Temozolomide protects against the progression of glioblastoma via SOX4 downregulation by inhibiting the LINC00470‐mediated transcription factor EGR2

Abstract Objective Temozolomide is extensively applied in chemotherapy for glioblastoma with unclear exact action mechanisms. This article seeks to address the potential molecular mechanisms in temozolomide therapy for glioblastoma involving LINC00470. Methods Bioinformatics analysis was conducted to predict the potential mechanism of LINC00470 in glioblastoma, which was validated by dual‐luciferase reporter, RIP, ChIP, and RNA pull‐down assays. LINC00470 expression and the predicted downstream transcription factor early growth response 2 (EGR2) were detected in the collected brain tissues from glioblastoma patients. Following temozolomide treatment and/or gain‐ and loss‐of‐function approaches in glioblastoma cells, cell viability, invasion, migration, cycle distribution, angiogenesis, autophagy, and apoptosis were measured. In addition, the expression of mesenchymal surface marker proteins was assessed by western blot. Tumor xenograft in nude mice was conducted for in vivo validation. Results Mechanistic analysis and bioinformatics analysis revealed that LINC00470 transcriptionally activated SRY‐related high‐mobility‐group box 4 (SOX4) through the transcription factor EGR2. LINC00470 and EGR2 were highly expressed in brain tissues of glioblastoma patients. LINC00470 and EGR2 mRNA expression gradually decreased with increasing concentrations of temozolomide in glioblastoma cells, and SOX4 expression was reduced in cells by temozolomide and LINC00470 knockdown. Temozolomide treatment induced cell cycle arrest, diminished cell viability, migration, invasion, and angiogenesis, and increased apoptosis and autophagy in glioblastoma, which was counteracted by overexpressing LINC00470 or SOX4 but was further promoted by LINC00470 knockdown. Temozolomide restrained glioblastoma growth and angiogenesis in vivo, while LINC00470 or SOX4 overexpression nullified but LINC00470 knockdown further facilitated these trends. Conclusion Conclusively, temozolomide repressed glioblastoma progression by repressing the LINC00470/EGR2/SOX4 axis.


| INTRODUC TI ON
Glioblastoma, which is believed to be generated by progenitor or neuroglial stem cells, has the highest degree of malignancy among gliomas and the highest prevalence among brain tumors. 1,2 Glioblastoma exhibits cell and nuclear rudimentary, microvascular proliferation, a high degree of cellularity, necrosis, and mitosis under the microscope. 3 Glioblastoma appears in any age group with increasing incidence with age (median age of diagnosis is around 65 years old) and is more frequent in men. 4 As of 2005, the Stupp protocol, consisting of surgery excision combined with adjuvant temozolomide chemotherapy and radiotherapy, has served as the care standard for glioblastoma. 5 In contrast to radiotherapy treatment alone, incorporation with temozolomide resulted in improvement in overall and progression-free survival of patients with glioblastoma. 6 Temozolomide is an orally administered front-line chemotherapy drug for patients with glioblastoma, whose action mode is primarily based on alkylating guanine residues N-7 or O-6 or adenine residues N-3 within DNA, thus resulting in mismatching in the subsequent replication of DNA and in turn leading to cell cycle arrest, senescence, apoptosis, autophagy, and death. 7,8 Although the action mechanism of temozolomide in glioblastoma has been extensively researched, the concrete molecular mechanism of its impact in glioblastoma warrants in-depth exploration.
It is well known that various dysregulated long noncoding RNAs (lncRNAs) are linked to glioblastoma progression with its involvement in nearly all tumor characteristics, comprising cell proliferation, invasion, angiogenesis, migration, severity, stemness, recurrence, and chemoresistance. 9 LINC00470, a novel lncRNA localized at chromosome 18p11.32, is already proposed to serve as an oncogene in melanoma and endometrial cancer. [10][11][12] LINC00470 was described as an oncogenic lncRNA in glioblastoma and a novel AKT activator that induces malignant features of glioblastoma, including cell proliferation and autophagy. 13 Bioinformatics analysis in our research predicted that LINC00470 orchestrated a transcription factor, early growth response 2 (EGR2), and that SRY-related high-mobility-group box 4 (SOX4) was a downstream target gene of EGR2. EGR2 is a transcription factor that participates in various cell processes, such as cell proliferation and cell cycle. 14 EGR2 was demonstrated to serve as a carcinogenic gene in renal cell cancer by modulating cancer cell proliferation and migration. 15 A prior publication reported that EGR2 knockdown curtailed cell invasion in glioma. 16 EGR2 may promote glioblastoma in view of these findings. SOX4 is a carcinogenic gene that enhances stemness, cancer cell migration, metastasis, epithelial-mesenchymal transition, survival, and angiogenesis. 17 Ikushima et al. observed that SOX4 was overexpressed in gliomainitiating cells and was a key element in maintaining the stemness of glioma-initiating cells. 18 Therefore, we hypothesized that temozolomide may delay the development of glioblastoma by inhibiting the transcriptional activation of SOX4 through LINC00470-regulated EGR2. This study is dedicated to delve into the impact of the relationship between temozolomide and the LINC00470/EGR2/SOX4 axis on glioblastoma progression, thus presenting a fresh theoretical basis for the treatment of glioblastoma with temozolomide.

