Down-regulation of Smad4 enhances proliferation and invasion of colorectal carcinoma HCT116 cells and up-regulates Id2

Abstract

The aim of this study was to determine whether the suppression of Smad4 by short hairpin RNA (shRNA) regulates the proliferation and invasion of colorectal carcinoma HCT116 cells and Id2 expression. The Smad4-shRNA expression vectors were constructed and stably transfected to HCT116 cells. The expression of mRNA and protein of Smad4 and Id2 was detected using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Cellular proliferation inhibitory activity was determined by methyl thiazolyl tetrazolium (MTT) assay. Transwell assay was used to detect the effect of the inhibition of Smad4-shRNA on migration and invasion. The Smad4-shRNA vector, which inhibited Smad4 expression, was constructed and successfully transfected to HCT116 cells. The levels of mRNA and protein expression of Smad4 were markedly decreased following transfection of shRNA compared with the control groups (P<0.05). The abilities of proliferation, migration and invasion were increased following transfection of shRNA (P<0.05). The expression of Id2 was increased following transfection of shRNA (P<0.05). For the Smad4-down-regulated HCT116 cells, treated with or without BMP7 (25 ng/ml), no difference was found. shRNA-mediated silencing of Smad4 was able to enhance the abilities of proliferation, migration and invasion in the HCT116 cell line. Therefore, Smad4 may act as a tumor-suppressor gene in colorectal carcinoma.