E2F transcription factor 1 elevates cyclin D1 expression by suppressing transcription of microRNA‐107 to augment progression of glioma

Abstract Background Dysregulation of microRNAs has been frequently implicated in the progression of human diseases, including glioma. This study aims to explore the interaction between E2F transcription factor 1 (E2F1) and miR‐107 in the progression of glioma. Methods Expression of miR‐107 in glioma tissues and cells was examined. Putative binding sites between E2F1 and the promoter region of miR‐107, and between miR‐107 and cyclin D1 (CCND1) mRNA were predicted via bioinformatic systems and validated via chromatin immunoprecipitation and luciferase reporter gene assays. Altered expression of miR‐107, E2F1, and CCND1 was introduced in A172 and T98G cells to examine their roles in cell growth and the activity of the Wnt/β‐catenin signaling. In vivo experiments were performed by injecting cells in nude mice. Results miR‐107 was poorly expressed, whereas E2F1 and CCND1 were highly expressed in glioma tissues and cells. E2F1 bound to the promoter region of miR‐107 to induce transcriptional repression, and miR‐107 directly bound to CCND1 mRNA to reduce its expression. Overexpression of miR‐107 reduced proliferation, migration and invasion, and augmented apoptosis of glioma cells, and it reduced activity of the Wnt/β‐catenin pathway. The anti‐tumorigenic roles of miR‐107 were blocked by E2F1 or CCND1 overexpression. Similar results were reproduced in vivo where miR‐107 overexpression or E2F1 inhibition blocked tumor growth in nude mice. Conclusion This study suggested that E2F1 reduces miR‐107 transcription to induce CCND1 upregulation, which leads to progression of glioma via Wnt/β‐catenin signaling activation.

mainly include surgery, radiotherapy, adjuvant chemotherapy, targeted therapy, and immunotherapy (Weller et al., 2013). However, prognosis of patients with glioma, especially for those with GBM, remains unfavorable, and the median survival time of these patients is 15-23 months (Ostrom et al., 2014;Shergalis et al., 2018). There has been an urgent need for the development of more effective therapeutic options for glioma.
The molecular profiling of cancer has arisen due to the clinical values of key molecules in diagnosis, prognosis, and therapy of patients (Diamandis & Aldape, 2017). MicroRNAs (miRNAs) are a subclass of short non-coding RNAs approximately 22 nucleotides long and they are emerging molecules in the field of cancer research owing to their involvements in fundamental cellular processes and potent regulation on target mRNAs (Harrandah et al., 2018). The prognostic and diagnostic values of miRNAs in glioma have aroused increasing concerns (Zhou et al., 2018). miR-107 has been demonstrated as a candidate tumor suppressor in several human malignancies such as colorectal cancer (Fu et al., 2019) and cervical cancer . Moreover, miR-107 has been reported to be poorly expressed in glioma cells (Zhen et al., 2019), and downregulation of miR-107 was correlated with invasiveness, proliferation, and stem-like properties of glioma cells (Wu et al., 2020;Yang et al., 2017). This body of evidence suggested that miR-107 may serve as a tumor suppressor in glioma. E2F transcription factor 1 (E2F1) is a member of the E2Fs family of transcription factors that mediate transcription activity of genes implicated in development, differentiation, proliferation, and apoptosis (Muller et al., 2001). Downregulation of E2F1 by miRNAs has been observed to be associated with reduced proliferation of glioma cells (Huang & Chi, 2019;. Interestingly, miR-107 was found to bind to E2F1 mRNA to regulate its expression (Carroll et al., 2012). Moreover, in addition to serving as miRNA targets, as a transcription factor, E2F1 can also regulate the transcription activity of specific transcripts including miRNA (Aguilar et al., 2021;. The integrated bioinformatic analyses in the present study suggested that E2F1 had a potential binding relationship with the promoter region of miR-107 which in turn had a binding sequence with the 3ʹuntranslated region (3ʹUTR) of cyclin D1 (CCND1) mRNA. CCND1 has recently been revealed to be highly expressed in glioma and reduced cancer cell apoptosis (Sun et al., 2020). Therefore, this study hypothesized that there might be an E2F1/miR-107/CCND1 axis involved in the pathogenesis of glioma. Altered expression of these molecules was induced in glioma cells for in vitro and in vivo experiments to validate this hypothesis.