| Ethical statements
This study that involved humans was ratified by the Medical Ethics

Committee of The Second Xiangya Hospital of Central South
University, and all patients signed an informed consent form. All animal experiments complied with the regulations and codes of practice for laboratory animal management and ethical requirements related to laboratory animals.

| Clinical specimens
Brain tissues were harvested from 30 patients with glioblastoma at The Second Xiangya Hospital of Central South University between November 2016 and November 2017 and normal brain tissue sections (10 cases) (traumatic brain tissue) collected as controls. All samples were rapidly frozen in liquid nitrogen until further use. Inclusion criteria were as follows: patients with complete clinical information (including general condition, pathological diagnosis, treatment plan, survival, and follow-up information) and without combined history of other malignancies. Exclusion criteria were as follows: patients with incomplete clinical data (no pathological diagnosis and immunohistochemistry [IHC] result) or the combined history of other malignancies.

| Immunohistochemistry
The prepared sections were placed in boiling water with ethylene diamine tetraacetic acid (EDTA) buffer (pH = 9.0) for 10 min antigen retrieval, naturally cooled, and then washed 3 times with phosphatebuffered saline (PBS). The sections were incubated in 3% hydrogen peroxide solution for 10 min to block endogenous peroxidase, washed 3 times in PBS, and blocked in serum for 30 min. Then, the sections were probed overnight at 4°C with 50 μL sealing solutionprepared primary antibodies (Abcam) against Ki67 (ab15580, 1:100), angiogenesis, apoptosis, autophagy, early growth response 2, glioblastoma, LINC00470, SRYrelated high-mobility-group box 4, temozolomide vascular endothelial growth factor (VEGF, ab32152, 1:100), light chain 3 (LC3, ab232940, 1:100), and p62 (ab109012, 1:100), followed by three PBS washes. After being probed with secondary antibody for 0.5 h at room temperature, the sections were washed 3 times with PBS, developed with diaminobenzidine, and stained for 3 min with hematoxylin. Subsequently, the sections underwent 1-3 s of color separation with 1% ethanol hydrochloride and running water washing. Afterward, the sections were returned to blue with 0.6% ammonia and washed with running water. Finally, the sections were dehydrated with gradient ethanol, transparentized with xylene, sealed with neutral resin, and observed and photographed by an ordinary optical microscope.

