Upregulated long non-coding RNA SBF2-AS1 promotes proliferation in esophageal squamous cell carcinoma

Abstract

Esophageal cancer is one of the most common types of malignant tumors located within the digestive system, with >50% of esophageal cancer cases worldwide occurring in China. Recent studies have demonstrated that long non-coding RNAs (lncRNAs) are frequently dysregulated in cancer; however, few lncRNAs have been characterized in esophageal squamous cell carcinoma (ESCC). In the present study, a novel lncRNA, SET-binding factor 2 (SBF2) antisense RNA1 (SBF2-AS1) was exhibited in ESCC. Expression levels of SBF2-AS1 in ESCC and adjacent non-cancerous tissues were detected using the reverse transcription-quantitative polymerase chain reaction. SBF2-AS1 was knocked down, and proliferation, migration, invasion, apoptosis and the cell cycle were examined in ESCC cells. Results identified that SBF2-AS1 was significantly upregulated in ESCC compared with adjacent non-cancerous tissues (fold increase, 8.82; P=0.023). The SBF2-AS1 expression level was significantly increased in patients who had a smoking (9.927 vs. 4.507; P=0.030) and/or drinking (10.938 vs. 4.232; P=0.032) history. Patients with a large tumor size exhibited increased SBF2-AS1 expression (≥4 vs. <4 cm, 14.898 vs. 5.435; P=0.018). Patients with advanced ESCC exhibited increased upregulation of SBF2-AS1 [tumor-node-metastasis (TNM) I-II vs. TNM III-IV, 1.302 vs. 15.475; P<0.01]. SBF2-AS1 was also silenced using small interfering RNA. Cell proliferative and invasive ability were significantly inhibited (P<0.05) following SBF2-AS1 silencing, the cell cycle was arrested in the G₂ phase; however, there was no significant difference in the proportion of apoptotic cells. Gene Set Enrichment Analysis revealed that genes associated with cell cycle biological processes, including the cancer suppressor gene cyclin-dependent kinase 1A (CDKN1A), were significantly associated with SBF2-AS1 in ESCC tissues. Further validation confirmed that CDKN1A expression levels were increased in ECA-109 cells following SBF2-AS1 silencing. The results of the present study demonstrate that SBF2-AS1 is significantly upregulated in ESCC, and that silencing of SBF2-AS1 inhibits the proliferative and invasive ability of ESCC cells. SBF2-AS1 may be a novel biomarker and therefore a potential therapeutic target for ESCC.

Keywords: SET-binding factor 2 antisense RNA1; esophageal squamous cell carcinoma; long non-coding RNA; proliferation.

Figure 1.

Analysis of SBF2-AS1 expression in ESCC tissues and associated clinical parameters. (A) SBF2-AS1 was detected in 51 pairs of ESCC tissues using RT-qPCR. Levels of SBF2-AS1 in ESCC tissues were significantly increased compared with those in non-tumor tissues (P=0.023). SBF2-AS1 was upregulated in groups with (B) drinking history (P=0.030), (C) smoking history (P=0.032), (D) tumor size (P=0.018), and (E) TNM stage (P<0.001). (F) Kaplan-Meier estimator curve demonstrates the survival rate of patients with different SBF2-AS1 expression levels. The difference between groups was statistically significant (P=0.016; Mantel-Cox test). *P<0.05, **P<0.01 vs. control groups. Results are presented as the mean ± standard error of the mean. TNM, tumor-node-metastasis; SBF2-AS1, SET-binding factor 2 antisense RNA1; ESCC, esophageal squamous cell carcinoma; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 2.

Expression of SBF2-AS1 in ESCC cell lines demonstrates that SBF2-AS1 promotes proliferation of ESCC cell lines in vitro. (A) Upregulation of SBF2-AS1 in ESCC cells. (B) Localization of SBF2-AS1 in ESCC cells. The majority of SBF2-AS1 was located within the cytoplasm through the nuclear mass separation experiment. *P<0.05, **P<0.01. Results are presented as the mean ± standard error of the mean. SBF2-AS1, SET-binding factor 2 antisense RNA1; ESCC, esophageal squamous cell carcinoma.

Figure 3.

SBF2-AS1 promotes proliferation of ESCC cell lines in vitro. (A) si-SBF2-AS1 demonstrates increased inhibition efficiency. (B) CCK-8 kit demonstrated that silencing of SBF2-AS1 inhibited cell proliferation of ECA-109 cells. (C) Clone formation complements data generated from the CCK-8 kit. *P<0.05, **P<0.01 vs. NC group. Results are presented as the mean ± standard error of the mean. SBF2-AS1, SET-binding factor 2 antisense RNA1; ESCC, esophageal squamous cell carcinoma; CCK-8, Cell Counting Kit-8; si, small interfering; NC, negative control.

Figure 4.

SBF2-AS1 alters the cell cycle of ESCC cell lines in vitro. (A) ECA-109 cells transfected with si-SBF2-AS1 were blocked in the G₂ phase. (B) Flow cytometric analyses indicated that si-SBF2-AS1 did not affect apoptosis. *P<0.05. Results are presented as the mean ± standard error of the mean. si, small interfering; SBF2-AS1, SET-binding factor 2 antisense RNA1; ESCC, esophageal squamous cell carcinoma; NC, negative control.

Figure 5.

SBF2-AS1 promotes invasion and metastasis of ESCC cell lines in vitro. (A) Following silencing of SBF2-AS1, ESCC cells demonstrated a decrease in migratory ability. (B) Knocking down SBF2-AS1 inhibited the invasion of ECA-109 cells. **P<0.01. Results are presented as the mean ± standard error of the mean. SBF2-AS1, SET-binding factor 2 antisense RNA1; ESCC, esophageal squamous cell carcinoma; NC, negative control; siRNA, small interfering RNA.

Figure 6.

GSEA results indicate that genes associated with (A) cell cycle process, (B) mitotic cell cycle and (C) cell cycle phase are positively enriched with SBF2-AS1, and among these genes, CDKN1A was significantly negatively associated with SBF2-AS1 (indicated by the red arrow). Genes regulated by PRC2 were negatively associated with SBF2-AS1. (D) Silencing of SBF2-AS1 increased the CDKN1A expression level in ECA-109 cells. (E) Cyclin E expression was decreased following SBF2-AS1 silencing, which is consistent with the (F) upregulation of CDKN1A. (G) SBF2-AS1 was negatively associated with CDKN1A in the GSE53622 dataset (P<0.01 vs. NC groups). *P<0.05. GSEA, Gene Set Enrichment Analysis; SBF2-AS1, SET-binding factor 2 antisense RNA1; CDKN1A, cyclin-dependent kinase 1A; PRC2, Polycomb repressive complex 2; NC, negative control; siRNA, small interfering RNA.