DNMT1 blocks SOX21-repressed CKS2 transcription to promote gastric cancer progression

Abstract

Background: The dysregulation of SOXs is related to tumor invasion, metastasis, proliferation, apoptosis, and epithelial-mesenchymal transition. This research sought to investigate the function and mechanisms of SOX21 in gastric cancer (GC).

Methods: Multiple databases were included to determine the hub transcription factors in GC. In addition, RT-qPCR and Western blot were used to validate gene expression in tissues from GC patients. CCK-8, EdU, colony formation, wound healing, Transwell assays, and a xenograft tumor model were used to determine the function of SOX21 in GC. The targets of SOX21 were predicted and verified using ChIP, dual-luciferase reporter, and functional assays. SOX21 DNA methylation in GC cells was determined by qMSP. Rescue experiments were carried out in GC cells with DNMT1 silencing alone or in combination with SOX21 silencing.

Results: SOX21 was downregulated in GC tissues and cells. Ectopic expression of SOX21 inhibited cell growth, invasion, and migration, and induced apoptosis of GC cells. CKS2 was a target of SOX21, and overexpression of CKS2 promoted cell viability and mobility in GC cells overexpressing SOX21. The downregulation of SOX21 was related to the DNA hypermethylation catalyzed by DNMT1. The silencing of SOX21, by contrast, overturned the anti-tumor effects of sh-DNMT1 in vitro and in vivo.

Conclusion: Our data showed that DNMT1 overexpression upregulated CKS2 expression via hypermethylation of SOX21, thus promoting GC cell proliferation and growth, indicating that the DNMT1/SOX21/CKS2 axis could be a target for GC treatment.

Supplementary Information: The online version contains supplementary material available at 10.1186/s12885-025-14577-z.

Keywords: CKS2; DNMT1; Gastric cancer; Hypermethylation; SOX21.

Fig. 1

The hub TF SOX21 is downregulated in GC tissues and cell lines. A Differentially expressed genes in the GSE13911, GSE118916, and GSE208099 datasets. B The intersection of differentially expressed genes in the three datasets, GC-related genes in the Genecards database, and the human TF list. C The PPI network of 16 differentially expressed TF and the hub 10 genes. D The expression of SOX21 in the STAD-UALCAN dataset. E The mRNA expression of SOX21 in adjacent and tumor tissues in GC patients was examined using RT-qPCR (n = 13). F The protein expression of SOX21 in adjacent and tumor tissues in GC patients was analyzed using western blot analysis (n = 13). Paired t-tests were used to compare the data between the two groups. Data are expressed as means ± standard errors of the means

Fig. 2

Overexpression of SOX21 inhibits proliferation, migration, and invasion and promotes apoptosis in GC cells. A The protein expression of SOX21 in GC cells and GES-1 cells was examined using western blot analysis. B The mRNA expression of SOX21 in GC cells after infection with oe-NC or oe-SOX21 was examined using RT-qPCR. C The protein expression of SOX21 in GC cells after infection with oe-NC or oe-SOX21 was examined using western blot analysis. D The OD value of GC cells was read at 0, 24, 48, and 72 h using the CCK-8 assay. E The proliferation of GC cells was examined using colony formation assays. F The wound healing rate of GC cells at 12 h and 24 h after scratches. G The migration and invasion of GC cells were examined using the Transwell assay. H Detection of apoptosis in GC cells by TUNEL assay. Unpaired t-tests were used to compare the data between two groups, and ANOVA and Tukey’s post hoc test were used to compare the data between multiple groups. Data are expressed as means ± standard errors of the means of three independent experiments

Fig. 3

Overexpression of SOX21 inhibits GC growth in vivo. A The representative images of subcutaneous xenograft tumors in nude mice and the tumor growth curve within 25 d. B The weight of tumors formed by AGS cells with oe-NC or oe-SOX21 at day 25. C The expression of SOX21 in the tumor tissues was examined using western blot analysis. D The representative immunohistochemical images and positive cells of Ki67 and cleaved-caspase-3 in the tumor tissues formed by AGS cells with oe-NC or oe-SOX21. Unpaired t-tests were used to compare the data between two groups, and ANOVA and Tukey’s post hoc test were used to compare the data between multiple groups. Data are expressed as means ± standard errors of the means (n = 6)

Fig. 4

High expression of SOX21 transcriptionally suppresses CKS2 expression in GC cells. A The intersection of differentially expressed genes in the three datasets and GC-related genes in the Genecards database. B The KEGG enrichment analysis of 240 differentially expressed genes in the three datasets and GC-related genes in the Genecards database. C The PPI network of genes is enriched in the hsa05200: Pathways in cancer. D The expression of CKS2 in the STAD-UALCAN database. E The binding relation between SOX21 and CKS2 was predicted on the JASPAR website. F The negative correlation between SOX21 and CKS2 was predicted using the GEPIA database. G The mRNA expression of CKS2 in adjacent and tumor tissues in GC patients was examined using RT-qPCR (n = 13). H The protein expression of CKS2 in GC cells and GES-1 cells was examined using western blot analysis. I The binding relation between SOX21 and the CKS2 promoter was verified using a ChIP assay. J The binding relation between SOX21 and the CKS2 promoter was examined using luciferase assays. K The mRNA expression of CKS2 in GC cells after infection with oe-NC or oe-SOX21 was examined using RT-qPCR. L The protein expression of CKS2 in GC cells after infection with oe-NC or oe-SOX21 was examined using western blot analysis. Paired or unpaired t-tests were used to compare the data between two groups, and ANOVA and Tukey’s post hoc test were used to compare the data between multiple groups. Data are expressed as means ± standard errors of the means of three independent experiments

