MicroRNA-34c-5p exhibits anticancer properties in gastric cancer by targeting MAP2K1 to inhibit cell proliferation, migration, and invasion

Abstract

Purpose: Gastric cancer(GC)is one of the deadliest digestive tract tumors worldwide，existing studies suggest that dysregulated expression of microRNAs (miRNAs) plays an important role in the pathogenesis and progression of GC. This study aimed to investigate the expression, biological function, and downstream mechanism of miR-34c-5p in GC, provide new targets for gastric cancer diagnosis and treatment.

Methods: The expression of miR-34c-5p in GC tissues and cell lines was examined by RT-qPCR. Cell wound healing, transwell and cell cloning assays were used to detect the effect of miR-34c-5p on the migration and invasion abilities, respectively, of GC cells. Western blot was performed to detect the expression of related proteins. Bioinformatics analysis was used to predict the binding of MAP2K1 to miR-34c-5p and the targeting relationship was confirmed by dual luciferase reporter assay.

Results: The expression level of miR-34c-5p was significantly decreased in GC tissues and cell lines. miR-34c-5p overexpression inhibited migration, invasion, and colony formation of gastric cancer cells, the related protein E-cadherin expression was significantly increased and N-cadherin, vimentin, and PCNA expression were significantly decreased, while miR-34c-5p knockdown exerted the opposite effects. In addition, the targeting relationship between miR-34c-5p and MAP2K1 was predicted and confirmed, and further confirmed by rescue experiments that MAP2K1 alleviated the inhibitory effect of miR-34c-5p in GC.

Conclusion: MiR-34c-5p is lowly expressed in GC, and it can target MAP2K1 to exert inhibitory effects on GC proliferation, invasion, and migration. These findings provide a promising biomarker and a potential therapeutic target for gastric cancer.

Figure 1

MiR-34c-5p is downregulated in GC tissues and cells. (A) The expression of miR-34c-5p in 50 pairs of GC tissues and adjacent paracancerous tissues. (B) The expression of miR-34c-5p in normal gastric mucosa epithelial cell line (GES-1) and four gastric cancer cell lines (AGS, MKN-28, MKN-45, HGC-27). ∗P < 0.05, ∗∗P < 0.01.

Figure 2

miR-34c-5p inhibits the proliferation, migration, and invasion of GC cells. (A, B) miR-34c-5p expression in HGC-27or MKN-28 cells transfected with miR-34c-5p mimic, mimics-NC or miR-34c-5p inhibitors, or inhibitor-NC were detected by qRT-PCR. (C) Proliferation ability of HGC-27 and MKN-28 cells was assessed by using colony formation. (D) CCK-8 assay detects the effect of cell proliferation viability after miR-34c-5p overexpression or knockdown.(E) The migration ability of cells was evaluated by wound healing assay. (F) The migration and invasion ability of cells were evaluated by transwell assay. (G) Western blot detection of protein levels of E-cadherin, N-cadherin, Vimentin, PCNA after miR-34c-5p overexpression or knockdown. ∗P< 0.05, ∗∗P< 0.01, ∗∗P < 0.01.

Figure 3

MAP2K1 is the target of miR-34c-p. (A) The predicted binding sequence between MAP2K1 3'-UTR and miR-34c-5p. (B) Dual-luciferase reporter gene assay was used to verify the binding relationship between miR-34c-p and MAP2K1 3'-UTR. (C) Western blot was performed to detect the protein level of MAP2K1 in normal gastric mucosal cells GES-1 and GC cells HGC-27 and MKN-28. (D, E) Starbase database was used to analyze the expression of MAP2K1 in GC tissues and its correlation with miR-34c-5p. (F) The expression of MAP2K1 after overexpression or knockdown of miR-34c-5p. ∗∗P < 0.01. ∗∗∗P< 0.001, ns: no significance.

Figure 4

Overexpression of MAP2K1 attenuated the inhibitory effect of miR-34c-5p on GC cells. (A) RT-qPCR was used to detect MAP2K1 expression after co-transfection of PCMV-MAP2K1 with miR-34c-5p mimics or MAP2K1 siRNA with miR-34c-5p inhibitors. (B) Proliferation ability of GC cells was assessed after cotransfection of PCMV-MAP2K1 with miR-34c-5p mimics or MAP2K1 siRNA with miR-34c-5p inhibitor using colony formation. (C, D) The migration and invasion ability of cells were evaluated by transwell assay after cotransfection of PCMV-MAP2K1 with miR-34c-5p mimics or MAP2K1 siRNA with miR-34c-5p.