Long noncoding RNA FALEC inhibits proliferation and metastasis of tongue squamous cell carcinoma by epigenetically silencing ECM1 through EZH2

Abstract

Tongue squamous cell carcinoma (TSCC), the most common epithelial cancer identified in the oral cavity, has become one of the most common malignancies across the developing countries. Increasing evidence indicates that long non-coding RNAs (lncRNAs) serve as important regulators in cancer biology. The focally amplified long non-coding RNA in epithelial cancer (FALEC) was found downregulated in the tissues of tongue squamous cell carcinoma (TSCC) and was predicted to present a good prognosis by bioinformatics analysis. Experiments indicated that FALEC knockdown significantly increased the proliferation and migration of TSCC cells both in vitro and in vivo; however, FALEC overexpression repressed these malignant behaviors. RNA pull-down and RNA immunoprecipitation demonstrated that FALEC could recruit enhancer of zeste homolog 2 (EZH2) at the promoter regions of extracellular matrix protein 1 (ECM1), epigenetically repressing ECM1 expression. The data revealed that FALEC acted as a tumor suppressor in TSCC and may aid in developing a novel potential therapeutic strategy against TSCC.

Keywords: ECM1; EZH2; lncRNA FALEC; tongue squamous cell carcinoma.

Figure 1

FALEC is markedly downregulated in TSCC and predicts good prognosis. (A) Hierarchical clustering analysis of lncRNAs that were differentially expressed (fold change > 2; p < 0.05) in tongue squamous cell carcinoma (TSCC) and normal tissues in TCGA cohort. (B) Kaplan-Meier curves for OS of TSCC patients with high vs. low expression of FALEC in TCGA cohort. (C) The Sequencing result of FALEC expression in TSCC and normal tissues in TCGA cohort. (D) The Sequencing result of FALEC in TSCC tissues of different T classification and normal tissues. (E) The Sequencing result of FALEC in TSCC tissues of different Stage and normal tissues. (F) FALEC expression was analyzed in GSE51700. (G) FALEC expression was detected in TSCC tissues and paired normal adjacent tissues. (H) Relative FALEC expression in cell lines and normal tissues. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

FALEC silence promotes the proliferation and migration of TSCC cells. (A) RT-qPCR analysis of FALEC was determined in FALEC-silenced and control SCC-4 and SCC-25 cell lines. (B) CCK-8 assays were used to determine the viability of FALEC-silenced TSCC cell lines. (C) EdU assays were performed to determine the proliferation of TSCC cell lines when FALEC was knockdown. (D) Colony formation assays were used to determine the colony-forming ability of FALEC-silenced cells. Representative images (left) and average number of colonies (right) are shown. (E) Transwell assays showed that FALEC silencing promoted TSCC cells migration. Representative images (left) and average number of cells (right) are shown. Data are shown as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

FALEC overexpression inhibits the proliferation and migration of TSCC cells. (A) RT-qPCR analysis of FALEC was determined in FALEC-overexpression and control TSCC cell lines. (B) CCK-8 assays were used to determine the viability of FALEC-overexpressed TSCC cell lines. (C) EdU assays was performed to the proliferation of cell lines when FALEC was overexpressed. (D) Colony formation assays were used to determine the colony-forming ability of FALEC-overexpressed cells. Representative images (left) and average number of colonies (right) are shown. (E) Transwell assays showed that FALEC overexpression suppressed TSCC cell migration. Representative images (left) and average number of cells (right) are shown. Data are shown as means ± SD. *p < 0.05, **p < 0.01.

Figure 4

Overexpression of FALEC significantly reduces tumor growth in vivo. (A) Tumors removed from the Nude mice 7 weeks after injection of SCC-9 and CAL-27 cells stably transfected with pcDNA-FALEC or pcDNA, respectively. (B) Average weight of tumors derived from each group. (C) The tumor growth of FALEC-overexpression and control cells grafted mice were measured every 7 days, tumor growth curve was calculated. Representative images of tumors of each group (n = 6). (D)The expression of FALEC in grafted tumor tissues was analyzed by RT-qPCR. (E) Representative images of IHC staining of the grafted tumor. Data are shown as means ± SD. *p< 0.05, **p < 0.01***p < 0.001.

Figure 5

ECM1 is highly expressed in TSCC and negatively controlled by FALEC. (A) Human FALEC locus was shown using UCSC Genome Browser. There is a divergent mRNA in near from FALEC locus (B) RT-qPCR analysis of FALEC in the subcellular fractions of SCC-25 and SCC-4 cells. U6 and GAPDH acted as nuclear and cytoplasmic markers, respectively (n=3). (C) Correlation of FALEC expression and ECM1 expression in primary TSCC samples and normal samples. (D) RT-qPCR analysis of ECM1 in FALEC overexpressing or knockdown TSCC cell lines. (E) Western blotting of ECM1 in FALEC overexpressing or knockdown TSCC cell lines. (F) Representative immunostaining of ECM1 in FALEC overexpressing and control xenografted tumors. (G) RT-qPCR analysis of ECM1 in FALEC overexpressing and control xenografted tumors. (H) Representative immunostaining of ECM1 in TSCC tissues and adjacent normal tissues. (I) RT-qPCR analysis of ECM1 in TSCC tissues and adjacent normal tissues. Data are shown as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6

FALEC suppress TSCC progression by downregulating ECM1. SCC-9 and CAL-27 cells transfected with pcDNA/pcDNA-FALEC/pcDNA-ECM1 and cells transfected with pcDNA-FALEC followed by transfection with pcDNA-ECM1. After transfection, the cells were analyzed by CCK-8 assays (A), EdU assays (B), Colony formation assays (C) and transwell assays (D). Data are shown as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7

FALEC Binds with EZH2 to Epigenetically Silence ECM1, Inhibiting Cell Proliferation and Migration in TSCC Cell Lines. (A) Western Blotting of proteins from antisense FALEC and sense FALEC pull-down assays (n=3). (B) RNA immunoprecipitation (RIP) experiments were performed using the EZH2 antibody, and specific primers were used to detect FALEC (n=3). (C) ECM1 level was detected by RT-qPCR in the TSCC cell lines after overexpression or knockdown of EZH2. (D) ECM1 protein level was detected by western blotting in the TSCC cell lines after overexpression or knockdown of EZH2. (E, F) chromatin immunoprecipitation (ChIP) assays of EZH2 and H3K27me3 of the promoter region of the ECM1 locus after FALEC overexpression or knockdown. qPCR was performed to quantify the ChIP assays products. Enrichment was quantified relative to the input controls. IgG antibodies were used as a negative control. Data are shown as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.