ZBTB7A decreased in GBM cells and patients
We investigated the RNA expression of ZBTB7A in GBM patients using various databases. Profiling of various tumor samples and normal brain (NB) tissues from the TCGA and GTEx projects in the GEPIA confirmed that ZBTB7A expression was lower in GBM and lower-grade gliomas than in NB tissues (Fig. 1a–c). Analysis of TCGA-GBM and TCGA-GBMLGG datasets using the GlioVis database revealed that ZBTB7A expression was significantly decreased in GBM, and it was confirmed that the expression of ZBTB7A decreased in high-grade tumors. (Fig. 1d, e). In addition, as a result of profiling the expression of ZBTB7A in the Freije dataset (GSE4412) through GEO, the expression of ZBTB7A significantly decreased in GBM, as was observed in the TCGA-GBMLGG dataset (Fig. 1f). Furthermore, the prognosis of patients with GBM was poor in the ZBTB7A-decreased patients with GBM (Fig. 1g), as a result of analyzing the Kaplan–Meier survival graph using the TCGA-GBMLGG dataset. To determine whether the expression of ZBTB7A was decreased in GBM tissues compared to NB tissues based on the results of analysis of various databases, we performed IHC using tissue microarrays (TMAs). The expression of ZBTB7A was significantly decreased in GBM tissues (Fig. 1h). When the expression of ZBTB7A was confirmed using the CCLE database before the in vitro experiment, ZBTB7A expression was relatively decreased in LN229, LN18, A172, and U343 cells, and U118 and U87 cells showed relatively increased ZBTB7A expression (Fig. 1i). In addition, when the CCLE database was used to analyze ZBTB7A protein expression in the GBM cell line, nearly identical results were obtained (Fig. 1j). Based on these results, the expression of ZBTB7A was decreased in both GBM and GBM patients. As a result of decreased expression of ZBTB7A mRNA and protein, the prognosis of GBM patients has worsened.
ZBTB7A knockdown increases GBM tumorigenesis
Using the ZBTB7A shRNA lentiviral plasmid, we examined the tumorigenesis of U87 cells, a cell line with relatively high expression of ZBTB7A (Supplementary Fig. 1a; Control shRNA and ZBTB7A shZBTB7A#1–6). This allowed us to determine the probable role of ZBTB7A in GBM development. In addition, we made ZBTB7A overexpressed in LN229 and U343 cells, which have relatively low ZBTB7A expression. To investigate GBM tumorigenesis in ZBTB7A-depleted U87 cells, we measured the protein and mRNA expression levels of ZBTB7A (Fig. 2a, Supplementary Fig.1b). When the expression of ZBTB7A was knockdown, cell migration was significantly increased in ZBTB7A-knockdown U87 cells (Fig. 2b), and tumor invasion and colony formation also increased in ZBTB7A-knockdown U87 cells compared with control U87 cells (Fig. 2c, d). Additionally, we performed a proliferation assay. As a result, cell proliferation significantly increased in ZBTB7A-knockdown U87 cells and increased in ZBTB7A-overexpressed U343 cells (Fig. 3e). Therefore, our results suggest that ZBTB7A acts as a tumor suppressor in GBM.
To gain insight into the mechanism of action of ZBTB7A, we performed mRNA sequencing analysis. Of the 1124 genes, 597 were upregulated and 527 were downregulated (DEG analysis) (Fig. 2f). We also performed gene ontology analysis of 1124 upregulated and downregulated genes. These results showed that multicellular organisms were enriched for genes involved in processes, cell motility, and the movement of cellular components (Fig. 2g). GSEA analysis revealed that all genes involved in the three processes were more enriched than those in the control group when ZBTB7A was knocked down in GBM cells (Fig. 2h), suggesting that ZBTB7A is generally involved in biological processes related to cell motility. Thus, we further confirmed whether knockdown of ZBTB7A plays an important role in the EMT process due to increased cellular motility. When ZBTB7A was knocked down in GBM cells, the EMT process was induced, and the related core genes were enriched (Fig. 2i, j), indicating that ZBTB7A knockdown activates GBM growth-related genes for the development and progression of GBM.
