https://orcid.org/0009-0002-3300-3649
Figures & data
Figure 1. WYC-209 inhibited the cell survival, proliferation, colony growth, and motility of GC cells.
(a) The cell survival of GES-1 under 0, 1, 2, 4, 6, 8, 10 μM of WYC-209 treatment, HGC-27 under 0, 1, 2, 4, 6, 8, 10 μM of WYC-209 treatment and AGS cells under 0, 8, 16, 32, 40 μM of WYC-209 treatment was examined by MTT assay. The histogram represents the quantified results, as below. (b) The cell proliferation of HGC-27 and AGS cells under 8 μM of WYC-209 treatment for 24 h was examined by MTT assay. (c) The colony growth ability of HGC-27 and AGS cells under 8 μM of WYC-209 treatment was examined by colony formation assay. (d) The migration and invasion capacity of HGC-27 and AGS cells under 8 μM of WYC-209 treatment for 24 h was examined by transwell experiments. Results were analyzed based on three repeatable experiments.**P < .01, *p < .05.
Figure 2. WYC-209 suppressed EMT and tumor progression.
(a) The EMT-related protein levels expressed in HGC-27 and AGS cells were detected by WB assay after 8 μM of WYC-209 treatment for 24 h. The histogram represents the quantified results, as below. (b) The EMT-related mRNA levels expressed in HGC-27 and AGS cells were detected by qRT-PCR after 8 μM of WYC-209 treatment for 24 h. (c) The phosphorylation of STAT3, AKT, smad2, smad3, and β-catenin expression levels in HGC-27 and AGS cells with 8 μM of WYC-209 treated for 24 h were tested by WB assay. Results were analyzed based on three repeatable experiments. **P < .01, *p < .05.
Figure 3. WYC-209 induced the heterotypic down-regulation of WNT4.
(a, b&c) The total RNA of HGC-27 cells was extracted after 8 μM of WYC-209 treatment for 24 h, and then RNA-seq was performed to analyze differentially expressed genes (DEGs). The heatmap and the volcano plot were generated by the R package. (d&e) KEGG and GO analysis were carried out for the enriched biological processes and pathways. (f&g) FPKM and qRT-PCR validation of DEGs in HGC-27 cells based on RNA-seq results. (h) The qRT-PCR validation of WNT4 expression level in HGC-27 and AGS cells after 8 μM of WYC-209 treatment for 24 h. The histogram represents the quantified results, as below. (i) The WB validation of WNT4 expression level in HGC-27 and AGS cells after 8 μM of WYC-209 treatment for 24 h. Results were analyzed based on three repeatable experiments. **P < .01, *p < .05.
Figure 4. Overexpressing WNT4 breached the inhibitory effect of WYC-209 on cell proliferation, colony-forming, and motility.
(a&b) To investigate the role of WNT4, the WNT4 overexpressed HGC27 and AGS cells were constructed. After qRT-PCR validation of WNT4 expression level, 8μM of WYC-209 was used to treat HGC27 and AGS cells with WNT4 overexpressed or not for 24h. Then MTT, colony formation, and (c) transwell assay were performed to detect the cell proliferation, colony-forming, migration, and invasion abilities. The histogram represents the quantified results. Results were analyzed based on three repeatable experiments. **P<.01, *p<.05.
Figure 5. EMT and tumor progression inhibited by WYC-209 were abrogated by overexpressing WNT4.
(A&B) 8 μM of WYC-209 was used to treat HGC27 and AGS cells with WNT4 overexpressed or not for 24 h, then WB was carried out to examine the EMT-related proteins levels, the phosphorylation of STAT3, AKT, smad2, smad3 and β-catenin expression level in HGC-27 and AGS cells. The histogram represents the quantified results. Results were analyzed based on three repeatable experiments.**P < .01, *p < .05.
Figure 6. WYC-209 inhibited GC progression by down-regulating WNT4 via RARα.
Table 1. Sequences of qRT-PCR primers.
Table 2. Information on the primary antibodies.
Data availability statement
All data generated or analyzed during this study are shown in this published article.