Long non-coding RNA AL139002.1 promotes gastric cancer development by sponging microRNA-490-3p to regulate Hepatitis A Virus Cellular Receptor 1 expression

Abstract

Mounting evidence suggests that lncRNA regulates many important diseases. However, the biological role of most lncRNAs in gastric cancer (GC) remain unclear. In this paper, we determined differential expression of lncRNAs and predicted ceRNA networks in the GC database by bioinformatics analysis and validated in GC cells. The effect of lncRNA AL139002.1 on GC cells biological function was assessed by flow cytometry, CCK-8, colony formation, wound healing assay, transwell, western blot, and qRT-PCR. And the relationship of lncRNA AL139002.1 or HAVCR1 with miR-490-3p was verified by luciferase reporter assay. The results showed that lncRNA AL139002.1 was highly expressed in GC cells and lncRNA AL139002.1 knockdown induced apoptosis, while suppressed cell proliferation, migration, invasion, and EMT. Functional examining indicated that lncRNA AL139002.1 regulated HAVCR1 expression by competitively binding miR-490-3p. In addition, lncRNA AL139002.1/miR-490-3p/HAVCR1 regulated EMT and metastasis through MEK/ERK signaling. In conclusion, lncRNA AL139002.1 was highly expressed in GC cells, and lncRNA AL139002.1/miR-490-3p/HAVCR1 functioned critically in GC by regulating MEK/ERK signaling. Our findings demonstrated that lncRNA AL139002.1 served as a potential therapeutic and anti-metastatic biotarget for GC.

Keywords: Gastric cancer; havcr1; lncrna al139002.1; miR-490-3p.

Graphical abstract

Figure 1.

Bioinformatics prediction of lncRNA AL139002.1 and targeting miRNA/mRNA and validation. (a-f) Volcano plot and Heatmap of DElncRNAs, DEmiRNAs, and DEmRNAs in the TCGA database. (g) TCGA and miRcode database consistently predicted five mRNAs interacted with lncRNA. (h) LncRNA-miRNA-mRNA regulatory network. (i, j) Kaplan-Meier survival analysis showed the relationship between lncRNA AL139002.1 or HAVCR1 expression with the prognosis of GC patients

Figure 2.

LNCRNA AL139002.1 and HAVCR1 expression increased, miR-490-3p expression decreased in GC cell lines. qRT-RCR was applied to detect the relative expression of lncRNA AL139002.1 (a) and miR-490-3p (b) in GC cell lines (MKN-45, AGS, BGC823, SGC7901, MGC803, MKN74) and (GES-1). (c) Western blot of HAVCR1 proteins in GC cell lines (MKN-45, AGS, BGC823, SGC7901, MGC803, MKN74) and (GES-1)

Figure 3.

Silencing of lncRNA AL139002.1 regulated proliferation, apoptosis, migration, invasion and EMT of GC cells. LncRNA AL139002.1 was silenced in AGS and MGC-803 cells. (a) qRT-RCR was applied to detect the relative expression of lncRNA AL139002.1 in cells. (b, c) Proliferation and apoptosis assays of cells through CCK-8 assay and flow cytometry. (d) The colony formation of cells results. (e) Migration assay of cells by wound healing analysis (40×). (f) Invasion assay of cells by transwell assay. (g) Western blot of E-cadherin, vimentin and N-cadherin proteins in cells

Figure 4.

LncRNA AL139002.1 regulated HAVCR1 by sponging miR‑490‑3p. Relative luciferase activities of lncRNA AL139002.1-wt and lncRNA AL139002.1-mut (a) or HAVCR1-wt and HAVCR1-mut (b) reporter plasmid in AGS and MGC-803 cells co-treated with miR-490-3p mimic. (c) LncRNA AL139002.1 was silenced in AGS and MGC-803 cells. qRT-RCR was conducted to detect the relative expressions of miR-490-3p and HAVCR1. (d) Western blot of HAVCR1 protein in AGS and MGC-803 cells treated with LncRNA AL139002.1 and miR‑490‑3p mimic

Figure 5.

LncRNA AL139002.1 regulated the proliferation, apoptosis, migration, invasion and EMT of GC cells by miR‑490‑3p/HAVCR1. Silencing of lncRNA AL139002.1, miR‑490‑3p inhibitor and silencing of HAVCR1 were transfected into AGS and MGC-803 cells. (a, b) Proliferation and apoptosis assays of cells by CCK-8 assay and flow cytometry. (c) The results of the colony formation of cells. (d) Migration assay of cells by wound healing analysis (40×). (e) Invasion assay of cells by transwell assay. (f) Western blot of E-cadherin, vimentin and N-cadherin proteins in AGS and MGC-803 cells

Figure 6.

LncRNA AL139002.1/miR‑490‑3p/HAVCR1 regulated GC through MEK/ERK signaling. Silencing of lncRNA AL139002.1, miR‑490‑3p inhibitor and silencing of HAVCR1 were transfected into AGS and MGC-803 cells. (a, b)Western blot of MEK/ERK-related proteins in AGS and MGC-803 cells