Fig. 5

Overexpression of CKS2 impairs the ability of SOX21 to inhibit the proliferation of GC cells. A The mRNA expression of CKS2 in GC cells infected with oe-SOX21 + oe-NC or oe-SOX21 + oe-CKS2 was examined using RT-qPCR. B The protein expression of CKS2 in GC cells infected with oe-SOX21 + oe-NC or oe-SOX21 + oe-CKS2 was examined using western blot analysis. C The proliferation of GC cells was examined using colony formation assays. D The EdU-positive cells in GC cells overexpressing both SOX21 and CKS2. E The protein expression of E-Cadherin, N-Cadherin, and Vimentin in GC was examined using western blot analysis. F The migration and invasion of GC cells were examined using the Transwell assay. G Detection of apoptosis in GC cells by TUNEL assay. Unpaired t-tests were used to compare the data between two groups, and ANOVA and Tukey’s post hoc test were used to compare the data between multiple groups. Data are expressed as means ± standard errors of the means of three independent experiments

Fig. 6

High expression of DNMT1 promotes DNA methylation of the SOX21 promoter. A Prediction of CpG islands in the SOX21 promoter region using the MethPrimer database. B The methylation of the SOX21 promoter region was predicted on the Mexpress database. C The correlation between DNMT3A, DNMT1, DNMT3B, DNMT3L, and SOX21 methylation levels in GC from the Meth450 platform in the LinkOmics database using Spearman analysis. D DNMT1 expression was predicted in the STAD-UALCAN database. E The protein expression of DNMT1 in adjacent and tumor tissues in GC patients was examined using western blot analysis (n = 13). F The protein expression of DNMT1 in GC cells and GES-1 cells was examined using western blot analysis. G The mRNA expression of DNMT1 in GC cells infected with shRNAs targeting DNMT1 was examined using RT-qPCR. H The protein expression of DNMT1 in GC cells infected with shRNAs targeting DNMT1 was examined using western blot analysis. I The methylation level of the SOX21 promoter in GC cells infected with sh-NC or sh-DNMT1 #2 was examined using the MSP assay. J The binding relation between DNMT1 and the SOX21 promoter was verified using a ChIP assay. K The binding relation between DNMT1 and the SOX21 promoter was examined using luciferase assays. L The mRNA expression of SOX21 in GC cells infected with sh-DNMT1 #2 was examined using RT-qPCR. M The protein expression of SOX21 in GC cells infected with sh-DNMT1 #2 was examined using western blot analysis. Paired or unpaired t-tests were used to compare the data between two groups, and ANOVA and Tukey’s post hoc test were used to compare the data between multiple groups. Data are expressed as means ± standard errors of the means of three independent experiments

Fig. 7

Silencing of SOX21 abates the anti-proliferative and pro-apoptotic properties of sh-DNMT1 on GC cells. A The mRNA expression of SOX21 in GC cells infected with sh-DNMT1 + shRNAs targeting SOX21 was examined using RT-qPCR. B The protein expression of SOX21 in GC cells infected with sh-DNMT1 + shRNAs targeting SOX21 was examined using western blot analysis. C The EdU-positive GC cells after infection. D The OD value of GC cells was read at 0, 24, 48, and 72 h using the CCK-8 assay. E The migration and invasion of GC cells were examined using the Transwell assay. F Detection of apoptosis in GC cells by TUNEL assay. ANOVA and Tukey’s post hoc test were used to compare the data between multiple groups. Data are expressed as means ± standard errors of the means of three independent experiments

Fig. 8

DNMT1/SOX21/CKS2 axis is involved in GC progression in vivo. A The representative images of subcutaneous xenograft tumors in nude mice and the tumor growth curve within 25 d. B The weight of tumors formed by AGS cells with sh-NC, sh-DNMT1 #2 + sh-NC, sh-DNMT1 #2 + sh-SOX21 #3, oe-SOX21 + oe-NC, or oe-SOX21 + oe-CKS2 at day 25. C DNMT1, SOX21, and CKS2 expression in the tumor tissues was examined using western blot analysis. D The representative immunohistochemical images and positive cells of Ki67 and cleaved-caspase-3 in the tumor tissues formed by AGS cells. ANOVA and Tukey’s post hoc test were used to compare the data between multiple groups. Data are expressed as means ± standard errors of the means (n = 6)