MicroRNA-375 Functions as a Tumor-Suppressor Gene in Gastric Cancer by Targeting Recepteur d'Origine Nantais

Abstract

Emerging evidence supports a fundamental role for microRNAs (miRNA) in regulating cancer metastasis. Recently, microRNA-375 (miR-375) was reported to be downregulated in many types of cancers, including gastric cancer. Increase in the expression of Recepteur d'Origine Nantais (RON), a receptor tyrosine kinase, has been reported in tumors. However, the function of miR-375 and RON expression in gastric cancer metastasis has not been sufficiently studied. In silico analysis identified miR-375 binding sites in the 3'-untranslated regions (3'-UTR) of the RON-encoding gene. Expression of miR-375 resulted in reduced activity of a luciferase reporter containing the 3'-UTR fragments of RON-encoding mRNA, confirming that miR-375 directly targets the 3'-UTR of RON mRNA. Moreover, we found that overexpression of miR-375 inhibited mRNA and protein expression of RON, which was accompanied by the suppression of cell proliferation, migration, and invasion in gastric cancer AGS and MKN-28 cells. Ectopic miR-375 expression also induced G1 cell cycle arrest through a decrease in the expression of cyclin D1, cyclin D3, and in the phosphorylation of retinoblastoma (Rb). Knockdown of RON by RNAi, similar to miR-375 overexpression, suppressed tumorigenic properties and induced G1 arrest through a decrease in the expression of cyclin D1, cyclin D3, and in the phosphorylation of Rb. Thus, our study provides evidence that miR-375 acts as a suppressor of metastasis in gastric cancer by targeting RON, and might represent a new potential therapeutic target for gastric cancer.

Keywords: Recepteur d’Origine Nantais; gastric cancer; microRNA-375.

Figure 1

Recepteur d’Origine Nantais (RON) is a miR-375 target. (A) Schematic representation of the miR-375-binding sites in the RON Recepteur d’Origine Nantais (3′-UTR) region, as detected by TargetScan; The letters in red color indicates the corresponding potential targets sites of miR-375 in 3′-UTR of RON; (B) Luciferase assay in AGS and MKN cells. AGS and MKN-28 cells were co-transfected with a miR-375 mimic or negative control or RON siRNA and a vector containing the 3′-UTR of RON cloned downstream of a luciferase gene. Relative repression of the luciferase expression was standardized to the β-gal signal. * p < 0.05 versus negative control (AGS cell); ** p < 0.05 versus negative control (MKN-28 cell); The data represent the mean ± SD from triplicate measurements. NC—negative control.

Figure 2

Inverse relationship between the expression of miR-375 and RON in AGS and MKN-28 cells. (A) RON mRNA levels after transfection of AGS and MKN-28 cells with a miR-375 mimic, a miR-375 inhibitor, a negative control, or RON siRNA. Total RNA was extracted 48 h after transfection and mRNA levels were determined by qRT-PCR. * p < 0.05 versus negative control (AGS cell); ** p < 0.05 versus negative control (MKN-28 cell); The data represent the mean ± SD from triplicate measurements; (B) RON protein levels after transfection of AGS cells with a miR-375 mimic, a miR-375 inhibitor, a negative control, or RON siRNA. Total cell lysates were extracted 48 h after transfection and protein levels were determined by Western blot. * p < 0.05 versus negative control; The data represent the mean ± SD from triplicate measurements; (C) RON protein levels after transfection of MKN-28 cells with a miR-375 mimic, a miR-375 inhibitor, a negative control, or RON siRNA. Total cell lysates were extracted 48 h after transfection and protein levels were determined by Western blot. * p < 0.05 versus negative control; The data represent the mean ± SD from triplicate measurements.

Figure 3

miR-375 and RON regulate cell proliferation in AGS and MKN-28 human gastric cancer cells. (A) Brdu assay in AGS cells transfected with a miR-375 mimic, a negative control, or RON siRNA; (B) Brdu assay in AGS cells transfected with a miR-375 inhibitor or a negative control; (C) Brdu assay in MKN-28 cells transfected with a miR-375 mimic, a negative control, or RON siRNA; (D) Brdu assay in MKN-28 cells transfected with a miR-375 inhibitor or negative control. * p < 0.05; The data represent the mean ± SD from triplicate measurements.

Figure 4

miR-375 and RON regulate the cell cycle by decreasing cyclin D1 and cyclin D3 expression and retinoblastoma (Rb) phosphorylation in AGS and MKN-28 human gastric cancer cells. (A,B) Representative histograms show cell cycle distribution of AGS cells in the negative control, miR-375 mimic, and RON siRNA groups. * p < 0.05 versus negative control (miR-375 mimic); ** p < 0.05 versus negative control (RON siRNA); (C) Cyclin D1, cyclin D3, and phosphorylation of Rb were assessed by Western blot analysis 48 h after transfection with a miR-375 mimic, a miR-375 inhibitor, a negative control, or RON siRNA in AGS cells. * p < 0.05 versus negative control; The data represent the mean ± SD from triplicate measurements; (D) Cyclin D1, cyclin D3, and phosphorylation of Rb were assessed by Western blot analysis 48 h after transfection with a miR-375 mimic, a miR-375 inhibitor, a negative control, or RON siRNA in MKN-28 cells. * p < 0.05 versus negative control; The data represent the mean ± SD from triplicate measurements.

Figure 5

Overexpression of miR-375 or knockdown of RON inhibits cell migration in AGS and MKN-28 cells. (A,C) Wound healing assays were performed to evaluate the effect of miR-375 or RON on the migratory ability of AGS cells; (B,D) Wound healing assays were performed to evaluate the effect of miR-375 or RON on the migratory ability of MKN-28 cells. * p < 0.05 versus negative control; The data represent the mean ± SD from triplicate measurements. Scale bars: 100 μM in (A,B). The space between back lines in (A,B) were measured for the migratory ability of cells.

Figure 6

Overexpression of miR-375 or knockdown of RON inhibits cell invasion in AGS and MKN-28 cells. (A,C) A modified Boyden chamber assay was performed to evaluate the effect of miR-375 or RON on the invasive ability of AGS; (B,D) A modified Boyden chamber assay was performed to evaluate the effect of miR-375 or RON on the invasive ability of MKN-28 cells. * p < 0.05 versus negative control; The data represent the mean ± SD from triplicate measurements. Scale bars: 100 μM in (A,B).