MicroRNA-143 regulates collagen type III expression in stromal fibroblasts of scirrhous type gastric cancer

Abstract

Gastric cancer (GC) is one of the most common malignancies worldwide. In particular, scirrhous type GC is highly metastatic and is characterized clinically by rapid disease progression and poor prognosis. MicroRNAs (miRNAs) play crucial roles in cancer development and progression. In the present study, we identified several miRNAs that are expressed at higher levels in scirrhous type GC than in non-scirrhous type GC by miRNA microarray analysis. Among these, microRNA-143 (miR-143) expression was higher in scirrhous type GC than in non-scirrhous types of GC. In situ hybridization and quantitative RT-PCR analysis showed that miR-143 is expressed by stromal fibroblasts but not by cancer cells. In stromal cells, miR-143 enhanced collagen type III expression in normal gastric fibroblasts and cancer-associated fibroblasts through activation of transforming growth factor-β)/SMAD signaling. Furthermore, high miR-143 expression in GC was associated with worse cancer-specific mortality (P = 0.0141). Multivariate analysis revealed that miR-143 was an independent prognostic factor. Treatment of GC cell lines with 5-aza-2'-deoxycytidine restored the expression of miR-143, and precursor miR-143 caused the inhibition of cancer cell invasion. These data suggest that miR-143 regulates fibrosis of scirrhous type GC through induction of collagen expression in stromal fibroblasts and that miR-143 expression serves as a prognostic marker of GC.

Keywords: Collagen type III; fibroblast; gastric cancer; microRNA-143; transforming growth factor-β.

Fig. 1

MicroRNA-143 (miR-143) expression in gastric cancer (GC) and non-neoplastic tissue. (a) Expression levels of miR-143 in GC tissue samples (n = 20) were measured by quantitative RT-PCR analysis. (b) MicroRNA-143 expression levels in 30 formalin-fixed paraffin-embedded GC tissue samples and non-neoplastic tissue samples as determined by quantitative RT-PCR were compared. Statistical differences were evaluated using the Wilcoxon matched pair test. Bars and error bars indicated median and standard error.

Fig. 2

MicroRNA-143 (miR-143) expression in scirrhous type gastric cancer (GC) tissue and cell lines. In situ hybridization of miR-143 was carried out in combination with immunofluorescence staining in scirrhous type GC. MicroRNA-143 labeling was revealed by Cy3-conjugated streptavidin (red). (a) α-Smooth muscle actin (α-SMA), (b) vimentin, or (c) CAM5.2 labeling was revealed by FITC-conjugated secondary antibody (green). DNA was counterstained with DAPI (blue). MicroRNA-143 expression was localized to α-SMA-positive and vimentin-positive stromal fibroblasts. (d) MicroRNA-143 expression levels were evaluated in GC cell lines and fibroblasts. Bars and error bars indicate median and standard error, respectively. *P < 0.05. CaF, cancer-associated fibroblasts; NF, normal gastric fibroblasts.

Fig. 3

Regulation of collagen type III expression by microRNA-143 (miR-143). (a) Collagen type III expression was assessed by immunohistochemical analysis of scirrhous type gastric cancer (GC) tissue. (b) Collagen type III mRNA expression levels were evaluated in GC and fibroblasts. Normal gastric fibroblasts and cancer-associated fibroblasts (NF-38 and CaF-38, respectively) were transfected with negative control miRNA or precursor miR-143 or miR-143 inhibitor, and (c) quantitative RT-PCR, (d) Western blot, and (e) cell staining were carried out for collagen type III expression. Scale bars: 50 μm. (f) Proliferation activity after coculture of HSC-44PE GC cells and CaF-38 with miR-143 inhibitor or negative control. Proliferation activity of HSC-44PE was assessed by percentages of BrdU/CAM5.2-positive cells. (g) Proliferation activity after coculture of HSC-44PE and CaF-38 with COL3A1 siRNA or negative control. Collagen type III expression level was determined by Western blot analysis. Results are mean ± SE of triplicate measurements. *P < 0.05.

Fig. 4

Effect of transforming growth factor-β (TGF-β) on microRNA-143 (miR-143) and collagen type III expression. (a) Quantitative RT-PCR analysis of miR-143 and collagen type III mRNA in normal gastric fibroblasts (NF-38) and cancer-associated fibroblasts (CaF-38) after the indicated times of TGF-β1 treatment. (b) MicroRNA-143 expression levels in NF-38 and CaF-38 in the absence or presence of TGF-β1 with or without miR-143 inhibitor. (c) Collagen type III mRNA expression levels in NF-38 and CaF-38 in the absence or presence of TGF-β with or without miR-143 inhibitor. (d) Expression levels of TGF-β family signal components were determined by Western blot analysis. (e) Quantitative RT-PCR analysis of miR-143 and collagen type III mRNA in NF-38 and CaF-38 with SMAD4 siRNA. Results are mean ± SE of triplicate measurements. *P < 0.05.

Fig. 5

Relation between microRNA-143 (miR-143) expression and patient prognosis. (a) Expression levels of miR-143 in formalin-fixed paraffin-embedded gastric cancer (GC) tissue samples (n = 68) were measured by quantitative RT-PCR analysis. (b) Cancer-specific survival of 68 patients with GC based on expression levels of miR-143 was examined. The expression levels of miR-143 were divided into two groups, high and low expression of miR-143, based on one-third of the miR-143 expression level (cut-off line = one-third of miR-143 expression level in this group).