p53-Induced LINC00893 Regulates RBFOX2 Stability to Suppress Gastric Cancer Progression

Abstract

Long noncoding RNAs (lncRNAs) have been reported to regulate diverse tumorigenic processes. However, little is known about long intergenic non-protein coding RNA 00893 (LINC00893) and its role in gastric cancer (GC). Herein we investigated its biological functions and molecular mechanism in GC. LINC00893 was decreased in GC tissues but significantly elevated in AGS cells after treatment with Nutlin-3. In GC patients, it was found that low expression of LINC00893 was correlated with tumor growth, metastasis and poor survival. Functionally, overexpression of LINC00893 suppressed the proliferation, migration and invasion of GC cells. Mechanistically, LINC00893 regulated the expression of epithelial-mesenchymal transition (EMT)-related proteins by binding to RNA binding fox-1 homolog 2 (RBFOX2) and promoting its ubiquitin-mediated degradation, thus suppressing the EMT and related functions of GC. In addition, the transcription factor p53 can regulate the expression of LINC00893 in an indirect way. Taken together, these results suggested that LINC00893 regulated by p53 repressed GC proliferation, migration and invasion by functioning as a binding site for RBFOX2 to regulate its stability and the expression of EMT-related proteins. LINC00893 acts as a tumor-inhibiting lncRNA that is induced by p53 in GC and regulates EMT by binding to RBFOX2, thus providing a novel experimental basis for the clinical treatment of GC.

Keywords: LINC00893; RBFOX2; epithelial-mesenchymal transition; gastric cancer; p53.

FIGURE 1

Identification of p53-regulated tumor suppressor lncRNAs in gastric cancer (A) Heat map is shown for the differentially expressed lncRNAs identified by RNA sequencing between three gastric cancer tissues and paired normal tissues (B,C) Isogenic TP53-WT AGS cells were untreated or treated with Nutlin-3 for 12 h, RT-qPCR and immunoblotting was performed for p53 and the loading control GAPDH (D) Heat map is shown for the differentially expressed lncRNAs identified by RNA sequencing in duplicate from AGS cells untreated or treated with nutlin-3 for 12 h. Upregulated genes are shown in red and downregulated genes in green (E,F) Venn diagram showing the overlap between the transcriptomes downregulated in gastric cancer tissues and the transcriptomes upregulated after nutlin-3 treatment of AGS cells.

FIGURE 2

LINC00893 is significantly low-expressed in gastric cancer and regulated by p53 (A) Analysis of LINC00893 expression in unpaired GC (T = 408) and normal tissues (N = 211) in the GEPIA (p < 0.05) (B) Relative LINC00893 expression in 50 paired fresh frozen normal gastric tissues and gastric cancer tissues (C) Maximum CSF scores of LINC00893 as well as other coding and noncoding RNAs determined by analysis with PhyloCSF (D) Kaplan-Meier survival curve indicated the lower expression of LINC00893 associated with poorer survival rates (E,F) p53 and p21 expression in AGS treated with gradient nutlin-3 or 5-FU (G,H) p53 and p21 expression in AGS after transfection of TP53 siRNA or overexpression plasmid (I,J) LINC00893 expression in AGS treated with gradient nutlin-3 or 5-FU (K,L) LINC00893 expression in AGS after transfection of TP53 siRNA or overexpression plasmid. *p < 0.05, ****p < 0.0001, all RT-qPCR results were normalized to GAPDH.

FIGURE 3

LINC00893 inhibits GC cell proliferation, migration, invasion and EMT in vitro (A) LINC00893 was overexpressed or knocked down in AGS and MKN28 cells (B,C) The effects of LINC00893 overexpression or knockdown on proliferation and plate colony-forming ability were measured in GC cells (D,E) The effects of LINC00893 overexpression or knockdown on migration and invasion were detected in GC cells (F) RNA-seq and Gene Set Enrichment Analysis (GSEA) of AGS treated with vector or LINC00893 overexpressed plasmid (G) Relative expression of E-cadherin, N-cadherin and Vimentin in GC cells after LINC00893 were either overexpressed or knocked down. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4

LINC00893 inhibits the growth and metastasis of gastric cancer cells in vivo (A) LINC00893 stable-overexpressed MKN45 cells was constructed (B) Representative images of subcutaneous xenograft tumors from the two group (C) Representative images of H&E and IHC with anti-Ki67 of the subcutaneous xenograft tumors (D) Quantification of tumor volumes in NC or LINC00893 overexpressed MKN45 cells in xenograft mouse models (E) Tumor weights were analyzed (F) Representative images of lung metastasis of LINC00893 overexpression groups and control group (G) Representative hematoxylin and eosin (H&E) staining results of corresponding lung metastatic nodules (H) Statistical analysis of numbers of metastatic nodules in the lung. ***p < 0.001.

FIGURE 5

Identification of RBFOX2 as a binding partner for LINC00893 (A) RT-qPCR analysis from nuclear and cytoplasmic fractions of GC cells; the cytoplasmic GAPDH mRNA and the nuclear lncRNA MALAT1 were used as controls (B) Subcellular localization of LINC00893 detected by FISH. Scale bar: 20 μm (C) The secondary structure of LINC00893 was predicted (http://rna.tbi.univie.ac.at). The red color indicates strong confidence for the prediction of each base (D) The potential binding areas between LINC00893 and RBFOX2 were predicted using the catRAPID database (E) SDS-PAGE of proteins purified from CHIRP assay using biotinylated LINC00893 or antisense RNA (F) Western blotting analyses following CHIRP assays in AGS cells confirmed the interaction between LINC00893 and RBFOX2 (G) RIP assay followed by RT-qPCR suggested LINC00893 binds to RBFOX2 (H) Representative images of immunofluorescence staining for RBFOX2 expression in MKN28 cells after transfection with LINC00893 overexpression or empty vectors. Scale bar: 20 μm (I) Western blotting to analyze total level of RBFOX2 after LINC00893 were overexpressed or knocked down in GC cells (J) A CHX treatment was administered to assess RBFOX2 degradation in AGS and MKN28 cells (K) MG-132 abolished the downregulation of RBFOX2 protein expression induced by LINC00893 overexpression in AGS and MKN28 cells (L) Ubiquitination assays revealed that LINC00893 overexpression increased the level of the ubiquitinated RBFOX2 protein in AGS and MKN28 cells.

FIGURE 6

RBFOX2 promotes malignant behavior and EMT in GC cells in vitro (A,B) The effects of RBFOX2 knockdown or overexpression on proliferation and plate colony-forming ability were measured in GC cells (C,D) The effects of RBFOX2 knockdown or overexpression on migration and invasion were detected (E) Relative expression of E-cadherin, N-cadherin and Vimentin was detected by western blotting in gastric cancer cells after RBFOX2 were either knocked down or overexpressed (F) Survival rates analysis of between RBFOX2 and GC were analyzed by KM Plotter database (https://kmplot.com). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 7

LINC00893 suppresses the proliferation and metastasis of GC cells through RBFOX2 (A,B) The effects of LINC00893 overexpression or knockdown on proliferation and plate colony-forming ability were rescued by RBFOX2 in GC cells (C,D) The effects of LINC00893 overexpression or knockdown on migration and invasion ability were rescued by RBFOX2 in GC cells (E,F) The effects of LINC00893 overexpression or knockdown on E-cadherin, N-cadherin and Vimentin were rescued by RBFOX2 in GC cells.