3.1Expression and Prognosis Analysis of ZFHX4-AS1 in Pan − Cancer
In comparing data from 33 tumor species, we observed significant up-regulation of ZFHX4-AS1 in 11 tumors using R software, such as GBM, LGG,LUAD,STAD,LUSC,LIHC,OV,PAAD,UCS,KIRP,THYM. We observed significant down-regulation in 17 kinds of tumors, such as BRCA,CESC,ESCA,COAD,PRAD,KIRC,SKCM,BLCA,THCA,READ,TGCT,ACC,KICH,CHOL,DLBC,LAML,UCEC(Fig. 1A).
According to our analysis of the relationship between ZFHX4-AS1 expression and tumour prognosis, 11 tumour types with high expression of ZFHX4-AS1 had poor prognosis, whereas 2 tumour types with low expression had poor prognosis(Fig. 1B).
In order to clarify the role of ZFHX4-AS1 in the occurrence and development of gliomas, we analyzed the relationship between ZFHX4-AS1 and clinical characteristics in the TCGA database, including gender, WHO grade, 1p19q deletion, and status of IDH. It was shown that ZFHX4-AS1 expression levels are also different when 1p19q deletions(Fig. 1C) and IDH mutations differ(Fig. 1D). Gender, and tumor grade did not show statistically significant differences(Fig. 1E-F).
3.2 ZFHX4-AS1 is Highly Expressed in Tissues and Cells
For clarification of ZFHX4-AS1 expression in glioma tissues and normal brain tissues, we performed RT-PCR analysis of the collected tissues. In comparison to normal brain tissues, ZFHX4-AS1 expression was significantly increased in glioma tissues(Fig. 2A), particularly WHO IV glioma tissues(Fig. 2B). Further, RT-PCR analysis of six glioma cell lines (U251, U138, U87, A172, LN229, T98G) and NHA cell lines demonstrated that glioma cells expressed ZFHX4-AS1 at significantly higher levels than NHA cells(Fig. 2C).
3.3 ZFHX4-AS1 Promotes Glioma Cells Proliferation in Vitro
Several studies have been conducted on the role of ZFHX4-AS1 in glioma cells. siRNAs were used to suppress ZFHX4-AS1 expression, and plasmids were used to overexpress it. By conducting RT-PCR, we determined that ZFHX4-AS1 had been effectively suppressed and overexpressed(Fig. 2D-E). Based on colony formation, CCK-8 assay, and EdU assay, it had been concluded that ZFHX4-AS1 silencing inhibits glioma cell proliferation, whereas ZFHX4-AS1 expression promotes proliferation(Fig. 2F-H).
3.4 ZFHX4-AS1 Promotesthe Growth of Glioma Cells in Vivo
U251 cells which were transfected with oe-ZFHX4-AS1 or oe-NC were stereotactically implanted in nude mice.We measured tumor’s volume every seven days, extracted tumor tissue after 28 days for weighing. The results showed that the tumor’sgrowth rate was significantly stimulated by overexpression of ZFHX4-AS1(Fig. 2I). It was found that the oe-ZFHX4-AS1 group had 410.54 mm3 volume compared to 125.99mm3 volume in the control group, while the oe-ZFHX4-AS1 group had 451.52 mg mass compared to 114.72mg mass in the control group. According to the experimental results, overexpression of ZFHX4-AS1 stimulates tumour growth to a significant degree.
3.5 ZFHX4-AS1 promotes Glioma Cells Invasion and Migration
To investigate the role of ZFHX4-AS1 in the invasion and migration of glioma cells, we performed healing assays, transwell assays. According to the results of the study, knockdown of ZFHX4-AS1 significantly inhibited the migration and invasion of glioma cells. Meanwhile, the migration and invasion ability of glioma cells was significantly enhanced after overexpression of ZFHX4-AS1. These results indicated that ZFHX4-AS1 promotes the invasion and migration process of glioma cells and has a stimulating effect on them(Fig. 3A-C).
3.6 Glioma Cells Express ZFHX4-AS1 in Both Their Nucleus and Cytoplasm
In this study, we used FISH and subcellular fractionation assays to determine the subcellular localization of ZFHX4-AS1 with the aim of exploring its mechanism of effect on malignant biological behavior. According to the results, both the nuclear and cell cytoplasms were found to express ZFHX4-AS1(Fig. 4A).
