Zyxin inhibits the epithelial-mesenchymal transition process in gastric cancer by upregulating SIRT1

Abstract

Tumor development relies on the stemness of cancer stem cells, which is regulated by environmental cues. Previous studies have shown that zyxin can inhibit the expression of genes for embryonic stem cell status. In the present study, the expression levels of zyxin protein in cancer tissues and adjacent noncancerous tissues from 73 gastric cancer patients with different clinical stages were analyzed by Western blot. We showed that the relative expression levels of zyxin in gastric cancer tissues (cancer tissues/adjacent tissues) were significantly downregulated in advanced clinical stages. Overexpression of zyxin inhibited the stemness and epithelial-mesenchymal transition (EMT) processes in gastric cancer cells. Zyxin also inhibited the proliferation, migration, and invasion but increased the sensitivity of cancer cells to drugs. Overexpression of zyxin in MKN45 cells inhibited tumor growth in nude mice. We show that the interactions between zyxin and SIRT1 led to the upregulation of SIRT1, reduced acetylation levels of histone H3 K9 and K23, decreased transcription levels of SNAI 1/2, and inhibition of the EMT process. This study demonstrated that zyxin negatively regulates the progression of gastric cancer by inhibiting the stemness of cancer stem cells and EMT. Our findings shed new light on the pathogenesis of gastric cancer.

Keywords: EMT;SIRT1; acetylation; gastric cancer; zyxin.

FIGURE 1

The relative expression of zyxin (cancer tissue/paracancerous tissue) was significantly decreased in patients with advanced gastric cancer. (A) Western blot analysis of relative expression of zyxin in gastric cancer patient samples at different clinical stages. T represents the gastric cancer area, N represents the adjacent noncancerous area. (B and C) Relative expression of 73 pairs of gastric cancer patient samples. Relative expression = the normalized value of cancer tissue/the normalized value of adjacent tissue. *p < 0.05, ***p < 0.001.

FIGURE 2

The expression of cancer stem cell marker, EMT and zyxin in gastric cancer cell lines MKN45, N87, and AGS. (A–C) RT‐qPCR analysis of CD44, OCT4, and NANOG mRNA. (D) Immunofluorescence staining of EMT markers E‐Cadherin and vimentin. Scale bar: 50 μm. (E) RT‐qPCR analysis of zyxin mRNA levels. (F) Western blot analysis of zyxin. (G) Immunofluorescence staining of zyxin. Scale bar: 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3

Zyxin overexpression inhibits the expression of EMT‐related markers. (A and B) RT‐qPCR and Western blot to verify the overexpression of zyxin in MKN45 cells. (C) RT‐qPCR analysis of the mRNA expression levels of E‐Cadherin and vimentin. (D) Western blot analysis of the expression of E‐Cadherin and vimentin. (E) Immunofluorescence staining of E‐Cadherin and vimentin in MKN45‐Zyxin and control MKN45‐Lv5 cells, scale bar: 100 μm. (F) RT‐qPCR analysis of E‐Cadherin and vimentin mRNA expression levels in N87‐Zyxin and control N87‐Lv5 cells. (G) RT‐qPCR analysis of EMT‐related genes in MKN45‐Zyxin and control MKN45‐Lv5 cells. Blue: MKN45‐Lv5, Red: MKN45‐Zyxin. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4

Overexpression of zyxin downregulates CD44 and upregulates CD24. (A) RT‐qPCR analysis of CD44 and OCT4 in N87‐Zyxin cells and control N87‐Lv5 cells. (B) RT‐qPCR analysis of CD44 and OCT4 in MKN45‐Zyxin cells and control MKN45‐Lv5 cells. (C) flow cytometric analysis of CD44 and CD24. (D) immunofluorescence staining of CD44 and CD24 in MKN45‐Zyxin and control MKN45‐Lv5 cells. Scale bar: 100 μm. (E) The Hoechst staining and flow cytometry were used to analyze the side population proportion in MKN45‐Lv5 and MKN45‐Zyxin cells. **p < 0.01.

FIGURE 5

Zyxin overexpression inhibits the migration and invasion of gastric cancer cells. (A and B) Transwell assay to assess the migration and invasion ability of MKN45‐Zyxin and control MKN45‐Lv5 cells, scale bar: 50 μm. (C) The migration ability of N87‐Zyxin and control N87‐Lv5 cells was analyzed by wound‐healing assay. (D) The wound width was quantified by Image J software to show the migration ability of cells. (E) Peritoneal tumor development at varying time points in nude mice following injection with MKN45‐Lv5 or MKN45‐Zyxin.**p < 0.01, ***p < 0.001.

FIGURE 6

Zyxin overexpression inhibits the growth and drug resistance of gastric cancer cells. (A) The ability of MKN45‐Zyxin and control MKN45‐Lv5 cells to proliferate in vitro was analyzed by CCK8 reagent. (B) The anchorage‐independent growth of MKN45‐Zyxin and control MKN45‐Lv5 cells was detected by using a soft agar colony formation assay. The scale bar in the left panel represents 2 mm, and the scale bar in the right panel represents 100 μm. (C) The sensitivity of MKN45‐Zyxin and control MKN45‐Lv5 cells to the chemotherapeutic drugs 5‐FU (4 mM) and (D) etoposide (80 μM) was analyzed by CCK8 reagent. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 7

Zyxin overexpression inhibits tumor growth in nude mouse xenograft model. (A) MKN45‐Zyxin and control MKN45‐Lv5 cells were injected subcutaneously in nude mice. The tumor volumes were measured on day 21. (B and C) On day 21, the tumors were taken out, excised, and weighed. N = 6/group. (D) Immunohistochemical staining was performed using an anti‐CD44 antibody. Six individual panels represent tumors from six different nude mice in the same group. Scale bar: 20 μm. (E and F) The image of tumor cell gradient dilution subcutaneous inoculation tumorigenesis experiment. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 8

Zyxin inhibits EMT by upregulating SIRT1. (A and B) RT‐qPCR and Western blot analysis of the expression of SIRT1 in MKN45‐Zyxin and control MKN45‐Lv5 cells; (C) Western blot analysis of zyxin and SIRT1 in protein products immunoprecipitated by antibodies against HA‐tag and SIRT1 or control IgG. Left panel: proteins were pulled down by HA antibody, then analyzed by Western blot using antibodies against zyxin or SIRT1; right panel: proteins were pulled down by SIRT1 antibody, then analyzed by Western blot using antibodies against zyxin or SIRT1. (D) Immunofluorescence staining of zyxin and SIRT1 in MKN45 cells. Scale bar: 50 μm. (E) RT‐qPCR analysis of the relative expression of HDAC1 mRNA in MKN45‐Zyxin and control MKN45‐Lv5 cells. (F) Western blot analysis of acetylation levels of H3 K9, K14, K23, and K27 sites in MKN45‐Zyxin and control MKN45‐Lv5 cells. (G) RT‐qPCR detection of the relative mRNA expression levels of SNAI1 and SNAI2 in MKN45‐Lv5, MKN45‐Zyxin, and MKN45‐Zyxin cells treated with NAM. *p < 0.05, **p < 0.01, ***p < 0.001.