Figure 1.
Expression of epithelial–mesenchymal transition (EMT)-related genes and programmed cell death factor 4 (PDCD4) were regulated in human gastric cancer tissues.
(A) Hematoxylin and eosin staining confirmed preoperative diagnosis and histological grade. Representative immunohistochemical staining of TWIST1, SNAIL, PDCD4, vimentin and E-cadherin distinct in adjacent normal tissue and high differentiation cancer tissue and middle/low differentiation cancer tissue. (B–E) Quantification of TWIST1-, SNAIL-, PDCD4-, vimentin- and E-cadherin- expression in normal and cancer tissues. The total number of samples is 30 and each point represents 1 sample. Horizontal medium bar is mean and whiskers are SD. *P<0.05 and **P<0.01 versus control.
Table 1.
Association between TWIST1 or PDCD4 expression and clinicopathological features in gastric cancer patients.
Table 2.
Association between expression of Twist1 or PDCD4 in gastric cancer.
Figure 2.
Expression of TWIST1, SNAIL, PDCD4, vimentin and E-cadherin were regulated by H. pylori cytotoxin-associated gene A (CagA) in gastric cancer cells.
CagA full-length plasmid was transfected into AGS, SGC-7901 and BGC-823 cells for 48 h,and cells were harvested for mRNA and western blot analysis. (A–C) Quantitative RT-PCR analysis of mRNA level of PDCD4, EMT-related transcription factors (TWIST1 and SNAIL) and cell markers(vimentin and E-cadherin)in 3 gastric cancer cell lines. All values were normalized to β- actin mRNA in the same sample. (D) Western blot analysis of protein level of CagA, TWIST1, SNAIL, PDCD4, vimentin and E-cadherin in control or CagA-transfected cells. (E–G) Western blot densitometry was quantified (ImageJ) and the level of the indicated protein was normalized to β-actin. Data shown represent mean±SD of 3 independent experiments. * P<0.05,** P<0.01 versus control.
Figure 3.
PDCD4 regulated EMT-related transcription factors and cell markers in gastric cancer cell lines.
Plasmids of pEGFP-C1 and pEGFP-C1-PDCD4 were respectively transfected into AGS, SGC-7901 and BGC-823 cells for 48 h, and then cells were harvested for isolating total cellular RNA or protein. (A–C) qRT-PCR analysis of mRNA level of TWIST1, SNAIL, PDCD4, vimentin and E-cadherin in AGS, SGC-7901 and BGC-823 cells. All values were normalized to β-actin mRNA in the same sample. (D) Western blot analysis of 5 target proteins expression in control cells and PDCD4 overexpression cells. (E–G) Western blot results were quantified (ImageJ) and the level of the target protein was normalized to β-actin. Data are mean±SD from 3 independent experiments. * P<0.05,** P<0.01 versus pEGFP-C1 con.
Figure 4.
H. pylori CagA promoted EMT in part by regulating PDCD4.
All experiments were divided into three groups: 1) pCDNA3.1+pEGFP-C1 (control), 2) pCDNA3.1-CagA+pEGFP-C1, 3) pCDNA3.1-CagA+pEGFP-C1-PDCD4. According to various groups, the corresponding plasmids were transfected into AGS, SGC-7901 and BGC-823 cells. Then cells were harvested for mRNA and western blot analysis. (A–C) qRT-PCR analysis of mRNA level of TWIST1, SNAIL, PDCD4, vimentin and E-cadherin in gastric cancer cell lines. All values were normalized to β- actin mRNA in the same sample. (D) Western blot analysis of CagA, TWIST1, SNAIL, PDCD4, vimentin and E-cadherin protein expression in all cells. (E–G) Western blot results were quantified (ImageJ) and normalized to β-actin. Data shown represent mean±SD from 3 independent experiments. * P<0.05, ** P<0.01 versus con, ΔP<0.05,ΔΔP<0.01 versus pEGFP + CagA.
Figure 5.
Cell migration assays of PDCD4 overexpression downregulating CagA-induced EMT biological phenomena.
(A) The experiment was divided into three groups: 1) pCDNA3.1+pEGFP-C1 (control), 2) pCDNA3.1-CagA+pEGFP-C1, 3) pCDNA3.1-CagA + pEGFP-C1-PDCD4.The corresponding plasmids were transfected into SGC-7901 cells according to various groups. The invasion and metastasis ability of cells was analyzed by matrigel invasion assay. (B) Quantitative the migrated cell numbers of SGC-7901 in three groups. Data shown represent mean±SD from 3 experiments. * P<0.05 versus con, ΔP<0.05 versus pEGFP + CagA.
Figure 6.
Schematic representation of the signaling pathways involved in the H. pylori CagA -induced epithelial-mesenchymal transition in gastric cancer cells.
H. pylori virulence factors CagA suppressed PDCD4 expression in three gastric cancer cell lines, while the decline of PDCD4 could accelerate transcription factor TWIST1 expression. And then the latter regulates epithelial cell marker (E-cadherin) and mesenchymal cell marker (vimentin). Ultimately,epithelial-mesenchymal transition in gastric cancer cells was induced in the process.