3.7 The Nearby Gene ZFHX4 is Highly Expressed in Glioma
Using the UCSC database, we were able to determine that ZFHX4 is a nearby gene to ZFHX4-AS1(Fig. 4.B). As a result of the data from the TCGA and GTEx databases, ZFHX4 expression exhibited a significant increase in glioma tissues(Fig. 4C), and ZFHX4 expression was positively correlated with ZFHX4-AS1(Fig. 4D). The expression of ZFHX4 in tissue and cell lines was examined in order to determine its functional significance.According to the results, there was a high expression level of ZFHX4 in glioma tissues and cells, and the level of expression of ZFHX4 increases with the progression of the disease(Fig. 5A).
3.8 ZFHX4 Promotes the Proliferation, Invasion and Migration in Glioma Cells
We used siRNA to knock down ZFHX4 expression and overexpressed it by plasmid to investigate its role in glioma.RT-PCR and Western Blot assays were performed to confirm ZFHX4 was successfully silenced and overexpressed in glioma cell lines(Fig. 5B). Based on colony formation assay(Fig. 5C), CCK-8 assay(Fig. 5D) and EdU assay(Fig. 5E), it was found that silencing ZFHX4 inhibited glioma cell proliferation, while enhanced ZFHX4 expression increased cell proliferation.In addition,we conducted healing assay(Fig. 6A),cell invasion(Fig. 6B) and migration(Fig. 6C) experiments and found that knocking down of ZFHX4 significantly decreased the migration and invasion abilities of glioma cells as compared to control cells. Meanwhile, it was found that overexpression of ZFHX4 significantly enhanced the migratory and invasive abilities of cells.
3.9 ZFHX4 Promotes the Growth of Glioma Cells In Vivo
The tumourigenicity of ZFHX4 was investigated in our experiments as well. A significant increase in tumor growth rate was observed after ZFHX4 was over expressed on days 7, 14, 21 and 28 after implantation. The final volume of the oe-ZFHX4 group was 1228.85 mm3 compared to 166.3 mm3 in the control group, and the mass of the oe-ZFHX4 group was 804.62 mg compared to 157.9 mg in the control group. It appears that ZFHX4 stimulates the proliferation of glioma cells in vivo(Fig. 6D).
3.10 ZFHX4‑AS1 Modulates Proliferation, Invasion and Migration of Glioma Cells via Regulating its Nearby Gene ZFHX4
Following the knockdown of ZFHX4-AS1 and the overexpression of ZFHX4-AS1, there were substantial changes in the expression of ZFHX4 in glioma cells as determined by RT-PCR(Fig. 7A) and Western Blot(Fig. 7B) analysis. The results demonstrated that ZFHX4 expression decreased and increased in glioma cells following knockdown and overexpression of ZFHX4-AS1. We also detected ZFHX4-AS1 expression in glioma cells that overexpressed and knocked down ZFHX4. The expression of ZFHX4-AS1 did not change with the change in ZFHX4, which suggests that ZFHX4-AS1 and ZFHX4 may have a one-way regulatory relationship(Fig. 7C). In addition, silencing ZFHX4 followed by overexpression of ZFHX4-AS1 restored ZFHX4 expression in glioma cells(Fig. 7D). Overexpression of ZFHX4 followed by silencing of ZFHX4-AS1 rescued the change in proliferation (Fig. 5C-E), migration and invasive ability(Fig. 6A-C) associated with ZFHX4 overexpression.The results of these studies confirmed that ZFHX4-AS1 modulates the proliferation, invasion, and migration of glioma cells through its nearby gene ZFHX4.
3.11 Analysis of Differentially Expressed Genes
We investigated the differentially expressed genes (DEGs) among the highly and lowly ZFHX4 expressed groups in TCGA to determine the mechanisms by which ZFHX4 regulates glioma development and occurrence.We used |log2 folding change(FC)|> 1 and p < 0.05 as a screening criterion, identifying 2129 genes down-regulated and 111 genes up-regulated(Fig. 8A). Based on the enrichment analysis of GO database,DEGs was enriched primarily for humoral immune response mediated by circulating immunoglobulins; humoral immune response;complement activation, classical pathway; blood micropartical; immunoglobulin complexes,circulating; immunoglobulin complexes;receptor ligand activity; immunoglobulin receptor binding;antigen binding (Fig. 8B). According to the enrichment analysis of the KEGG database, DEGS was mainly enriched in Cytokine-cytokine receptor,Viral protein interaction with cytokine and cytokine receptor,IL-17 signaling pathway(Fig. 8C). We used GSEA to predict ZFHX4's potential carcinogenic pathway.A total of 54 datasets satisfy FDR (q-value) < 0.25 and p.adjust < 0.05. Several classical tumor-related signal pathways were significantly related to the expression of ZFHX4 including the MAPK family signal cascade and FCERI-mediated NF-κB activation(Fig. 8D).
