Long Non-Coding RNA LINC01089 Enhances the Development of Gastric Cancer by Sponging miR-145-5p to Mediate SOX9 Expression

Abstract

Background: Long non-coding RNAs (lncRNAs) have potential regulatory effects in oncogenesis. Previous studies showed that several lncRNAs could participate in the progression of gastric cancer (GC). However, the specific biological mechanisms in GC are still unclear. We analyzed an lncRNA microarray of GC and selected LINC01089 for study.

Methods: LINC01089 expression in GC was tested by qRT-PCR. GC cell proliferation was assessed using CCK-8 and EdU assays. Cell invasion was assessed using the Transwell assay. A dual-luciferase reporter gene assay and bioinformatics assay were performed to detect potential targets of LINC01089. Additionally, RNA immunoprecipitation and Western blot assays were performed to clarify their interactions and roles in the regulation of GC progression.

Results: High LINC01089 expression was observed in GC cells. LINC01089 overexpression notably expedited cell migration, proliferation, and invasion. LINC01089 positively regulated SOX9 expression by competitively binding to microRNA (miR-145-5p).

Conclusion: LINC01089 competitively binds to miR-145-5p to mediate SOX9 expression. LINC01089 may participate in the progression of GC.

Keywords: ceRNA; epigenetics; lncRNA; migration; proliferation.

Figure 1

Characteristics and expression of LINC01089 in GC cells and tissues. (A) Heatmap shows the differential expression lncRNAs in GC tissues adjacent normal tissue samples from GSE96964 Microarray. (B) LINC01089 expression in GC tissues and adjacent normal tissue samples (n= 50). (C) LINC01089 expression in GC cell lines (MKN45, MGC-803, BGC-823 and AGS) and normal human gastric mucosal cell line GES-1 as determined by qRT-PCR. (D) Kaplan–Meier analysis for overall survival of GC patients stratified by LINC01089 expression. *P value < 0.05, **P value < 0.01 and ***P value < 0.001.

Figure 2

Regulation effects of LINC01089 on proliferative, migratory and invasive abilities of GC cells. (A) qRT-PCR for LINC01089 expression in cells after transfected with si-LINC01089 or LINC01089 overexpression vector. (B–D) Proliferation of BGC-823 post transfection with LINC01089 siRNA and AGS cells post transfection with LINC01089 overexpression vector from CCK-8 assay and EdU assay. (E and F) Migration of BGC-823 post transfection with LINC01089 siRNA and AGS cells post transfection with LINC01089 overexpression vector. (G and H) Invasion of BGC-823 post transfection with LINC01089 siRNA and AGS cells post transfection with LINC01089 overexpression vector as shown by Transwell assay. All the data were from three individual experiments and shown as mean ± SD. *P<0.05, **P value < 0.01, ***P value < 0.001, OE: overexpression, si: siRNA.

Figure 3

Direct interaction of LINC01089 with miR-145-5p. (A) Cytoplasmic and nuclear level of LINC01089 in BGC-823 and AGS cells were determined by qRT-PCR. (B) MiR-145-5p expression in GC tissues and adjacent normal tissues. (C) Correlation analysis of LINC01089 and miR-145-5p expression in GC samples. (D) The binding sequences of miR-145-5p and LINC01089. (E) Dual-luciferase reporter gene assay in HEK293T cells post transfection with miR-NC or miR-145-5p mimics. (F) Amount of LINC01089 and miR-145-5p in BGC-823 and AGS cells as detected by RIP experiments. **P value < 0.01, ***P value < 0.001, WT: wild type, MUT: mutant type.

Figure 4

SOX9 is directly targeted by miR-145-5p. (A) Supposed miRNA binding sites in SOX9 sequences. (B) Dual-luciferase reporter gene assay. (C) SOX9 expression in GC tissues and adjacent normal tissues. (D) Western Blot for protein level of SOX9 in GC tissues and adjacent normal tissues. (E) Correlation analysis of SOX9 and miR-145-5p expression in GC samples. (F) Correlation analysis of LINC01089 and SOX9 expression in GC samples. All the data are from three individual experiments and shown as mean ± SD. **P value < 0.01, ***P value < 0.001, WT: wild type, MUT: mutant type.

Figure 5

LINC01089/miR-145-5p axis is key for SOX9 expression. (A) Transfection efficiency of miR-145-5p inhibitor and miR-145-5p mimics. (B) Transfection of miR-145-5p inhibitor with or without LINC01089 siRNA into BGC-823 cells and qRT-PCR evaluation for RNA level of SOX9. (C) Transfection of AGS cells with miR-145-5p mimics with or without LINC01089 overexpression plasmid and relative RNA levels of SOX9 as detected by qRT-PCR. (D) Western blot of SOX9 protein level after treatment, GAPDH as the control. (E) Relative protein level of SOX9 for transfection with miR-145-5p mimics and reversion by LINC01089 expression plasmid. (F) Relative RNA level of SOX9 for transfection with LINC01089 MUT overexpression plasmid or LINC01089 WT overexpression plasmid. (G) Relative protein level of SOX9 for transfection with LINC01089 WT overexpression plasmid or LINC01089 MUT overexpression plasmid. All the data were from three individual experiments and shown as mean ± SD. *P value < 0.05, **P value < 0.01, ***P value < 0.001, ns: no significant difference, WT: wild type, MUT: mutant type, OE: overexpression, si: siRNA, inh: inhibitor.

Figure 6

LINC01089 regulates cell functions by miR-145-5p. (A, B) Proliferation of BGC-823 and AGS cells as determined by CCK-8 assay. (C, D) Proliferation of BGC-823 and AGS cells as determined by EdU assay. (E, F) Invasion of BGC-823 and AGS cells post different transfections. All the data were from three individual experiments and reported as mean ± SD. *P value < 0.05, **P value < 0.01, OE: overexpression, si: siRNA, inh: inhibitor.

Figure 7

LINC01089 regulates GC in vivo. (A) Xenograft tumors. (B) Tumor volumes in both LINC01089 knockdown and control groups measured at an interval of seven days. (C) Tumor weights measured after tumor dissection. *P value < 0.05, sh: shRNA.