EPB41L5 is the target gene of ZBTB7A in GBM cells
Next, we analyzed the genes related to these three processes in ZBTB7A knockdown GBM cells. The upregulated genes overlapped as ZBTB7A was knocked down in three processes, and 18 genes were upregulated in all processes (Fig. 3a). The expression of 18 overlapping genes was confirmed using a heat map (Fig. 3b). Therefore, 18 genes were upregulated when ZBTB7A expression was low, suggesting that there is a reverse correlation between the expression of ZBTB7A and the 18 target genes. To confirm the mRNA sequencing data, the mRNA expression levels of 18 target genes were analyzed in ZBTB7A knockdown and overexpression cells using qPCR (Fig. 3c, d). In addition, correlation analysis was performed on the TCGA-GBM dataset to confirm that the 18 target genes obtained from our mRNA-seq data had a reverse correlation with ZBTB7A, even in patient samples. We showed that the reverse correlation of MDK, IL33, and EPB41L5 with ZBTB7A was confirmed for three of the 18 target genes (Fig. 3e, Supplementary Fig. 2a). Therefore, we examined whether the mRNA levels of MDK, IL33, and EPB41L5 increased in ZBTB7A shRNA-expressing GBM cells. As shown in Fig. 3f, the mRNA expression of MDK, IL33, and EPB41L5 was inversely correlated with that of ZBTB7A. In addition, the expression of EPB41L5 mRNA was significantly decreased in ZBTB7A overexpressed GBM cells, but not in MDK and IL33 cells (Fig. 3g). Additionally, we confirmed that ZBTB7A and EPB41L5 had a reverse correlation at the protein level in ZBTB7A knockdown U87 cells or ZBTB7A overexpressed LN229 and U343 cells, respectively (Fig. 3h, i, Supplementary Fig. 2b, c). Furthermore, the protein expression levels of ZBTB7A and EPB41L5 were inversely correlated in GBM cell lines (Fig. 3j). The expression of EPB41L5 in tissues of patients with GBM (GBM TMAs) was observed using IHC staining, and the expression of EPB41L5 increased in the high-grade GBM tissues (Fig. 3k, l). Taken together, ZBTB7A controls GBM tumorigenesis by regulating EPB41L5 expression.
ZBTB7A overexpression decreases GBM tumorigenesis through the transcriptional inhibition of EPB41L5
To confirm the previous results, we expressed ZBTB7A in LN229 and U343 cells, cells with relatively low expression levels of ZBTB7A. The protein and mRNA expression of ZBTB7A were measured in ZBTB7A-expressing LN229 and U343 cells (Fig. 4a, Supplementary Fig. 3a; overexpressed cells: pCDH-ZBTB7A and control cells: pCDH-CTR). To analyze the biological function of ZBTB7A in GBM cells, we performed various cell-based assays, such as cell migration, invasion, and colony-forming assay in ZBTB7A-overexpressed LN229 and U343 cells. As shown in Fig. 4b, cell migration was decreased in ZBTB7A-overexpressed GBM cells compared to that in control cells. The invasiveness and colony formation ability of GBM cells were significantly decreased in ZBTB7A-overexpressed GBM cells compared to control cells (Fig. 4c, d), indicating that ZBTB7A inhibits tumorigenesis in GBM cells.
