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Screening for Long Noncoding RNAs Associated With Oral Squamous Cell Carcinoma Reveals the Potentially Oncogenic Actions of DLEU1

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Screening for Long Noncoding RNAs Associated With Oral Squamous Cell Carcinoma Reveals the Potentially Oncogenic Actions of DLEU1

Koyo Nishiyama et al. Cell Death Dis.

Abstract

Recent studies have shown that long noncoding RNAs (lncRNAs) have pivotal roles in human malignancies, although their significance in oral squamous cell carcinoma (OSCC) is not fully understood. In the present study, we identified lncRNAs functionally associated with OSCC. By analyzing RNA-seq datasets obtained from primary head and neck squamous cell carcinoma (HNSCC), we identified 15 lncRNAs aberrantly expressed in cancer tissues. We then validated their expression in 18 OSCC cell lines using qRT-PCR and identified 6 lncRNAs frequently overexpressed in OSCC. Among those, we found that knocking down DLEU1 (deleted in lymphocytic leukemia 1) strongly suppressed OSCC cell proliferation. DLEU1 knockdown also suppressed migration, invasion, and xenograft formation by OSCC cells, which is suggestive of its oncogenic functionality. Microarray analysis revealed that DLEU1 knockdown significantly affects expression of a number of cancer-related genes in OSCC cells, including HAS3, CD44, and TP63, suggesting that DLEU1 regulates HA-CD44 signaling. Expression of DLEU1 was elevated in 71% of primary OSCC tissues, and high DLEU1 expression was associated with shorter overall survival of HNSCC patients. These data suggest that elevated DLEU1 expression contributes to OSCC development, and that DLEU1 may be a useful therapeutic target in OSCC.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Screening for aberrantly expressed lncRNAs in OSCC.
a Workflow of the screening to identify lncRNAs associated with OSCC. b Results of qRT-PCR for the 6 selected lncRNAs in 18 OSCC cell lines and a specimen of normal tongue. Results are normalized to the expression levels in normal tongue. Shown are means of three replications; error bars represent SDs
Fig. 2
Fig. 2. Effects of lncRNA knockdown on OSCC cell proliferation.
a Summary of cell viability assays in the indicated OSCC cell lines with lncRNA knockdown. Cells were transfected with a siRNA targeting each lncRNA or a negative control, and cell viabilities were assessed 96 h after transfection. Shown are means of nine replications; error bars represent SDs. b qRT-PCR showing DLEU1 knockdown in OSCC cell lines. Cells were transfected with control siRNA (siCtrl) or siRNAs targeting DLEU1 and were harvested 72 h after transfection. c Cell viability assays in OSCC cell lines with DLEU1 knockdown. Cells were transfected with the indicated siRNAs, and cell viabilities were assessed at the indicated times. Shown are means of nine replications; error bars represent SDs. **P < 0.01, ***P < 0.001
Fig. 3
Fig. 3. Functional analysis of DLEU1 in OSCC cells.
a, b Results of migration (a) and invasion (b) assays with HSC-3 cells transfected with siRNAs targeting DLEU1 or control siRNA (siCtrl). Representative results are on the left, and summarized results are on the right. Shown are means of five random microscopic fields per membrane; error bars represent SDs. c Results of wound healing assays using HSC-3 cells transfected with the indicated siRNAs. Representative results are on the left, and summarized results are on the right. Shown are means of three replications; error bars represent SDs. d, e Results of cell cycle (d) and apoptosis (e) analyses in HSC-3 cells. Cells were transfected with the indicated siRNAs, after which cell cycle and apoptosis were assessed at the indicated time points. Summarized results of three replications are shown; error bars represent SDs. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 4
Fig. 4. Effects of DLEU1 knockdown on xenograft tumor formation by OSCC cells in mice.
a Tumor growth in nude mice injected with HSC-3 cells transfected with siRNA targeting DLEU1 or control siRNA (siCtrl). Intratumoral injection of siRNAs was also performed at the indicated times. Shown are means of four replications; error bars represent SDs. b Resected tumors (upper) and tumor weights (lower) in the indicated groups. Shown are means of four replications; error bars represent SDs. c Representative views of hematoxylin and eosin (left) and Ki-67 (right) staining in tumors treated with the indicated siRNAs. Scale bars, 200 µm. d Summary of Ki-67-positive cell counts. Shown are means of four replications; error bars represent SDs. **P < 0.01, ***P < 0.001
Fig. 5
Fig. 5. Effects of DLEU1 knockdown on gene expression profiles in OSCC cells.
a OSCC cell lines were transfected with siRNA targeting DLEU1 (siDLEU1-2) or control siRNA (siCtrl), after which microarray analysis was performed. A Venn diagram showing genes whose expression was altered by DLEU1 knockdown (>2-fold) in the indicated OSCC cell lines is on the left. A heatmap for the expression of 963 selected probe sets is on the right. b Results of GO (left) and pathway analyses (right) of the 963 probes. c qRT-PCR for cancer-related genes in HSC-3 cells transfected with the indicated siRNAs. Shown are means of three replications; error bars represent SDs
Fig. 6
Fig. 6. Elevated expression of DLEU1 in primary tumors.
a Summaries of DLEU1 expression in normal tissue and primary HNSCC tumors in TCGA datasets. b Kaplan–Meier curve showing the effect of DLEU1 expression on survival of HNSCC patients (n = 505). c Summarized qRT-PCR results for DLEU1 in primary tumors (n = 29) and adjacent normal tissues (n = 17) obtained from Japanese OSCC patients. d qRT-PCR for DLEU1 in paired normal tissue and primary tumors. ***P < 0.001

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