Sample collection
Glioma tissue samples were obtained from the neurosurgery department of the Second Affiliated Hospital of Nanchang University. Samples from 23 patients with glioma treated from January 2018 to August 2019 were included. All samples were confirmed as glioma tissues by pathological examination. Another 10 normal brain tissues collected from patients who underwent intracranial surgery for craniocerebral injury were collected as control samples. All tissue samples were instantly frozen in liquid nitrogen and stored at −80°C until further use.

Immunochemistry
The tissue samples were embedded in paraffin, cut into 5-μm sections,

Cell culture and treatment
A normal brain glial cell line Heb was procured from Jennio-bio miR-107 + oe-NC group, miR-107 + oe-E2F1 group, and sh-E2F1 + oe-CCND1 group. In detail, glioma cells in good growth condition were digested in trypsin and resuspended to 5 × 10 4 cells/ml. The cell suspension was cultured in 6-well plates (2 ml per well) at 37°C overnight.
After that, the cells in each well were transfected with the LV packagescarried target plasmids in 1 ml culture medium (MOI: 5-10). Interference of two target genes was achieved by infecting cells with two LV packages concomitantly. After 48 h, the supernatant was collected.
The LV particles in the supernatant were diluted, and the virus titer was examined. Exponentially growing virus was collected. Stably transfected cell colonies were screened by puromycin resistance, and the stable transfection was further validated by enzyme digestion and electrophoresis, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and sequencing.

Chromatin immunoprecipitation (ChIP)-qPCR
The glioma cells were fixed in methanol for 10 min for DNA-protein crosslinking. The cells were fractured via ultrasonication to obtain chromatin fragments. The fragments were centrifuged at 4°C for 10 min at 12,000 rpm to collect the supernatant into two tubes. The tube was incubated with anti-IgG (negative control) (1:2500; Abcam Cat#: ab6785; RRID: AB_955241; Abcam Inc. ) or anti-E2F1 (1:1000, Abcam Cat#: ab112580; RRID: none; Abcam Inc.) at 4°C overnight. The DNA-protein complex was precipitated using Protein A agarose and was centrifuged for 5 min at 12,000 rpm to discard the supernatant.
The non-specific binding complexes were washed away, and the specific binding ones were de-crosslinked at 65°C overnight. The DNA fragments were extracted using phenol/chloroform, purified, and collected. Enriched fragments in the miR-107 promoter were examined by qPCR.

Immunofluorescence staining
The glioma cells were seeded on slides and fixed with 4% paraformaldehyde (PFA) for 10 min. Thereafter, the cells were incubated with

Transwell assay
Cell migration and invasion were examined using transwell chambers

Flow cytometry
An Annexin V-phycoerythrin (PE)/7-aminoactinomycin D (7-AAD) kit (BD Biosciences) was used to examine cell apoptosis. The 7-AAD is a standard flow cytometry probe used to distinguish living and nonliving cells. In short, the glioma cells were resuspended in 1 × binding buffer and incubated with Annexin V-PE/7-AAD in the dark at 25°C for 15 min. Apoptosis of cells was then analyzed using a FACS Canto™II flow cytometer (BD Biosciences) within 1 h.

Dual luciferase reporter gene assay
The specific binding sequence between miR-107 and CCND1 mRNA was predicted from the bioinformatic system LNCIPEDIA (https: //lncipedia.org/), and the binding relationship was validated by a was examined using Image J software.