3.12 ZFHX4 Binds to SOX2 and Promotes its Expression, thereby Contributing to the Malignant Biological Behavior of Glioma Cells
We analyzed the proteins interacting with ZFHX4 by BioGRID. Eight key proteins with strong interactions were identified, including SOX2, CHD3, CHD4, PAX6, CIC, NFIX, KPNA1 and TEAD1, which were positively correlated with the expression of ZFHX4(Fig. 8E-F). Upon examining the relative expression of these eight genes, it was discovered that they were all highly expressed in glioma(Fig. 8G). As a follow-up study, we selected SOX2, which had the highest relative expression and differential expression. And in glioma, SOX2 has a strong positive interaction with ZFHX4(Fig. 8H).
We also verified the expression of SOX2 in glioma tissues and cells. Based on the results of the RT-PCR, SOX2 is highly expressed in glioma tissues(Fig. 9A), and its expression increases with tumor grade(Fig. 9B). A high level of SOX2 expression was also observed in glioma cells(Fig. 9C).
We knocked down SOX2 expression using siRNA and overexpressed it via plasmid in glioma cells in order to ascertain the role of SOX2 in glioma.The knockdown as well as the overexpression of SOX2 were both successfully verified(Fig. 9D-E). It was determined that SOX2 silencing inhibited proliferation of glioma cells, whereas increased SOX2 expression enhanced proliferation of glioma cells by colony formation assay(Fig. 9F), CCK-8 assay(Fig. 9G), and EdU assay(Fig. 9H). The knockdown of SOX2 significantly reduced glioma cell migration and invasion in wound healing assays(Fig. 10A), transwell assays(Fig. 10B-C).In contrast to cells in the control group, cells overexpressing SOX2 showed greater migration and invasion.
We confirmed that SOX2 and ZFHX4 can bind to each other in glioma cells by co-immunoprecipitation(Fig. 11A). Following the knockdown of ZFHX4 and the overexpression of ZFHX4, there were substantial changes in the expression of SOX2 in glioma cells as determined by RT-PCR (Fig. 11B)and Western Blot(Fig. 11C)analysis.The results demonstrated that SOX2 expression decreased and increased in glioma cells following knockdown and overexpression of ZFHX4.We also detected ZFHX4 expression in glioma cells that overexpressed and knocked down SOX2. The expression of SOX2 in glioma cells also changed after knockdown or overexpression of ZFHX4, and the trend was the same as that of ZFHX4(Fig. 11D-E).Furthermore, overexpression of SOX2 followed by knockdown of ZFHX4 restores the malignant biological behaviour of glioma cells, such as proliferation, migration and invasion(Fig. 10A-C).In addition, the above findings further confirmed that ZFHX4 contributes significantly to modulating proliferation, invasion, and metastatic processes in glioma cells through SOX2.
Based on analysis of DEGs in SOX2 high and low expression groups, we attempted to clarify the mechanism by which SOX2 regulates glioma development and occurrence. We screened 4175 down-regulated genes and 199 up-regulated genes(Fig. 12A). Based on the enrichment analysis of GO database, DEGs was enriched primarily for humoral immune response mediated by circulating immunoglobulins; complement activation, classical pathway; humoral immune response;external side of plasma membrane; immunoglobulin complexes,circulating; immunoglobulin complexes;receptor ligand activity; immunoglobulin receptor binding;antigen binding (Fig. 12B). According to the enrichment analysis of the KEGG database, DEGs was mainly enriched in neuroactive ligand-receptor interaction;cytokine-cytokine receptor interaction;viral protein interaction with cytokine and cytokine receptor (Fig. 12C). We used GSEA to predict SOX2's potential carcinogenic pathway. A total of 311 datasets satisfy FDR (qvalue) < 0.25 and p.adjust < 0.05. Several classical tumor-related signal pathways are significantly related to the expression of SOX2 including the Calcium signaling pathway;JAK-STAT signaling pathway; Chemokine signaling pathway (Fig. 12D).We then examined the JAK-STAT signaling pathway and found that the phosphorylation ratios of JAK1 and STAT3, key factors in the JAK-STAT signaling pathway, were altered upon both knockdown and overexpression of SOX2, and that overexpression of ZFHX4-AS1 after knockdown of SOX2 partially restored these changes(Fig. 12E).