| Cell culture and treatment
Temozolomide were attained from Sigma-Aldrich (T2577). LN229 and U87 cells (iCell Bioscience) were cultured with high-glucose Dulbecco's modified Eagle's medium (DMEM; Gibco) encompassing 10% fetal bovine serum, and GL261 + luc cells (the mouse glioma cells with luciferase gene; iCell Bioscience) were cultured in the GL261 + luc cell-specific medium (iCell-0059a-001b, iCell Bioscience). All of the above media contained 100 U/mL penicillin and 100 μg/mL streptomycin, and all of the cells were cultured in a constant temperature incubator (37°C, 5% CO 2 , and 95% humidity). Cells in the logarithmic phase were harvested for the experiments.  Table S1. After transfection, cells were added to serum-free DMEM for 48-h further culture in a 5% CO 2 and 37°C constant temperature incubator.

| Cell counting kit-8 assay
Glioma cells were seeded onto 96-well plates at a density of 4000-6000 cells per well, followed by supplementation of fresh medium with the appropriate concentration of reagents in the next day.
Following 48-h incubation, cell viability was evaluated using cell counting kit (CCK)-8 kits (KeyGEN Biotech) as per the protocols.

| Quantitative reverse transcription polymerase chain reaction
Total RNA was extracted following the instructions of the Trizol kits (Invitrogen) and reversely transcribed into cDNA using the PrimeScript RT kit (RR037A; Takara). The samples were subjected to fluorescent PCR on a real-time quantitative fluorescent PCR instrument system (ABI7500; Applied Biosystems) in accordance with the manuals of the TB Green® Premix Ex Taq™ II kit (RR820A, TaKaRa).
The relative expression of each target gene was calculated using the 2 −ΔΔCt method with β-actin as an internal reference. Each experiment was conducted with three replicates. The relevant primers (Table 1) were designed by Sangon Biotechnology.

| Western blot
LN229 and U87 cells were lysed with enhanced radioimmunoprecipitation assay (RIPA) lysis solution encompassing protease inhibitors (BOSTER) to obtain proteins, followed by protein concentration determination using the bicinchoninic acid protein quantification kit (BOSTER). After protein separation using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, the separated proteins were electrotransferred onto polyvinylidene fluoride membranes. horseradish peroxidase-labeled secondary antibody and then incubated for 1 min at room temperature with electrogenerated chemiluminescence (ECL) working solution (EMD Millipore). Following the removal of the excess ECL reagent, membranes were sealed with cling film and developed and fixed by 5-10 min of exposure to X-ray film in the dark box. The grayscale quantification of bands in western blot images was performed using the Image J analysis software (National Institutes of Health) with β-actin as an internal reference.
The experiment was repeated 3 times each.

| Chromatin immunoprecipitation assay
After treatment of glioma cells with 4% formaldehyde (the final concentration of 1%), the cells were broken by ultrasonication and in- Immunoglobulin G (IgG, ab172730, 1:100, Abcam) served as a negative control (NC). The experiment was replicated 3 times.

| Dual-luciferase reporter assay
Artificially synthesized SOX4 promoter sequence fragments were inserted into the pMIR-reporter (Promega) after enzyme digestion.
Next, the correctly sequenced luciferase reporter plasmids were respectively cotransfected with LV-NC, LV-LIN00470, LV-EGR2, si-NC, and si-LINC00470 into U87 cells. Following 48 h of transfection, the cells were lysed and centrifuged for 3-5 min, and the supernatant was obtained and analyzed for luciferase activity in the cell extracts using the dual-luciferase reporter assay system (Promega).
The luciferase activity was measured on a fluorescence detector (Promega) with the ratio of the target luciferase activity to the internal reference luciferase activity as the relative luciferase activity.
The parallel experiment was repeated 3 times.
The three most likely binding sites of EGR2 protein to SOX4 DNA were identified through the JASPAR (http://jaspar.gener eg.net/) website. Subsequently, recombinant luciferase reporter vectors of truncated or mutant binding sites were constructed and cotransfected with the EGR2 expression vector into U87 cells for a dual luciferase reporter assay to verify the specific site of EGR2 protein binding to SOX4 DNA. The exact method steps were the same as mentioned earlier.