To confirm that ZBTB7A had the same inhibitory effect on GBM cell migration, invasion, and colonization, control cells (pCDH-CTR) and ZBTB7A-expressing U343 cells (pCDH- ZBTB7A) were injected subcutaneously into nude mice and the tumor weight was monitored every 2 days for 6 weeks. As expected, tumor growth on ZBTB7A-expressing U343 cells was further reduced compared to that in control cells (Fig. 4e, Supplementary Fig. 3b). In addition, we confirmed whether the protein expression of ZBTB7A was normal in the ZBTB7A-expressing tumor using western blotting (Fig. 4f) and whether the tumor volume and weights were suppressed in ZBTB7A-expressing U343 cells compared to the control cells (Fig. 4g, h). The survival rate was also higher in ZBTB7A-expressing U343 cells than in the control group (Supplementary Fig. 3c).
We analyzed the target genes of ZBTB7A in ZBTB7A-knockdown cancer cells using the ChIP-Atlas database, and predicted that the target gene binds to suppress tumorigenesis as a transcription factor. We found that EPB41L5, a protein essential for cell migration and involved in EMT (Supplementary Fig. 3d). In a previous study, we reported that the expression of EPB41L5 induced by TGF-b1 promotes the migration and invasion of gastric cancer cells by TGF-b signaling34. Therefore, we conducted the following experiment to determine whether ZBTB7A, a transcription factor, regulates transcriptional activity by targeting EPB41L5 in GBM cells. The protein expression of EPB41L5 and ZBTB7A was observed in mouse tumor tissues injected with control U343 cells and ZBTB7A-expressing U343 cells using IHC analysis. The expression of EPB41L5 was significantly decreased in ZBTB7A-overexpressed tumor tissues (Fig. 4i, j). Based on these results, we suggest that ZBTB7A acts as a transcriptional repressor that inhibits tumorigenesis by targeting EPB41L5 in GBM.
ZBTB7A binds on the EPB41L5 promoter to repress the transcription activity
To investigate whether ZBTB7A and EPB41L5 are inversely correlated, we performed a promoter assay to confirm that ZBTB7A binds to the promoter of EPB41L5 as a transcription factor and negatively regulates its transcriptional activity. The binding motif sequence of ZBTB7A was confirmed using the JASPAR database, and the region where ZBTB7A binds was predicted in the 1 kb region (-900 to 100) of the EPB41L5 promoter through the EPD database (Supplementary Fig. 4a, b). We also obtained overlapping results from the JASPAR and EPD databases for four regions that were significant for ZBTB7A binding in the EPB41L5 promoter (Fig. 5a). In fact, the 1 kb region of the EPB41L5 promoter was cloned into the pGL4.21 vector to perform the luciferase assay, and the ZBTB7A ORF was cloned into the Myc-tagged pSG5 vector for co-transfection in U343 cells with relatively low ZBTB7A expression. ZBTB7A was transfected in a dose-dependent manner, and we confirmed a gradual decrease in the transcription activity of EPB41L5 (Fig. 5b). To analyze transcriptional activity, the 1 kb full-length EPB41L5 (-900 to 100: #1, -500 to 100: #2, -150 to 100: #3, and -40 to 100: #4) promoter regions were truncated. Furthermore, we measured transcriptional activity through the transfection of EPB41L5 promoter-deleted mutants (EPB41L5 promoter truncated constructs) in ZBTB7A-overexpressed U343 cells or control U343 cells. The transcriptional activity of EPB41L5 promoter wild-type/deletion mutants was significantly reduced in ZBTB7A-overexpressed U343 cells compared to the control group, and when the -150 to 100 promoter regions (#3) were truncated, the transcriptional activity was drastically reduced in the ZBTB7A-overexpressed U343 cells and control cells (Fig. 5c). Based on these results, we confirmed that the promoter region, including the EPB41L5 #3 (-150 to 100) position, was a significant ZBTB7A binding region.
