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. 2019 Mar 22:10:255.
doi: 10.3389/fgene.2019.00255. eCollection 2019.

Downregulation of Long Non-coding RNA FALEC Inhibits Gastric Cancer Cell Migration and Invasion Through Impairing ECM1 Expression by Exerting Its Enhancer-Like Function

Huazhang Wu  1 , Fengchang Qiao  2 , Yunli Zhao  3 , Shouwei Wu  1 , Minjie Hu  1 , Tao Wu  1 , Fuxin Huang  1 , Wenjing Chen  1 , Dengzhong Sun  4 , Mulin Liu  4 , Jinsong Zhao  5
Affiliations

Downregulation of Long Non-coding RNA FALEC Inhibits Gastric Cancer Cell Migration and Invasion Through Impairing ECM1 Expression by Exerting Its Enhancer-Like Function

Huazhang Wu et al. Front Genet. .

Abstract

Long non-coding RNAs (lncRNAs) have been shown to play important roles in many human diseases. However, their functions and mechanisms in tumorigenesis and development remain largely unknown. Here, we demonstrated that focally amplified lncRNA in epithelial cancer (FALEC) was upregulated and significantly correlated with lymph node metastasis, TNM stage in gastric cancer (GC). Further experiments revealed that FALEC knockdown significantly inhibited GC cells migration and invasion in vitro. Mechanistic investigations demonstrated that small interfering RNA-induced silencing of FALEC decreased expression of the nearby gene extracellular matrix protein 1 (ECM1) in cis. Additionally, ECM1 and FALEC expression were positively correlated, and high levels of ECM1 predicted shorter survival time in GC patients. Our results suggest that the downregulation of FALEC significantly inhibited the migration and invasion of GC cells through impairing ECM1 expression by exerting an enhancer-like function. Our work provides valuable information and a novel promising target for developing new therapeutic strategies in GC.

Keywords: ECM1; FALEC; gastric cancer; lncRNA; migration and invasion.

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Figures

FIGURE 1
FIGURE 1
The expression of FALEC was upregulated in GC cell lines and tissues. (A) Relative expression of FALEC in GC cell lines and immortalized normal gastric epithelial cell line (GES-1) was examined by qRT-PCR. (B) The fold change (log2) of FALEC expression between 60 pairs of GC and adjacent normal tissues was measured by qRT-PCR. (C) FALEC expression in GC tissues and paired adjacent normal tissues were examined by qRT-PCR (n = 60), T (Tumor) and N (Non-tumor). (D) Patients with lymph node metastasis showed significantly higher levels of FALEC compared with no lymph node metastasis. ∗P < 0.05, ∗∗P < 0.01.
FIGURE 2
FIGURE 2
Knockdown of FALEC inhibited the cell migration and invasion in GC cells. (A,B) The interference efficiency of three siRNAs and two ASOs targeting to FALEC was analyzed by qRT-PCR in HGC-27 and MGC-803 cells. (C) The knockdown efficiency was measured by qRT-PCR when cell scratch and transwell assays were performed. (D) Knockdown of FALEC attenuated cell migration in HGC-27(left) and MGC-803 (right) cell lines. Images were acquired at 0 and 36 h after scratches were generated. Depletion of FALEC significantly inhibited HGC-27 (E) and MGC-803 (F) cells migration and invasion. Representative images of transwell results were taken under 100× original magnifications. The numbers of migrated or invaded cells were counted in five fields under the microscope from three independent experiments. ∗P < 0.05, ∗∗P < 0.01.
FIGURE 3
FIGURE 3
FALEC modulated ECM1 expression by exerting its enhancer-like function in cis. (A) The relative loci of genes adjacent to FALEC. The scale bar represents 100 kb. (B) Depletion of FALEC specifically decreased expression of protein-coding gene ECM1 in HGC-27 and MGC-803 cells. (C) siRNA-induced knockdown of the ECM1 did not affect the transcription of its neighboring genes. ∗∗P < 0.01. (D) Diagram of the FALEC DNA sequence cloned down-stream of Firefly luciferase in a pGL3-Promoter vector (pGL3-Promoter-FALEC-Reverse). (E) FALEC inserts result in a significant enhancement of transcription in GC cells, and depletion of FALEC resulted in a significant decrease in transcriptional enhancement compared with control in HGC-27 and MGC-803 cells. ∗P < 0.05, ∗∗P < 0.01.
FIGURE 4
FIGURE 4
FALEC knockdown decreased migration and invasion partly by down regulating ECM1 in GC cells. (A,B) Relative expression of ECM1 was examined after ECM1 silencing alone or simultaneously silencing FALEC and ECM1 in HGC-27 and MGC-803 cells by qRT-PCR and western blotting assay. Compared with control, ECM1 knockdown significantly impaired cell migration and invasion ability of HGC-27 and MGC-803 cells (C,D). Similarly, the knockdown of FALEC and ECM1 simultaneously resulted in significant decreased the migration and invasion in GC cells than sciencing ECM1 alone or control group (C,D). (E) ECM1 overexpression markedly restored the abrogated migration and invasion of GC cells inhabited by FALEC.
FIGURE 5
FIGURE 5
Elevated ECM1 expressions is significantly positively associated with increased FALEC expression in patients with GC. (A) ECM1 expression in GC tumor tissues was significantly higher than paired adjacent non-tumor samples, and a remarkable positive correlation was found between ECM1 and FALEC mRNA expression in GC by using GEPIA database (B). (C,D) shows a box plot that ECM1 expression in different histological subtypes and stages in GC patients using UALCAN database. Kaplan-Meier survival plots shown that GC patients with high ECM1 expression have reduced lower overall (E) and progression-free survival (F) rates compared with patients with low expression level of FALEC. ∗P < 0.05, ∗∗P < 0.01.
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