| RNA immunoprecipitation assay
The binding of LIN00470 to EGR2 was tested using the RNA immunoprecipitation (RIP) kit (EMD Millipore). Cells were washed with

| RNA pull-down
Biotinylated LIN00470 and U6 RNA were mixed with proteins from nuclear extracts of cancer cells. The biotinylated LIN00470-protein complex was purified using streptavidin-agarose beads (Thermo Fisher Scientific). Proteins were then eluted from the RNA-protein complex for immunoblotting using EGR2 antibodies.

| Cell cycle analysis
The cultured glioma cells were centrifuged at 250 g for 5 min, washed with prechilled PBS, and fixed overnight at −20°C with 70% ethanol. The fixed cells were washed with PBS for 10 min, treated with RNAase A for 30 min, and then incubated with propidium iodide at room temperature for 30 min. BD FACSCalibur™ Flow Cytometer (BD Biosciences) was adopted to assess the cell cycle of each specific sample following the manufacturer's protocols. The experiment was replicated 3 times.

| Transwell assay
Glioma cells were starved in serum-free medium for 24 h, digested, rinsed twice with PBS, and resuspended in serum-free DMEM containing 10 g/L BSA (Sigma-Aldrich) to adjust the cell density to 3 × 10 4 cells/mL. Experiments were performed using Transwell

| Matrix gel-based in vitro endothelial tube formation assay
As described in a prior study, 19 endothelial cell tube formation was assessed using matrix gel-coated slides. Experimental results were photographed using an Eclipse Ti microscope with a DS-Fi1 camera (Nikon) at 40× magnifications, and the total area of endothelial cellformed vessels in each lumen was calculated using NIS-Elements-Basic Research software (Nikon) and presented as angiogenesis score. The experiment was replicated 3 times.

| Flow cytometry for apoptosis analysis
Cells were centrifuged at 112 g for 10 min, followed by three PBS washes. The cell suspension was fixed with 70% ethanol (prechilled at −20°C) for 1 h and centrifuged to remove the fixing solution.
Then, the cells were rinsed and mixed with PBS to make a single cell suspension of 1 × 10 7 cells/mL. An Annexin V-fluorescein isothiocyanate apoptosis assay kit (Beyotime) was applied for the detection of apoptosis, and a FACSCanto II flow cytometer system for the calculation of apoptosis percentage. Finally, the FlowJo 7.6.1 Software (BD Biosciences) was utilized for the analysis of flow cytometry results. The experiment was replicated 3 times.

| Tumor xenograft in nude mice
Thirty specific pathogen-free (SPF) grade male BALB/c-nu nude mice

| TdT-mediated dUTP-biotin nick endlabeling staining
After completion of all the aforementioned animal experiments, the mice were euthanized under deep anesthesia and the brains were carefully extracted. The brain tissues were fixed in 4% paraformaldehyde and

| Statistical analysis
GraphPad prism8 software was applied for statistical analysis, and all data were summarized as mean ± standard deviation. The normal distribution of data was assessed with the Shapiro-Wilk test, and all data conformed to normal distribution. After normal distribution test, the T test was performed for comparisons between the two groups, and the one-way analysis of variance test was applied for comparisons among multiple groups, with Tukey's multiple comparisons test for post hoc multiple comparisons, except for the results whose specific methods of analysis were described. The p s < 0.05 were considered a statistically significant difference.

