miR-644a Inhibits Cellular Proliferation and Invasion via Suppression of CtBP1 in Gastric Cancer Cells

Abstract

Epithelial-mesenchymal transition (EMT) is one of the most important mechanisms in the metastasis of various cancers, including gastric cancer (GC). In this study, we explored the putative significance of miR-644a and its role in EMT-mediated metastasis of GC. We first detected the expression of miR-644a in a cohort of 107 GC tissues using quantitative RT-PCR. The expression of miR-644a was suppressed in GC tissues and was associated with a later clinical stage and tumor metastasis. Restoring the expression of miR-644a could significantly suppress the migration and invasion of HGC-27 and SGC-7901 cells, which might be correlated to its suppressive effect on the EMT process. We also found that carboxyl-terminal-binding protein 1 (CtBP1) was a putative target gene of miR-644a in GC and might be involved in the suppressive effect. Collectively, through targeting CtBP1-mediated suppression of the EMT process, miR-644a might suppress the tumor metastasis of GC cells.

Figure 1

Expression level of miR-644a in gastric cancer tissues. (A) The expression level of miR644a was detected in 107 pairs of GC tissues using TaqMan quantitative RT-PCR. Data are shown as log₁₀ of relative ratio change of esophageal cancer tissues relative to normal tissues. (B) The mean level of miR-644a expression in GC cancerous and noncancerous tissues. The expression of miR-644a was suppressed in GC tissues. (C, D) The expression of miR-644a was suppressed in cancer tissues in patients at a later clinical stage (stage III + IV vs. stage I + II) and metastasis. The expression of miR-644a was normalized to small nuclear RNA U6. **p < 0.01 compared with scramble group.

Figure 2

The effects of miR-644a on GC cell proliferation and invasion. (A) The level of miR-644a expression was detected in HGC-27 and SGC-7901 cells upon transfection with miR-644a or scramble mimic by quantitative RT-PCR. (B, C) The effects of miR-644a on cell migration and invasion of HGC-27 and SGC-7901 cells were performed by Transwell assay upon transfection with miR-644a. (Left) Cells passing through the chambers. (Right) The relative number of cells passing through per field. (D) A cell proliferation assay of GC cells was performed after transfection with miR-644a or scramble mimic by using CCK-8. (E) Western blot analysis of the expression of epithelial and mesenchymal marker proteins in GC cells transfected with miR-644a or scramble mimic was performed. Upon transfection, the epithelial marker E-cadherin was increased, and the mesenchymal markers vimentin and N-cadherin were suppressed. (F) Immunofluorescence assays further explored the expression of markers mentioned above. *p < 0.05, **p < 0.01 compared with the scramble group.

Figure 3

CtBP1 is a putative target gene of miR-644a in GC cells. (A) Schematic representation of CtBP1 3′-UTR showing the putative miR-644a target site. (B) A quantitative RT-PCR assay was performed to detect the expression of CtBP1 upon transfection with miR-644a or scramble mimic. (C) Relative luciferase activity of the indicated CtBP1 reporter construct in GC cells, cotransfected with miR-644a or scramble mimic, is shown. (D) Western blot analysis of the expression of CtBP1 protein in HGC-27 and SGC-7901 cells transfected with miR-644a or scramble mimic was performed. **p < 0.01 compared with the scramble group.

Figure 4

Knockdown of CtBP1 suppressed the proliferation and invasion of GC cells. (A) The level of CtBP1 mRNA expression was detected in HGC-27 and SGC-7901 cells upon transfection with si-CtBP1 and relative control. (B) The expression of CtBP1 protein level as well as relative markers was detected. The expression of CtBP1 and its downstream genes N-cadherin, as well as vimentin, was suppressed, while the expression of E-cadherin and β-catenin was upregulated. (C) The proliferation was suppressed upon transfection with si-CtBP1. (D) The invasion was suppressed upon transfection with si-CtBP1. **p < 0.01 compared with the scramble group.