Upregulation of circRNA_100395 sponges miR-142-3p to inhibit gastric cancer progression by targeting the PI3K/AKT axis

Abstract

Gastric cancer (GC) has a high morbidity and mortality, hence it is very important to elucidate the molecular pathogenesis mechanism of GC progression in order to find new treatment strategies. The present study aimed to explore the biological function of circular RNA_100395 (circRNA_100395) in GC. The expression level of circRNA_100395 in GC tissues, as well as normal epithelial cells and various gastric cancer cell lines, was detected using reverse transcription-quantitative PCR. Cell Counting Kit-8, EdU assay, flow cytometry and Transwell assays were performed to investigate cell proliferation, apoptosis, migration and invasion, respectively. A dual-luciferase reporter assay was performed to detect the correlation between circRNA_100395 and micro (mi)RNA-142-3p. Western blotting was performed to elucidate the potential regulatory mechanism. circRNA_100395 expression was found to be increased in GC tissues and cell lines. However, miR-142-3p expression was significantly reduced. Besides, low expression levels of circRNA_100395 were associated with poor tumor differentiation, advanced Tumor-Node-Metastasis stage, lymph node metastasis and shorter overall survival time. Moreover, overexpression of circRNA_100395 suppressed cell proliferation, increased the apoptosis rate and suppressed cell invasion and migration by inhibiting the PI3K/AKT signaling pathway. These findings also showed that miRNA-142-3p rescued the antitumor effects induced by circRNA_100395-overexpression. cirRNA_100395-overexpression had antitumor effects via regulating the miR-142-3p signaling pathway, which might be a promising treatment target for GC.

Keywords: cell proliferation; circRNA_100395; gastric cancer; invasion; miR-142-3p; migration.

Figure 1.

Expression pattern of circRNA_100395 in GC. (A) Expression of circRNA_100395 in GC tissues. (B) circRNA_100395 was downregulated (74.1%, 40/54) in GC. Relationship between circRNA_100395 expression and (C) tumor differentiation, (D) TMN stage and (E) lymph node metastasis. (F) Kaplan-Meier curves of overall survival of patients with GC. **P<0.01 and ***P<0.001. circRNA, circular RNA; GC, gastric cancer.

Figure 2.

Upregulation of circRNA_100395 inhibits GC cell proliferation. (A) Expression of circRNA_100395 in GC cells. Successful generation of circRNA_100395-overexpression in (B) HGC-27 and (C) AGS cells. Cell proliferation of (D) HGC-27 and (E) AGS with circRNA_100395-overexpression determined via a Cell Counting Kit-8 assay. EdU assay of (F) HGC-27 and (G) AGS cells after upregulating circRNA_100395 expressions. ***P<0.001 vs. GES-1 cells or OE-Vector. circRNA, circular RNA; GC, gastric cancer; OE, overexpression.

Figure 3.

Upregulation of circRNA_100395 increases apoptosis rate and inhibits cell invasion and migration in gastric cancer. Apoptosis flow cytometry analysis of (A) HGC-27 and (B) AGS cells following circRNA_100395 upregulation. (C) Cell migration and (D) invasion images of HGC-27 and AGS cells. **P<0.01 and ***P<0.001 vs. respective control. circRNA, circular RNA; OE, overexpression.

Figure 4.

Upregulation of circRNA_100395 inhibits the PTEN-PI3K/AKT signaling pathway. Western blot analysis of PTEN, PI3K, total AKT and p-AKT expression in (A) HGC-27 and (B) AGS cells when circRNA_100395 was overexpressed. circRNA, circular RNA; OE, overexpression; p-, phosphorylated.

Figure 5.

miR-142-3p is a downstream regulatory gene of circRNA_100395. (A) Predicted 3′ untranslated region binding sites of circRNA_100395 on miR-142-3p. (B) miR-142-3p was significantly downregulated in patients with GC. (C) Expression of miR-142-3p in GC tissues. (D) Correlation between miR-142-3p and circRNA_100395 expression. (E) Relative luciferase activity of 293T cells after co-transfection. (F) Expression of miR-142-3p in circRNA_100395-overexpressed AGS cells after treatment with miR-142-3p inhibitors. **P<0.01 and ***P<0.001 vs. respective control. circRNA, circular RNA; OE, overexpression; miR, microRNA; hsa, Homo sapien; NC, negative control.

Figure 6.

Upregulation of miR-142-3p rescues antitumor effects induced by overexpression of circRNA_100395. (A) Cell proliferation ability of circ_100395-overexpressed AGS cells. (B) Apoptosis rate of circ_100395-overexpressed AGS cells. (C) Cell migration and invasion of circ_100395-overexpressed AGS cells. (D) Expression of PI3K/AKT signaling pathway-related proteins in circ_100395-overexpressed AGS cells after the addition of miR-135a-5p. ***P<0.001 vs. respective control. circRNA, circular RNA; OE, overexpression; NC, negative control; p-, phosphorylated.