| Temozolomide restrained glioblastoma cell survival through inhibition of LINC00470-regulated EGR2
Quantitative reverse transcription polymerase chain reaction (qRT-PCR) results of brain tissue samples attained from clinical patients with glioblastoma exhibited that the brain tissues of patient with glioblastoma had highly expressed LINC00470 ( Figure 1A). Also, our results demonstrated that temozolomide treatment dramatically de-  Figure 1J). Moreover, the results in Figure 1C also described that LINC00470 expression in temozolomide-treated  Figure 2A). Thus, 28 candidate target genes were attained and analyzed for gene interactions to construct a gene interaction network graph ( Figure 2B), followed by counting of the degree value of each gene ( Figure 2C). The results demonstrated that six genes, including SOX4, were in a relatively core position in the gene interaction network graph, and SOX4 had the largest upregulation fold in GSE50161 among these six genes (Table S2).
Next, chromatin immunoprecipitation (ChIP) assays presented that knockdown of LINC00740 in LN229 and U87 cells resulted in a striking reduction in SOX4 promoter enrichment, and the same trend was observed for SOX4 promoter enrichment in temozolomidetreated cells ( Figure 2D). As revealed in qRT-PCR and western blot experiments, knockdown of LINC00470 or temozolomide treatment markedly diminished SOX4 expression in LN229 and U87 cells ( Figure 2E,F). To confirm the regulation of LINC00470 on SOX4, SOX4 promoter sequence was cloned into pMIR reporter, and the luciferase activity was assessed by the dual-luciferase reporter assay after cotransfection of pMIR-reporter plasmid with si-LINC00470 or LV-LINC00470 into U87 cells. The results displayed that overexpression of LINC00470 appreciably enhanced but knockdown of LINC00470 evidently decreased luciferase activity ( Figure 2G).
The relationship between LINC00470 and EGR2 was further clarified by RIP experiments, which demonstrated that the EGR2 antibody enriched more LINC00470 than the IgG antibody ( Figure 2H).
Further RNA-pulldown assay also exhibited that LINC00470 specifically bound to EGR2 ( Figure 2I).
Meanwhile, the presence of the EGR2 binding structural domain in the SOX4 promoter region (nt: 1704-1718; 1722-1736) was identified by the JASPAR database (Table S3). Then, the trun-   Figure 3D). In addition, LINC00470 or SOX4 overexpression also annulled temozolomidediminished angiogenesis ( Figure 3E). In addition, it was also found that combination of temozolomide treatment with LINC00470 knockdown was more effective than temozolomide treatment alone was appreciably reduced in tumor tissues following temozolomide treatment or the simultaneous treatment of temozolomide and si-LINC00470, while there were reverse trends in mice treated with F I G U R E 2 LINC00470 increases SOX4 expression via EGR2. (A) Intersection of prominently upregulated genes in GSE50161 and predicted results of target genes of EGR2 was exhibited. (B) Interaction analysis on candidate target genes of EGR2 was detailed, in which each circle represented a gene and the linkage between the circles indicated the existence of interaction between genes (the more genes linkage, the higher the degree value). (C) The degree value statistics of candidate target genes were displayed. The horizontal coordinate indicated the degree value and the vertical coordinate indicated the gene name. (D) SOX4 promoter enrichment in cells was measured by ChIP assay. (E) SOX4 mRNA expression in cells was detected by qRT-PCR. (F) SOX4 protein expression in cells was evaluated by western blot. (G) The luciferase activity of SOX4 after overexpression or knockdown of LINC00470 was evaluated by dual-luciferase reporter assay.

| Temozolomide restricted cell proliferation, migration, invasion, and angiogenesis in glioblastoma by inhibiting the LINC00470/EGR2/SOX4 axis
(H) The association between LINC00470 and EGR2 was assessed by RIP assay. (I) The interaction of LINC00470 and EGR2 was determined by RNA pull-down assay. (J) The relative luciferase activity after transfection with different lengths of SOX4 promoters and overexpression of EGR2 in U87 cells was tested by dual-luciferase reporter assay. (K) The relative luciferase activity after transfection of wild and mutant SOX4 promoters and overexpression of EGR2 in U87 cells was examined by dual-luciferase reporter assay. (L) SOX4 promoter enrichment in cells was assessed by ChIP assay. *p < 0.05, compared with the control, IgG, or LV-NC group. # p < 0.05, compared with the si-NC group. Data were derived from three independent replicate experiments and compared using one-way analysis of variance with Tukey's test for post hoc multiple comparisons. EGR2, early growth response 2; LV, lentiviral vectors for overexpression; si, small interfering RNA; SOX4, SRY-related high-mobility-group box 4; TMZ, temozolomide.

