MicroRNA-335 acts as a metastasis suppressor in gastric cancer by targeting Bcl-w and specificity protein 1

Abstract

Aberrant expression of miR-335 has been frequently reported in cancer studies, suggesting that there is a close correlation between miR-335 and cancer during its development, progression, metastasis and prognosis. The expression of miR-335 in gastric cancer and its effects are not known. Relative expression of miR-335 in 4 gastric cancer cell lines and in 70 gastric cancer tissues was confirmed by real-time quantitative reverse transcriptase-PCR compared with controls. Transwell cell migration and Matrigel invasion assay in vitro and metastasis formation assay in vivo were used to examine the effects of miR-335 expression on gastric cancer cell invasion and metastasis. The effect of miR-335 expression on gastric cancer cell proliferation was estimated by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Luciferase reporter assay and western blot were used to examine the potential target genes and related pathways. Gene silencing with small-interfering RNA was used to examine the effects of target genes on gastric cancer cell invasion. miR-335 was dramatically downregulated in gastric cancer cell lines than in the normal gastric cell line GES-1. Low expression of miR-335 was significantly associated with lymph-node metastasis, poor pT stage, poor pN stage and invasion of lymphatic vessels. Overexpression of miR-335 suppressed gastric cancer cell invasion and metastasis in vitro and in vivo, but has no significant effects on cell proliferation. Furthermore, miR-335 might suppress gastric cancer invasion and metastasis by targeting Bcl-w and specificity protein 1 (SP1). Taken together, our results provide evidence that miR-335 might function as a metastasis suppressor in gastric cancer by targeting SP1 directly and indirectly through the Bcl-w-induced phosphoinositide 3-kinase-Akt-Sp1 pathway. miR-335 showing altered expression at different stages of gastric cancer could be a target for gastric cancer therapies and could be further developed as a potential prognostic factor.

Figure 1

The expression of miR-335 in tissues and cell lines. (a) Relative expression of miR-335 in four gastric cancer cell lines (MGC-803, SGC-7901, BGC-823 and AGS). Quantification of miR-335 was measured by qRT–PCR. Data are presented in gastric cancer cell lines relative to GES-1 (which is a normal gastric epithelial cell line that was chosen as a control). Results represent the means of the values. Bars indicate s.d. ** Refers to statistical significance between groups (P<0.01). (b) There is no statistical significance with miR-335 expression between gastric cancer tissues and their pair-matched adjacent non-tumor tissues (P=0.7). miR-335 was normalized by U6RNA. ΔC_T=C_T miR-335−C_T U6RNA. Results represent the means of the values. Bars indicate s.d. (c) Relative expression of miR-335 in 70 patients with gastric cancer. Quantification of miRNAs was measured by qRT–PCR. Data that were transformed to log2 values in gastric cancer tissues relative to their matched non-tumor adjacent tissues were divided into two groups of metastasis free and metastasis positive. The non-parametric test revealed that the low expression of miR-335 was significantly associated with lymph-node metastasis (P<0.001). (d) The non-parametric test demonstrated that patients with better pT stage had significantly higher miR-335 expression than did those with poor pT stage (P<0.05). Quantification of miRNAs was measured by qRT–PCR. Results represent the median of the values, which are the relative expressions of miR-335 associated with the pT stage.

Figure 2

miR-335 inhibits cell invasion in vitro. (a) Representative photomicrographs of transwell results for BGC-823 cells were taken under × 100 original magnification. (b) Representative photomicrographs of transwell results for SGC-7901 cells were taken under × 100 original magnification. (c) Representative photomicrographs of transwell results for AGS cells were taken under × 100 original magnification. (d) The numbers of miR-335-transfected BGC-823 cells passing through the Matrigel were significantly lower than those of NC-transfected and parental BGC-823 cells. The number of cells was counted in 16 independent symmetrical visual fields under the microscope ( × 400 original magnification) from three independent experiments. Results represent the means of the values from three independent experiments. Bars indicate s.d. * Refers to statistical significance between groups (P<0.05). (e) The numbers of miR-335-transfected SGC-7901 cells passing through the Matrigel were significantly lower than those of NC-transfected and parental SGC-7901 cells. The number of cells was counted in 16 independent symmetrical visual fields under the microscope ( × 200 original magnification) from three independent experiments. Results represent the means of the values from three independent experiments. Bars indicate s.d. * Refers to statistical significance between groups (P<0.05). (f) The numbers of anti-miR-335-transfected AGS cells passing through the Matrigel were significantly higher than those of NC-transfected and parental AGS cells. The number of cells was counted in 16 independent symmetrical visual fields under the microscope ( × 200 original magnification) from three independent experiments. Results represent the means of the values from three independent experiments. Bars indicate s.d. * Refers to statistical significance between groups (P<0.05).

Figure 3

miR-335 suppresses gastric cancer metastasis in vivo. (a) Representative photographs for lung tissues. Cell aggregates, which were observed by staining with dark nucleus, represent lung metastases. (b) The data are shown graphically with the number of lung metastases from each mouse. Results represent the means of the values. Bars indicate s.d. * Refers to statistical significance between groups (P<0.05).

Figure 4

SP1 and Bcl-w are potential targets of miR-335 in gastric cancer cells. (a) Analysis of luciferase activity of SP1 and SP1-MUT with miR-335 mimics or NCs in SGC-7901 cells. * Refers to statistical significance between groups (P<0.05). (b) Analysis of luciferase activity of Bcl-w and Bcl-w-MUT with miR-335 mimics or NCs in SGC-7901 cells. * Refers to statistical significance between groups (P<0.05). (c) mRNA expression analysis for SP1 in parental and transfected SGC-7901 cells by real-time qRT–PCR. No significant difference in the level of the endogenous SP1 mRNA was found in transfected and parental SGC-7901 cells normalized to an endogenous reference GAPDH mRNA. (d) mRNA expression analysis for Bcl-w in parental and transfected SGC-7901 cells by real-time qRT–PCR. No significant difference in the level of endogenous Bcl-w mRNA was found in transfected and parental SGC-7901 cells normalized to an endogenous reference GAPDH mRNA. (e) Protein expression analysis for SP1 and Bcl-w in miR-335-transfected, NC-transfected and parental SGC-7901 cells by western blot. A clear reduction in the level of the endogenous SP1 and Bcl-w proteins was demonstrated in miR-335-transfected SGC-7901 cells than in NC-transfected SGC-7901 cells normalized to an endogenous reference β-actin protein. (f) Protein expression analysis for SP1 and Bcl-w in anti-miR-335-transfected, anti-NC-transfected and parental SGC-7901 cells by western blot. The level of the endogenous SP1 and Bcl-w proteins was increased in anti-miR-335-transfected SGC-7901 cells than in anti-NC-transfected SGC-7901 cells normalized to an endogenous reference β-actin protein.