Xenograft tumors in nude mice
A172 cells stably transfected with NC mimic, miR-107 mimic, sh-NC + oe-NC, sh-E2F1 + oe-NC, sh-E2F1 + oe-CCND1 were resuspended in serum-free medium to 2 × 10 6 cells/ml. Twenty-five specific pathogenfree grade nude mice (4-6 weeks old, 17-20 g) procured from SLAC Laboratory Animal Co., Ltd. (Shanghai, China) were allocated into five groups, n = 5 in each. After animal anesthesia using diethyl ether and disinfection, 2 × 10 5 A172 cells (100 μl) with above transfections were injected into the right hemisphere of the mice via stereotactic injection. In short, the mice were anaesthetized by 80 mg/kg pentobarbital sodium (1%). The head was fixed, and the disinfected injection needle with cell suspension was placed in the surgical area above the mouse skull. An injection hole was drilled, and the injection site was located 0.62 mm in front of the bregma, 1.5 mm to the right of the midline, and 3.5 mm below the dura. The needle was placed into the injection hole, and the cell suspension was slowly injected into the brain tissue. After injection, the needle was taken out, and the wound was disinfected and glued with bone wax. On the 21st day after injection, the animals were euthanized by intraperitoneal injection of 150 mg/kg pentobarbital sodium. The volume of the xenograft tumors was calculated as follows: volume = length × width 2 /2, and the tumor weight was examined.

Statistical analysis
SPSS21.0 (IBM Corp. Armonk, NY, USA) was applied for data analysis. Measurement data were shown as the mean ± standard test was applied for comparison between every two groups. Differences among multiple groups were compared by the one-or two-way analysis of variance (ANOVA). p < .05 indicated that the difference was statistically significant.

miR-107 inhibits proliferation, migration and invasion, and promotes apoptosis of glioma cells
To examine the role of miR-107 in glioma, we first detected the miR-107 expression in the glioma tissues. As shown in Figure 1a Artificial overexpression of miR-107 was introduced into A172 and T98G cells via administration of LV-carried miR-107 mimic, and successful transfection was confirmed by RT-qPCR ( Figure 1c). Thereafter, the EdU labeling assay suggested that the DNA replication, namely, the proliferation ability of cells, was significantly reduced after miR-107 upregulation (Figure 1d). The transwell assay results showed that the migration and invasion abilities of both A172 and T98G cells were suppressed by miR-107 mimic versus mimic NC (Figure 1e,f). In addition, the apoptosis of A172 and T98G cells, according to the flow cytometry, was significantly reduced after miR-107 overexpression ( Figure 1g).

E2F1 suppresses miR-107 transcription
E2F1 has been reported as a cancer driver in glioma (Zhi et al., 2019).
The promoter sequence of miR-107 was obtained from UCSC (https: //genome.ucsc.edu/index.html). Intriguingly, E2F1 was predicted to own a binding relationship with the promoter region of miR-107 at the −16 to −26 bp site according to the ALGGEN system F I G U R E 2 E2F1 suppresses miR-107 transcription. (a) Potential binding sites between E2F1 and the promoter region of miR-107 (−16 to −23 bp and −19 to −26 bp) predicted on the ALGEEN system; (b) mRNA and protein levels of E2F1 in glioma tumor tissues and in normal tissues determined by RT-qPCR and western blot analysis, respectively; (c) mRNA and protein levels of E2F1 in normal brain glial cells (Heb) and in glioma cell lines (U251, A172 and T98G) examined by RT-qPCR and western blot analysis, respectively; (d) Expression of E2F1 mRNA and miR-107 in A172 and T98G cells after E2F1 overexpression examined by RT-qPCR; (e) enrichment of E2F1 at the −16 to −26 bp site at the promoter region examined by a ChIP-qPCR assay. Data were collected from three independent experiments and presented as mean ± SD. Differences were compared by unpaired t test (b), one-way ANOVA (c), or two-way ANOVA (d,e), *p < .05 versus Normal/Heb/oe-NC (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo) (Figure 2a). Therefore, we examined the expression of E2F1 in glioma tissues and normal brain tissues. It was found that the mRNA and protein expression of E2F1 was higher in tumor tissues than in normal tissues ( Figure 2b). In agreement with this, increased expression of E2F1 was found in U251, A172, and T98G cells compared to the Heb cells ( Figure 2c).
Overexpression of E2F1 was introduced in A172 and T98G cells, after which the miR-107 expression was significantly decreased (Figure 2d). The binding relationship between E2F1 and miR-107 was validated via a chromatin immunoprecipitation (ChIP)-qPCR assay. Importantly, an enrichment of E2F1 fragments was found at the −16 to −26 bp site at the promoter region of miR-107 (Figure 2e). These results indicated that E2F1 can bind to the promoter region of miR-107 to repress its transcription in glioma cells.