| Temozolomide treatment promoted apoptosis and autophagy of glioblastoma cells by inhibiting the LINC00470/EGR2/SOX4 axis in vivo and in vitro
This study further ascertained whether the regulation of temozolomide on cell apoptosis and autophagy in glioblastoma also in- Temozolomide, a type of alkylating drug authorized for glioblastoma anticancer therapy, is able to cross the blood-brain barrier damaging DNA and initiating cellular suicide. 23  proliferation, invasion, migration, and angiogenesis) and also facilitated cell apoptosis and autophagy in glioblastoma. As a hallmark of glioblastoma pathogenesis, angiogenesis is also an ongoing goal of glioblastoma treatment and intervention. 28 Similarly, an earlier study found that temozolomide treatment reduced the vascular density and vascular endothelial growth factor expression (which are strongly associated with angiogenesis) in mice with glioblastoma. 29 Although plenty of studies have focused on the molecular mechanisms underpinning temozolomide resistance and sensitivity in cancers, 30 to the family of early growth response genes, has been discovered to facilitate cell invasion in glioma. 16 SOX4 was implicated in the promotion of self-renewal and stemness in gliomas. 43 It was discovered in a prior work that the ALK pathway includes the SOX4, Sta3, Akt, and N-myc activities, which jointly facilitated cell proliferation and tumor neovascularization in nonhypoxic contexts in glioblastoma. 44  were also evidenced in nude mice.
In conclusion, our research innovatively and first explored the molecular mechanism of temozolomide in glioblastoma.
Specifically, temozolomide treatment restricted cell invasion, migration, and angiogenesis but facilitated cell autophagy and apoptosis in glioblastoma via the inactivation of the LINC00470/EGR2/ SOX4 axis ( Figure 6). However, this work is limited by the fact that the impacts of temozolomide via the LINC00470/EGR2/SOX4 axis on angiogenesis in vivo was not verified, which requires further research. This work intends to provide theoretical basis and fresh perspectives for glioblastoma management with temozolomide.
Thus, further research in the future is warranted whether the combination of temozolomide with the inhibitors of small molecules studied here can improve the efficiency of temozolomide therapy.

F I G U R E 4
Temozolomide treatment delays glioblastoma growth in nude mice via the inhibition of the LINC00470/EGR2/SOX4 axis. (A) Tumors extracted from mice were photographed. (B) Tumor volumes were measured at the times shown in the figure, n = 6 mice in each group; *p < 0.05, compared with the control group, # p < 0.05, compared with the TMZ group; and data were analyzed by two-way analysis of variance with LSD test for post hoc multiple comparisons. (C) Tumors from each group of nude mice were weighed. (D) Ki-67 expression in the tumor tissues of nude mice was measured by IHC. (E) VEGF expression in the tumor tissues of nude mice was evaluated by IHC. (F) The protein expression of cyclinD1 and CDK4 in nude mice was examined by western blot (data represent three independent replicate trials). (G) Tumor size in mice of intracranial in situ graft tumor model was determined by in vivo animal imaging, n = 6; *p < 0.05, compared with the control group; # p < 0.05, compared with the TMZ group. Except where noted, other data were analyzed using one-way analysis of variance with Tukey's test for post hoc multiple comparisons. CDK4, cyclin-dependent kinase 4; EGR2, early growth response 2; IHC, immunohistochemistry; LSD, least significant difference; LV, lentiviral plasmids for overexpression; si, small interfering RNA; SOX4, SRYrelated high-mobility-group box 4; TMZ, temozolomide; VEGF, vascular endothelial growth factor.

202104040152) and the Natural Science Foundation of Hunan
Province (2022JJ30800).

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.
F I G U R E 6 Molecular mechanism diagram. Temozolomide treatment suppressed glioblastoma progression by inhibiting LINC00470-regulated EGR2 and thus decreasing SOX4 expression.