets in PDAC with high HOXA11-AS expression. (F) Summarized results of GO analysis using genes upregulated in PDAC with high HOXA11-AS expression in TCGA dataset. ** p < 0.01, *** p < 0.001.

Figure 3

Functional analysis of HOXA11-AS in PDAC cells. (A) qRT-PCR analysis of HOXA11-AS in PDAC cells transfected with a control siRNA or siRNAs targeting HOXA11-AS (n = 3). (B) Cell viability assays with PDAC cells transfected with the indicated siRNAs (n = 6). (C,D) Cell migration (C) and invasion (D) assays with PDAC cells transfected with the indicated siRNAs. Representative results are shown on the left, summarized results on the right (n = 3). Error bars represent SDs. * p < 0.05, ** p < 0.01, *** p < 0.001, NS, not significant.

Figure 4

HOXA11-AS regulates IFNL and HMGB family genes in PDAC cells. (A) Results of RNA-seq in KP1-NL cells transfected with the indicated siRNAs. Shown is a heatmap of genes whose expression was altered (>1.5-fold) by HOXA11-AS knockdown. Representative genes are indicated on the right. (B) Summarized results of GSEA of the indicated gene sets using genes upregulated by HOXA11 AS knockdown. (C) GO analysis of genes upregulated by HOXA11-AS knockdown. (D–F) qRT-PCR analysis of IFNL1 (D), IFNL2 (E) and HMGB3 (F) in the indicated PDAC cell lines transfected with the indicated siRNAs (n = 3). Error bars represent SDs. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Functional analysis of HMGB3 in PDAC cells. (A) qRT-PCR analysis of HMGB3 in KP1-NL cells transfected with a control siRNA or siRNA targeting HMGB3 (n = 3). (B) Cell viability assays with KP1-NL cells transfected with the indicated siRNAs (n = 6). (C) Cell migration assays with KP1-NL cells transfected with the indicated siRNAs. Representative results are shown on the left, summarized results on the right (n = 3). (D) Summarized results of GSEA of indicated gene sets using genes upregulated in PDAC with high HMGB3 expression in TCGA dataset. (E) Results of GSEA of the indicated gene sets in PDAC with high HMGB3 expression. Error bars represent SDs. ** p < 0.01, *** p < 0.001.