KLF8 involves in TGF-beta-induced EMT and promotes invasion and migration in gastric cancer cells

Abstract

Purpose: Krüppel-like factor 8 (KLF8), a downstream transcription factor of transforming growth factor-β1 (TGF-β1), has a role in tumorigenesis, tumor progress and epithelial-to-mesenchymal transition (EMT) induction. Recent studies mainly focused on its role in breast cancer and hepatocellular carcinoma; however, little is studied in gastric cancer. Here, we aim to explore whether KLF8 is involved in TGF-β1-induced EMT in gastric cancer cells.

Methods: Western blot and real-time PCR assays were used to detect the expression of KLF8, E-cadherin and vimentin in gastric cancer cell line SGC7901 treated with or without TGF-β1. The lentivirus-mediated RNA interference technique was used to knock down the expression of KLF8 in gastric cancer cell line SGC7901. In vitro, the ability of cell migration and invasion were measured by transwell and wound healing assays; the cell motility was detected by high content screening assay.

Results: TGF-β1 could induce EMT via down-regulating E-cadherin and up-regulating vimentin expression in gastric cancer cells. Further study found that TGF-β1 could induce KLF8 expression at the protein and mRNA levels in gastric cancer cells (P < 0.05). Western blot and real-time PCR assays found that small interference RNA (siRNA)-mediated KLF8 silence blocked TGF-β1-induced EMT-like transformation and subsequently reversed the loss of E-cadherin and gain of vimentin. In vitro, inhibition of KLF8 decreased TGF-β1-prompted cell migration, invasion and motility.

Conclusions: KLF8, a transcription factor, is involved in TGF-β1-induced EMT in gastric cancer cells and may be a novel therapeutic target for the treatment of gastric cancer.

Fig. 1

a Western blot assay showed that in four pairs of gastric cancer cell lines (SGC7901, BGC823, MKN28 and MKN45) treated with or without TGF-β1, the E-cadherin expression was down-regulated, but there were no changes in MKN28 and MKN45 cells. Vimentin expression was up-regulated in four pairs. b The mRNA level of E-cadherin was down-regulated, and the mRNA level of vimentin was up-regulated in SGC7901 cells. c Morphologic changes in SGC7901 and NC cells were treated with TGF-β1, but there were no changes in siKLF8 cells. d Cell immunofluorescence assay showed that E-cadherin decreased and β-catenin translocated into nucleus in SGC7901 and NC cells after TGF-β1 treatment

Fig. 2

Effect of TGF-β1 (10 ng/ml) and KLF8 siRNA on KLF8 expression in SGC7901 cells. a, b KLF8 expression in SGC7901 cells treated with or without TGF-β1(10 ng/ml). c, d The levels of KLF8 mRNA and protein were effectively suppressed by KLF8 siRNA in SGC7901 cells. e, f Effect of KLF8 siRNA on TGF-β1-induced KLF8 production in SGC7901 cells. The cells were treated with or without TGF-β1(10 ng/ml) for 24 h. In Western blot, β-actin was used as a control; in real-time PCR, GAPDH was used as a control. **P < 0.05 versus 0 h; ^# P < 0.05 versus 7901 or NC; *P < 0.05 versus &; ^& P < 0.05 versus ^; ^## P < 0.05 versus ^; ^## p > 0.05 versus * 7901, SGC7901 cells; NC negative control; siKLF8 KLF8-RNAi lentiviral vector (Lenti-siRNA/KLF8)

Fig. 3

Decreased KLF8 expression in gastric cancer cells is highly correlated with the increase in E-cadherin expression and the decrease in vimentin expression. The cells were stably transfected with lentivirus and then treated with or without TGF-β1 (10 ng/ml) for 24 h. a, b Correlation between the increase in KLF8 expression and the decrease in E-cadherin expression. c, d The increased vimentin expression in human gastric cancer cells. The expression of E-cadherin and vimentin was detected by Western blot and RT-PCR. *P < 0.05 versus &; ^& P < 0.05 versus ^; ^# P < 0.05 versus ^; ^# p > 0.05 versus *

Fig. 4

Role of KLF8 in invasive and migratory abilities of cells treated with or without TGF-β1 (10 ng/ml). a, b There was highly cell migratory ability in SGC7901 and NC cells treated with TGF-β1. c, d Cell invasion was examined with Matrigel-coated transwell chamber; there is low invasive ability in siKLF8 cells. e, f Confluent SGC7901 cells were wounded with a pipette tip and then treated with or without TGF-β1 (10 ng/ml). Cell migration to the wound area was monitored by microscopy for 48 h post-wound. *P < 0.05 versus &; ^& P < 0.05 versus ^; ^# P < 0.05 versus ^; ^# p > 0.05 versus *

Fig. 5

Role of KLF8 in the cell motility which was treated with or without TGF-β1 (10 ng/ml). Cell mobility was examined by Cellomics Array Scan VTI 1700 plus. The relative distance of cell mobility was calculated with this instrument. *P < 0.05 versus &; ^# P < 0.05 versus &; *P < 0.05 versus #