Expression level of microRNA-200c is associated with cell morphology in vitro and histological differentiation through regulation of ZEB1/2 and E-cadherin in gastric carcinoma

Abstract

Scirrhous type gastric cancer is characterized by diffuse infiltration of poorly differentiated adenocarcinoma cells and poor prognosis. Although association of poorly differentiated histology with reduction in E-cadherin expression, as well as association of microRNA (miR)-200c with E-cadherin through regulation of ZEB1/2, has been reported, participation of miR-200c in gastric carcinogenesis is not fully understood. We used 6 cell lines originating from gastric cancers, and investigated levels of miR-200c along with its target mRNAs ZEB1/2 and E-cadherin by qRT-PCR. ZEB1 and E-cadherin protein expression was also assessed via western blotting. Furthermore, we investigated the expression levels of miR‑200c by in situ hybridization, along with the expression of ZEB1 and E-cadherin by immunohistochemistry, in 97 gastric adenocarcinoma tissues. Inverse correlation between miR‑200c and ZEB1 levels were obtained by qRT-PCR in cell lines (P<0.05). Cell lines with low miR-200c and high ZEB1 exhibited low E-cadherin expression in both qRT-PCR and western blotting, and exhibited spindle-shaped morphology, in contrast to round cell morphology in those cell lines with high miR-200c levels. Inverse correlations were also obtained between miR-200c and ZEB1 as well as between ZEB1 and E-cadherin levels in tissue samples (P<0.001). Cancer tissues with low miR-200c, high ZEB1, and low E-cadherin expression were associated with poorly differentiated histology, in contrast to tubular form in cancers with high miR-200c expression levels (P<0.001). Our data revealed that downregulation of miR-200c primarily regulated cell morphology by downregulation of E-cadherin through upregulation of ZEB1, leading to poorly differentiated histology in gastric cancer.

Figure 1.

The level of miR-200c and respective target mRNAs in gastric carcinoma cell lines. (A) Gastric carcinoma cell lines were arranged according to the expression level of miR-200c. The expression levels of (B) ZEB1, (C) ZEB2 and (D) E-cadherin expression in gastric carcinoma cell lines arranged according to the level of miR-200c expression.

Figure 2.

Expression of ZEB1, E-cadherin and β-actin proteins in gastric carcinoma cell lines, presented by western blot analysis performed on 10 µg of the cell extract.

Figure 3.

Six gastric carcinoma cell lines arranged according to expression the level of miR-200c. This order is compared with the origin (primary cancer or metastatic cancer), differentiation state (tubular or poorly differentiated), and shape (round/intermediate/spindle) of the cell lines.

Figure 4.

Representative photomicrographs including in situ hybridization and immunohistochemistry. (A and B) Staining with H&E and staining for (C and D) miR-200c, (E and F) ZEB1 and (G and H) E-cadherin in (A, C, E and G) tubular adenocarcinoma and (B, D, F and H) poorly differentiated adenocarcinoma. Original magnification, ×200. (A) Left side are normal glands, right side is carcinoma. (B) Left side is carcinoma, right side is normal foveolar epithelium. (C) Cytoplasmic granular staining in tubular adenocarcinoma was stronger than that found in normal glands. (D) Cytoplasmic granular staining in poorly differentiated adenocarcinoma was weaker than that observed in normal epithelium. (E) Nuclear immunostaining was negative in tubular adenocarcinoma. (F) Nuclear immunostaining was weak but positive in poorly differentiated adenocarcinoma. (G) Cytomembranous immunostaining in tubular adenocarcinoma was as strong as that found in normal glands. (H) Cytomembranous immunostaining in poorly differentiated adenocarcinoma was weaker than that observed in normal epithelium.

Figure 5.

Upregulation/downregulation of miR-200c affects cell morphology and histology.