To further confirm whether the EPB41L5 #3 (-150 to 100) position is the ZBTB7A binding region, the EPB41L5 promoter construct (#3-WT) or the specific binding sequence deleted form of the EPB41L5 promoter construct (#3-MT) was transfected in the ZBTB7A-overexpressed U343 cells or control cells, and the promoter activity was measured using a luciferase assay. The transcription activity of the ZBTB7A binding sequence deleted EPB41L5 promoter (#3-MT) was not significantly different between ZBTB7A-expressing U343 cells and control cells compared with the wild-type EPB41L5 promoter (#3-WT) (Fig. 5d). Therefore, we tested whether ZBTB7A inhibits transcription by binding to region #3 of the EPB41L5 promoter using a ChIP assay. ZBTB7A was significantly recruited to the #3 region of the EPB41L5 promoter in ZBTB7A-expressing U343 cells compared to control cells (Fig. 5e). Additionally, we investigated whether binding to the EPB41L5 promoter region through site-direct mutagenesis of R399L in the zinc finger domain of ZBTB7A. ChIP assay was performed in U343 cells transfected with the empty control vector, wild-type (wt) Myc-ZBTB7A, and mutant type (mt) Myc-ZBTB7A. mtZBTB7AR399L failed to bind to the #3 position of the EPB41L5 promoter (Fig. 5f). We also confirmed that mtZBTB7AR399L failed to repress the transcriptional activity of EPB41L5. We examined the transcriptional activity in GBM cell lines LN229, U343, U118, and U87 transfected with the empty vector, wtZBTB7A, and mtZBTB7A (Fig. 5g). As expected, mtZBTB7AR399L did not inhibit the transcriptional repression of EPB41L5 compared to wtZBTB7A, and the wt/mt Myc-ZBTB7A protein was normally expressed in all cells. (Supplementary Fig. 4c). Thus, we suggest that ZBTB7A negatively regulates the transcriptional activity of EPB41L5 by binding to the ZBTB7A #3 position of the EPB41L5 promoter.
ZBTB7A knockdown increases GBM tumorigenesis through the transcriptional activation of EPB41L5
Next, we performed a luciferase assay to determine whether transcriptional activation of EPB41L5 was increased by ZBTB7A knockdown. The transcriptional activation of EPB41L5 was greatly increased in the -150 to 100 region (#3) of the EPB41L5 promoter by ZBTB7A knockdown, but not in the #3 mutant promoter (Fig. 6a). In addition, mRNA expression of EPB41L5 and ZBTB7A was confirmed in shZBTB7A-expressing U87 cells, and the transcription activity of EPB41L5 was measured in shZBTB7A-expressing U87 cells that target the 3’UTR EPB41L5 promoter, mtZBTB7A expression greatly induced the transcription activity of EPB41L5. The activities of wtZBTB7A and the control were similar (Fig. 6b, c, Supplementary Fig. 4d). Additionally, a ChIP assay was performed in shZBTB7A-expressing U87 cells co-transfected with the EPB41L5 promoter and wtZBTB7A or mtZBTB7A.
In the previous results, mtZBTB7A failed to recruit the EPB41L5 promoter, and wtZBTB7A did not recruit the #3 mutation region of the EPB41L5 promoter (Fig. 6d). Therefore, we monitored the development and progression of shZBTB7A-expressing GBM cells in an orthotopic xenograft mouse model. Control U87 and shZBTB7A knockdown U87 cells were injected into the right corpus striatum of the mouse brain. As a result of observation for 4 weeks, the GBM tumor significantly increased in a mouse injected with shZBTB7A-expressing U87 cells compared to the control mouse group, and the protein expression of ZBTB7A and EPB41L5 in GBM tumor tissues was inversely correlated (Fig. 6e).
We observed the expression of EMT markers (N-cadherin, b-catenin, and Vimentin) together with EPB41L5 (Fig. 6f). Similarly, the protein and mRNA expression of N-cadherin, b-catenin, and Vimentin was confirmed in shZBTB7A U87 cells, and both protein and mRNA levels were increased (Fig. 6g, h). Taken together, our results show that knockdown of ZBTB7A induces transcriptional activation of EPB41L5 and increases GBM tumorigenesis via the EMT process.