miR-107 targets CCND1 mRNA
According to the bioinformatic analysis in the StarBase system (http:// starbase.sysu.edu.cn/), miR-107 was predicted to have a specific binding site with CCND1 mRNA (Figure 3a), which has been recently reported to be upregulated in glioma (Sun et al., 2020). We surmised that miR-107 possibly regulates CCND1 expression to mediate glioma progression.
Therefore, we examined the expression of CCND1 in glioma samples. The RT-qPCR and immunochemistry (IHC) staining results indicated that the level of CCND1 was higher in glioma tissues than in normal tissues (Figure 3b,c). In cells, the RT-qPCR and western blot assays suggested that the mRNA and protein expression of CCND1 were higher in glioma cell lines (U251, A172, T98G) than in Heb cells ( Figure 3d).

F I G U R E 3 miR-107 targets CCND1 mRNA. (a)
Binding sequence between miR-107 and CCND1 mRNA predicted on the StarBase system; (b) mRNA and (c) protein levels of CCND1 in normal brain tissues (n = 10) and glioma tissues (n = 23) examined by RT-qPCR and IHC staining, respectively; (d) mRNA and protein levels of CCND1 in normal brain glial cells (Heb) and in glioma cell lines (U251, A172, and T98G) examined by RT-qPCR and western blot analysis, respectively; (e) Binding relationship between miR-107 and CCND1 mRNA validated through a luciferase assay; (f) mRNA expression of CCND1 in A172 and T98G cells after oe-E2F1 or miR-107 mimic transfection examined by RT-qPCR. Data were collected from three independent experiments and presented as mean ± SD. Differences were compared by unpaired t test (b and c), one-way ANOVA (d and f), or two-way ANOVA (e), *p < .05 versus Normal/Heb/oe-NC; #p < .05 versus NC mimic The binding relationship between miR-107 and CCND1 mRNA was validated using a luciferase assay (Figure 3e). It was found that miR-107 mimic significantly reduced the luciferase activity of CCND1-3ʹUTR-WT vector in 293T cells, whereas it had no effect on the activity of the CCND1-3ʹUTR-MUT luciferase vector.
The RT-qPCR results also indicated that the level of CCND1 mRNA was increased in A172 and T98G cells in the setting of E2F1 overexpression. In addition, compared to NC mimic, transfection of miR-107 mimic significantly reduced the CCND1 expression in the glioma cells ( Figure 3f).

