Epigenetic Silencing of STAT3-Targeted miR-193a, by Constitutive Activation of JAK/STAT Signaling, Leads to Tumor Progression Through Overexpression of YWHAZ in Gastric Cancer

Abstract

Purpose: The purpose of this study was to identify genes that were epigenetically silenced by STAT3 in gastric cancer.

Methods: MBDcap-Seq and expression microarray were performed to identify genes that were epigenetically silenced in AGS gastric cancer cell lines depleted of STAT3. Cell lines and animal experiments were performed to investigate proliferation and metastasis of miR-193a and YWHAZ in gastric cancer cell lines. Bisulfite pyrosequencing and tissue microarray were performed to investigate the promoter methylation of miR-193a and expression of STAT3, YWHAZ in patients with gastritis (n = 8) and gastric cancer (n = 71). Quantitative methylation-specific PCR was performed to examine miR-193a promoter methylation in cell-free DNA of serum samples in gastric cancer patients (n = 19).

Results: As compared with parental cells, depletion of STAT3 resulted in demethylation of a putative STAT3 target, miR-193a, in AGS gastric cancer cells. Although bisulfite pyrosequencing and epigenetic treatment confirmed that miR-193a was epigenetically silenced in gastric cancer cell lines, ChIP-PCR found that it may be indirectly affected by STAT3. Ectopic expression of miR-193a in AGS cells inhibited proliferation and migration of gastric cancer cells. Further expression microarray and bioinformatics analysis identified YWHAZ as one of the target of miR-193a in AGS gastric cancer cells, such that depletion of YWHAZ reduced migration in AGS cells, while its overexpression increased invasion in MKN45 cells in vitro and in vivo. Clinically, bisulfite pyrosequencing revealed that promoter methylation of miR-193a was significantly higher in human gastric cancer tissues (n = 11) as compared to gastritis (n = 8, p < 0.05). Patients infected with H. pylori showed a significantly higher miR-193a methylation than those without H. pylori infection (p < 0.05). Tissue microarray also showed a positive trend between STAT3 and YWHAZ expression in gastric cancer patients (n = 60). Patients with serum miR-193a methylation was associated with shorter overall survival than those without methylation (p < 0.05).

Conclusions: Constitutive activation of JAK/STAT signaling may confer epigenetic silencing of the STAT3 indirect target and tumor suppressor microRNA, miR-193a in gastric cancer. Transcriptional suppression of miR-193a may led to overexpression of YWHAZ resulting in tumor progression. Targeted inhibition of STAT3 may be a novel therapeutic strategy against gastric cancer.

Keywords: STAT3; YWHAZ; epigenetics; gastric cancer; miR-193a.

Figure 1

Identification of miR-193a as a STAT3-mediated hypermethylated target in gastric cancer cells. AGS gastric cancer cells infected with lentivirus expression control (AGS/shGFP) or shRNA against STAT3 (AGS/shSTAT3) were used to perform methylation and expression analysis. (A) Global methylation analysis of the parental AGS/shGFP or AGS/shSTAT3, by MBDcap-Seq. Left panel, heatmap showing differential methylated regions (DMR) in AGS/shSTA3 and AGS/shGFP cells. Right panel, representative histogram showing methylation level of chromosome region around miR-193a (chr17:29886200-29888000) in AGS control and STAT3 depleted cells. (B) Bisulfite sequencing of miR-193a methylation in AGS control and STAT3 depleted cells. Upper panel, schematic diagram showing the genomic map of miR-193a promoter, with the corresponding locations of CpG sites and the putative STAT3 binding site (SBE, blue box). The location of miR-193a (red line) and region for bisulfite pyrosequencing analysis (enlarged region) is also shown. Lower panel, scatter plot showing the methylation level of the seven CpG sites being interrogated in AGS control (AGS/shGFP) and STAT3 depleted cells (AGS/shSTAT3). (C) ChIP-PCR showing the binding of STAT3 to SBE1, SBE2 of the promoter region of miR-193a. Negative control region (Ctrl, -400 upstream of TSS) and positive control (GATA6) were also shown. Relative expression and methylation level of mature miR-193a in a panel of gastric cancer cell lines was also determined by (D) quantitative RT-PCR and (E) bisulfite pyrosequencing. Relative expression level of miR-193a in AGS cells treated with (F) epigenetic modifiers (DNMT inhibitor, 5aza, and/or HDAC inhibitor, TSA), (G) shRNA against STAT3 (shSTAT3), or (H) a specific STAT3 inhibitor (JSI-124). Each bar represents mean ± SD of duplicate experiments (*p < 0.05, **p < 0.01; ***p < 0.005).

