MicroRNA-125a-5p inhibits invasion and metastasis of gastric cancer cells by targeting BRMS1 expression

Abstract

Accumulating studies have demonstrated microRNAs (miRNAs/miRs) have an important role in multiple processes of human malignant tumor development and progression. Decreased expression of miR-125a-5p has been observed in several types of cancer, including gastric cancer (GC). However, the mechanism and exact function of miR-125a-5p in GC have not been largely elucidated. In the present study, reverse transcription-quantitative polymerase chain reaction indicated that the expression of miR-125a-5p was downregulated in GC tissues and cell lines compared with matched normal tissues (P<0.01) and normal gastric mucosa cell lines (P<0.01), respectively. Moreover, clinical pathological characteristics and Kaplan-Meier analysis indicated that a low expression of miR-125a-5p was not only associated with lymph metastasis, peritoneal dissemination and advanced tumor-node metastasis stage but also affected the prognosis of GC patients. Compared with miR-control-transfected GC cells, markedly decreased migration and invasion was observed in GC cells that overexpress miR-125a-5p. By contrast, increased metastasis and invasion were observed in miR-125a-5p-knocked down cells compared with the control. Furthermore, luciferase reporter assays indicated that breast cancer metastasis suppressor 1 (BRMS1) was a direct target of miR-125a-5p. Notably, a positive correlation between the levels of BRMS1 and miR-125a-5p in GC tissues was observed, and BRMS1 expression was indicated to be regulated by miR-125a-5p in GC cells. In conclusion, miR-125a-5p may act as a tumor suppressor by targeting the metastasis-inhibitory gene, BRMS1. The data suggesting that BRMS1 is a potential target gene of miR-125a-5p, may provide novel insight into miRNA regulation of human gene expression, and a useful target for gene therapy of GC.

Keywords: Invasion and metastasis; breast cancer metastasis suppressor 1; gastric cancer; microRNA-125a-5p.

Figure 1.

miR-125a-5p was downregulated in GC and associated with the capacity to metastasize. (A) The expression of miR-125a-5p in three GC cell lines (BGC823, SGC7901 and HGC27) and two normal gastric cell lines (GES1 and HFE145) was analyzed by RT-qPCR. Values are shown as the mean ± standard deviation (n=3). *P<0.05, **P<0.01 compared with GES1 and HFE145. (B) RT-qPCR results showed that miR-125a-5p expression was decreased in primary cancer tissues compared with matched normal tissues (P<0.001). (C) Expression of miR-125a-5p in primary GC tissues with (n=14) and without peritoneal metastasis (n=68) (P=0.0421). (D) Kaplan-Meier curves indicate that the poor prognosis of GC patients was positively associated with miR-125a-5p expression level (P=0.0092). The expression of miR-125a-5p was normalized to the expression of U6 small nuclear RNA. Each experiment was repeated three times. GC, gastric cancer; miR, microRNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 2.

miR-125a-5p inhibits the migration and invasion of GC cells in vitro. (A) Transwell migration assays confirmed that the ectopic expression of miR-125a-5p inhibited the migratory ability of SGC7901 and HGC27 cells. (B) Transwell invasion assays confirmed that the ectopic expression of miR-125a-5p inhibited the invasive ability of SGC7901 and HGC27 cells. Transwell assays revealed that the knockdown of endogenous miR-125a-5p increased the (C) migratory and (D) invasive abilities of BGC823 cells. Representative micrographs and the relative number of cells are shown. The number of cells was counted from three independent experiments. The error bars represent standard deviations. Magnification, ×200. *P<0.05, **P<0.01 and ***P<0.001. GC, gastric cancer; miR, microRNA.

Figure 3.

Confirmation of predicted binding sites between miR-125a-5p and BRMS1. (A) The bioinformatics websites (microRNA.org, MicroCosm Targets, TargetScan Human and miRTar) predicted that the 5′-UTR of BRMS1 mRNA contains the binding sequences of miR-125a-5p. (B) Dual luciferase reporter assay performed in HGC27 cells. The average values of normalized 5′-UTR luciferase intensity were calculated from three independent experiments. Luciferase activity was significantly decreased in the group transfected with miR-125a-5p and Wt-BRMS1-5′UTR, compared with the other three groups, *P<0.05. BRMS1, breast cancer metastasis suppressor 1; CD, coding DNA sequence; miR, microRNA; MU, mutated; UTR, untranslated region; WT, wild-type.

Figure 4.

Expression of BRMS1 in GC tissues and cell lines, and positive correlation of BRMS1 expression with miR-125a-5p expression level. (A) Representative western blot result of GC tissues (n=6) and matched normal tissues (n=6). (B) Expression of BRMS1 in 82 GC tissues and matched normal tissues were detected by western blotting as shown by scatter plot (P<0.01). BRMS1 expression was normalized to β-actin expression. (C) Linear correlation analysis between the levels of BRMS1 protein and miR-125a-5p in GC tissues using Spearman's correlation analysis (n=82, r=0.5676; P<0.01). (D) The levels of BRMS1 protein in three gastric cancer cell lines (BGC823, SGC7901 and HGC27) and two normal gastric cell lines (GES1 and HFE145). β-actin was used as an internal loading control. BRMS1, breast cancer metastasis suppressor 1; GC, gastric cancer; miR, microRNA; N, normal tissues; T, gastric cancer tumor tissues.

Figure 5.

miR-125a-5p regulates the expression of BRMS1 in GC cell lines. (A) Compared with the expression of miR-125a-5p in miR-control-transfected controls, miR-125a-5p expression in miR-125a-5p plasmid-transfected cells was upregulated. By contrast, following the transfection of anti-miR-125a-5p, the miR-125a-5p expression in BGC823 cells was significantly suppressed. *P<0.05, **P<0.01. The expression of BRMS1 mRNA and protein in (B) SGC7901 and (C) HGC27 cells following the transfection of miR-NC or miR-125a-5p. *P<0.05 vs. Mock or miR-control groups. (D) Following transfection of BGC823 cells with anti-miR-125a-5p, the expression of BRMS1 mRNA and protein was significantly decreased compared with the expression in anti-miR-NC or mock groups. *P<0.05. For reverse transcription-quantitative polymerase chain reaction assays, β-actin was used as an internal control for expression of BRMS1, and the values indicate the mean ± standard deviation (n=3). For western blotting, β-actin served as an internal control. BRMS1, breast cancer metastasis suppressor 1; miR, microRNA; NC, negative control; si, small-interfering.

Figure 6.

Involvement of BRMS1 in miR-125a-5p-induced suppression of migration and invasion of GC cells. (A) BRMS1 mRNA levels in BGC823 cells analyzed by reverse transcription-quantitative polymerase chain reaction following transfection with si-BRMS1, anti-miR-125a-5p, BRMS1 and anti-miR-125a-5p. The error bars represent standard deviation values obtained from three independent experiments. *P<0.05, **P<0.01. The value was normalized by β-actin. (B) The levels of BRMS1 protein in BGC823 cells that were transfected with si-BRMS1, anti-miR-125a-5p, and a combination of BRMS1 and anti-miR-125a-5p as detected by western blotting. β-actin served as an internal control. (C) Migration and (D) invasion assays in BGC823 cells transfected with different plasmids. Values for cell numbers represent the mean ± standard deviation (n=3). *P<0.05, **P<0.01. BRMS1, breast cancer metastasis suppressor 1; GC, gastric cancer; miR, microRNA; si-small-interfering.