hsa_circ_0023409 Accelerates Gastric Cancer Cell Growth and Metastasis Through Regulating the IRS4/PI3K/AKT Pathway

Abstract

Gastric cancer (GC) is a big threat to human life and health. Circular RNAs (circRNAs), a subclass of noncoding RNAs, were reported to play a critical role in GC progression. Here, we investigated the role of a novel circRNA named hsa_circ_0023409 in GC and its mechanism. Hsa_circ_0023409 expression in GC and adjacent tissues was examined by quantitative real-time polymerase chain reaction and in situ hybridization. The functions of hsa_circ_0023409 in GC cells were assessed both in vitro and in vivo. Immunofluorescence staining was performed for the localization of hsa_circ_0023409 and miR-542-3p in cells. The interaction between hsa_circ_0023409 and miR-542-3p, and miR-542-3p and insulin receptor substrate 4 (IRS4) was detected by dual-luciferase reporter assay. The effect of hsa_circ_0023409, miR-542-3p, and IRS4 on IRS4/phosphatidylinositol 3-kinase (PI3K)/AKT pathway was detected by western blot. The results showed that hsa_circ_0023409 was mainly located in cytoplasm and highly expressed in GC tissues and cells. Moreover, hsa_circ_0023409 showed positive correlation with tumor size, histological grade, and tumor-node-metastasis staging of GC patients. Functional studies showed that hsa_circ_0023409 promoted cell viability, proliferation, migration, and invasion and suppressed apoptosis in GC. Mechanism studies demonstrated that hsa_circ_0023409 upregulated IRS4 via sponging miR-542-3p in GC cells. Furthermore, IRS4 overexpression activated the PI3K/AKT pathway and reversed the inhibitory effect of hsa_circ_0023409 knockdown on the PI3K/AKT pathway. Taken together, we prove that hsa_circ_0023409 activates IRS4/PI3K/AKT pathway by acting as a sponge for miR-542-3p, thus promoting GC progression, indicating that hsa_circ_0023409 may serve as a potential target for treatment of GC and prognosis of GC patients.

Keywords: IRS4/PI3K/AKT pathway; cell growth; cell metastasis; gastric cancer; hsa_circ_0023409.

Figure 1.

Hsa_circ_0023409 was upregulated in GC tissues and cells and associated with the prognosis of GC patients. (A) The mRNA expression levels of hsa_circ_0023409 in 87 paired GC tissues and adjacent normal tissues. (B) In situ hybridization analysis of hsa_circ_0023409 with locked nuclei acid probes in GC tissues and adjacent tissues. Scale bar =200 μm (100x) and 100 μm (200x). (C) Overall survival rate of GC patients with high hsa_circ_0023409 expression or low hsa_circ_0023409 expression. (D) The mRNA expression levels of hsa_circ_0023409 in GC cells (AGS, MKN45, HGC-27, SUN-1, and MKN7) and normal gastric cells (GES-1). (E) Relative mRNA expression levels of hsa_circ_0023409 and its linear RNA RNF 121 in HGC-27 and MKN45 cells after RNase R treatment. (F) Relative expression of hsa_circ_0023409 and glyceraldehyde 3-phosphate dehydrogenase in nucleus and cytoplasm in HGC-27 and MKN45 cells. Values are the mean ± standard deviation. **P < 0.01. GC: gastric cancer; mRNA: micro RNA.

Figure 2.

Hsa_circ_0023409 promoted the growth of GC cells. (A) The mRNA expression levels of hsa_circ_0023409 in MKN45 cells transfected with hsa_circ_0023409-overexpressed plasmid and in HGC-27 cells transfected with underexpressed plasmid. (B) Cell viability in MKN45 cells transfected with hsa_circ_0023409-overexpressed plasmid and in HGC-27 cells transfected with underexpressed plasmid for 0, 24, 48, 72, and 96 h. (C, D) The cell proliferation and apoptosis were measured in GC cells after hsa_circ_0023409 knockdown or upregulation. Values are the mean ± standard deviation. *P < 0.05 and **P < 0.01. circRNA: circular RNA; EdU: 5-ethynyl-2′-deoxyuridine; GC: gastric cancer; NC: negative control; shRNA: short hairpin RNA.

Figure 3.