Figure 2

Overexpression of miR-193a inhibits cell proliferation and migration in AGS gastric cancer cells. AGS cells were transfected with control or miR-193a overexpressing plasmid. (A) Relative expression of mature miR-193a in control (AGS/Control) or miR-193a (AGS/miR-193a) overexpressing AGS cells, as determined by quantitative real-time PCR. Proliferation and migration of AGS/Control and AGS/miR193a cells were determined by (B) colony formation assay and (C) wound healing assay. Right panel shows the quantitative analysis of the assay. Each bar represents mean ± SD of duplicate experiments (**p < 0.01).

Figure 3

Identification of YWHAZ as a novel miR193a target gene in gastric cancer. (A) Schematic diagram showing the experimental scheme of this study. AGS knockdown control (AGS/control) or STAT3 depleted cells (AGS/shSTAT3) were used to perform Illumina expression microarray. The scatter plot shows the fluorescence signal of each gene on the array in AGS/control vs AGS/shSTAT3. There were 1,125 genes showing expression changes of ≥1.5-fold. In additional, bioinformatic analysis using three microRNA databases was performed to predict potential miR-193a targets. There are 97 potential miR-193a targets. YWHAZ, a potential miR-193a target showing downregulation in AGS/shSTAT3 cells (as compared to control) and has been shown to be involved in metastasis, was selected for further analysis. (B) Relative expression of YWHAZ in AGS gastric cancer cells overexpressed with control or miR-193a-expressing plasmid. 3’UTR luciferase confirmed that miR-193a targets YWHAZ in (C1) AGS and (C2) MKN28 cells. (D) Relative expression of YWHAZ in a panel of gastric cancer cell lines. Each bar represents mean ± SD of duplicate experiments (*P < 0.05, **p < 0.01).

Figure 4

YWHAZ regulates proliferation, migration and invasion in gastric cancer cells. (A) Migration of AGS cells were infected with shRNA against GFP (shGFP, control) or YWHAZ (shYWHAZ) in AGS gastric cancer cells, as determined by wound healing assay. Right panel shows the quantitative analysis of the assay. MKN45 transfected control (pcDNA, MKNB45/Control) or YWHAZ expressing plasmid (MKN45/YWHAZ), were used to perform (B) colony formation assay, and (C) transwell invasion assay. Addition of TGF-β further enhanced the invasion ability of YWHAZ-overexpressing MKN45 cells. Quantitative analysis was shown in the right panel. Each bar represents mean ± SD of duplicate experiments (*P < 0.05, **p < 0.01; ***p < 0.005).

Figure 5

YWHAZ enhanced metastasis in MKN45 cells in vivo. MKN45 cells transfected with control (MKN45/Control) or plasmid expressing YWHAZ (MKN45/YWHAZ) were injected subcutaneously (s.c.) into nude mice (A–D) or intraperitoneally into NOD-SCID mice (E). (A) Representative images showing the tumor in an s.c. xenograft mouse model. Overexpression of YWHAZ demonstrated a similar tumor size (B, C) and weight (D) of the xenograft, as compared to control. (E) However, MKN45/YWHAZ cells demonstrated more intraperitoneal nodules (red arrow) as compared to control, after 5 weeks of injection (red arrow). H&E stain presented tumor cells obtained from the liver of NOD-SCID mice.

Figure 6

Infection of H. pylori is associated with miR-193a hypermethylation and increased YWHAZ expression in gastric cancer. (A, B) Scatter plot showing miR-193a methylation in patient tissue samples with gastritis (n = 8), and paired tumor adjacent normal and gastric cancer (n = 11). Red lines denote median. Infection of H.pylori is associated higher miR-193a methylation in gastritis and tumor adjacent normal. Immunohistochemistry of STAT3 and YWHAZ in gastric cancer tissue (n = 60) was performed using tissue microarray. (C) Scatter plot showing a positive trend between expression score of STAT3 and YWHAZ. (D) Tissues infected with H. pylori showed a higher expression of YWHAZ than those without H. pylori infection. (E) Representative photos showing low (STAT3^Lo, YWHAZ^Lo) and high (STAT3^Hi, YWHAZ^Hi) expression of STAT3 and YWHAZ in tissue microarray. (F) Kaplan-Meier analysis showed that patients with serum miR-193a methylation is associated with shorter overall survival as compared to samples without methylation (p < 0.05).