Hsa_circ_0023409 promoted the migration and invasion of GC cells. (A, B) Cell migration and invasion ability of GC was measured by cell scratch assay and transwell assay, respectively, after hsa_circ_0023409 knockdown or upregulation. Values are the mean ± standard deviation. *P < 0.05 and **P < 0.01. circRNA: circular RNA; GC: gastric cancer; NC: negative control; shRNA: short hairpin RNA.

Figure 4.

Hsa_circ_0023409 sponged miR-542-3p. (A) Binding sites of hsa_circ_0023409 to miR-542-3p predicted by Starbase v3.0. (B) Immunofluorescence staining of hsa_circ_0023409 and miR-542-3p in HGC-27 and MKN45 cells. Scale bar = 50 μm. Red represents hsa_circ_0023409 staining, green for miR-542-3p identification, and blue for nuclei identification. (C) The luciferase activity in HGC-27 and MKN45 cells transfected with miR-542-3p mimics or inhibitor. (D) Relative miR-542-3p expression levels in GC cells after hsa_circ_0023409 knockdown or overexpression. (E) The mRNA expression levels of miR-542-3p in 87 paired GC tissues and adjacent normal tissues and correlation analysis between hsa_circ_0023409 and miR-542-3p in GC (P < 0.0001). Values are the mean ± standard deviation. **P < 0.01. circRNA: circular RNA; GC: gastric cancer; MUT: mutant; shRNA: short hairpin RNA; WT: wild type.

Figure 5.

Hsa_circ_0023409 activated IRS4/PI3K/AKT pathway via sponging miR-542-3p. (A) Binding sites of miR-542-3p to IRS4 predicted by TargetScanHuman 7.2. (B) The luciferase activity in HGC-27 and MKN45 cells transfected with miR-542-3p mimics or inhibitor. (C) The protein levels of IRS4, Akt, and p-Akt in MKN45 cells transfected with miR-542-3p inhibitor and in HGC-27 cells transfected with miR-542-3p mimics. (D) The mRNA expression levels of IRS4 in 87 paired GC tissues and adjacent normal tissues. (E) Correlation analysis of IRS4 with hsa_circ_0023409 and miR-542-3p in GC. (F) The protein levels of IRS4, Akt, and p-Akt in hsa_circ_0023409 downregulated cells treated with miR-542-3p mimics or inhibitor. Values are the mean ± standard deviation. **P < 0.01 and ^## P < 0.01. circRNA: circular RNA; GC: gastric cancer; IRS4: insulin receptor substrate 4; NC: negative control; PI3K: phosphatidylinositol 3-kinase; UTR: untranslated region; WT: wild type.

Figure 6.

Hsa_circ_0023409 promoted the growth, migration, and invasion of GC cells via upregulating IRS4 level. (A) Cell viability in hsa_circ_0023409 downregulated cells treated with IRS4-overexpressed plasmid or its control for 0, 24, 48, 72, and 96 h. (B, C, and D) Cell proliferation, apoptosis, migration, and invasion in hsa_circ_0023409 downregulated cells treated with IRS4-overexpressed plasmid or its control. (E) The protein levels of IRS4, Akt, and p-Akt in hsa_circ_0023409-downregulated cells treated with IRS4-overexpressed plasmid or its control. Values are the mean ± standard deviation. **P < 0.01 and ^## P < 0.01. EdU: 5-ethynyl-2′-deoxyuridine; FITC: fluorescein isothiocyanate; GC: gastric cancer; IRS4: insulin receptor substrate 4; NC: negative control; OD: optical density; shRNA: short hairpin RNA.

Figure 7.

Knockdown of hsa_circ_0023409 inhibited tumor growth and lung metastasis in vivo. (A) Tumors from nude mouse treated with hsa_circ_0023409-overexpressed cells or its control. (B) Tumor volume and weight. (C) Immunohistochemical staining of IRS4, Ki-67, and p-AKT in tumor tissues. Scale bar =50 μm. (D) Hematoxylin and eosin staining of lung tissues and the number of lung metastatic nodules in lung tissues. Values are the mean ± standard deviation. **P < 0.01. HE: hematoxylin and eosin; IRS4: insulin receptor substrate 4; NC: negative control; shRNA: short hairpin RNA.