E2F1 regulates the miR-107/CCND1 axis to promote malignant behaviors of glioma cells
To further validate the interactions between E2F1, miR-107, and CCND1, the A172 and T98G cells were concomitantly transfected with F I G U R E 4 E2F1 regulates the miR-107/CCND1 axis to promote malignant behaviors of glioma cells. Expression of miR-107, E2F1 and CCND1 mRNA, and E2F1 and CCND1 protein in (a) A172 and (b) T98G cells determined by RT-qPCR and western blot analysis, respectively; (c) Proliferation of A172 and T98G cells determined by the EdU labeling assay; (d) Migration and (e) invasion abilities of A172 and T98G cells examined by the transwell assays; (f) Apoptosis rate of A172 and T98G cells determined by flow cytometry. Data were collected from three independent experiments and presented as mean ± SD. Differences were compared by one-way (c-f) or two-way ANOVA (a,b), *p < .05 versus miR-107 mimic +oe-NC miR-107 mimic and oe-E2F1 or oe-CCND1. Importantly, overexpression of E2F1 reduced the expression of miR-107 but increased the level of CCND1 in A172 and T98G cells. Concomitant transfection of oe-CCND1 did not alter the expression of E2F1 or miR-107, but only increased the expression of CCND1 in cells (Figure 4a,b).
The malignant behaviors of cells with miR-107 mimic and oe-E2F1/oe-CCND1 transfections were examined. The EdU labeling assay results suggested that the proliferation ability of A172 and T98G cells inhibited by miR-107 mimic was restored upon E2F1 or CCND1 overexpression (Figure 4c). Also, the migration and invasion potentials of cells were recovered by oe-E2F1 or oe-CCND1 according to the tran-swell assays (Figure 4d,e). Moreover, the flow cytometry results indicated that the miR-107-induced apoptosis in A172 and T98G cells was blocked after E2F1 or CCND1 upregulation (Figure 4f).

E2F1 mediates the Wnt/β-catenin signaling pathway
CCND1 is one of the downstream targets of the Wnt/β-catenin pathway, but it can also regulate the nuclear translocation of β-catenin . Here, we examined the nuclear translocation of F I G U R E 5 E2F1 mediates the Wnt/β-catenin signaling pathway. (a) Nuclear translocation of β-catenin in A172 and T98G cells examined by immunofluorescence staining; (b) TOP/FOP activity in A172 and T98G cells determined by the TOP/FOP flash assay; (c) Protein levels of Wnt10B and β-catenin in A172 and T98G cells detected by western blot analysis. Data were collected from three independent experiments and presented as mean ± SD. Differences were compared by one-way ANOVA (b) or two-way ANOVA (c), *p < .05 versus miR-107 mimic +oe-NC β-catenin using immunofluorescence staining and TOP/FOP activity using the TOP/FOP flash assay. As shown in Figure 5a,b, either overexpression of E2F1 or CCND1 increased the nuclear accumulation of β-catenin as well as the activity of TOP/FOP in A172 and T98G cells.
The nuclear levels of the Wnt/β-catenin signaling-related proteins Wnt10B and β-catenin were further examined by western blot analysis. In concert with the above results, increased nuclear protein levels of Wnt10B and β-catenin were detected in A172 and T98G cells transfected with miR-107 mimic + oe-E2F1 or miR-107 mimic + oe-CCND1 ( Figure 5c).

The functions of E2F1, miR-107, and CCND1 in tumorigenesis of glioma cells in vivo
To further examine the role of E2F1/miR-107/CCND1 axis in glioma progression, A172 cells stably transfected with NC mimic, miR-107 mimic, sh-NC + oe-NC, sh-E2F1 + oe-NC and sh-E2F1 + oe-CCND1 , and β-catenin in tumor tissues examined by western blot analysis. Data were collected from three independent experiments and presented as mean ± SD. In each group, n = 5. Differences were compared by one-way ANOVA (b,c) or two-way ANOVA (d,e), *p < .05 versus NC mimic; #p < .05 versus sh-NC + oe-NC; and p < .05 versus sh-E2F1 + oe-NC

DISCUSSION
Gliomas, which represent 80% of all malignant tumors in the CNS, are virtually incurable since the 5-year overall survival rate of the most common but aggressive type GBM is no more than 5%, even following the optimal treating combinations (Chen et al., 2012;Hombach-Klonisch et al., 2018). Researchers in this field have made significant efforts in identifying the molecular mechanisms involved in cancer progression. Here, this study reports that there might be an E2F1/miR-107/CCND1 axis that mediates the malignant development of glioma cells in vitro and in vivo with the implication of Wnt/β-catenin signaling.
MiRNAs have emerged as key molecules having important diagnostic and prognostic values in glioma (Mondal & Kulshreshtha, 2021;. Recent studies mainly focused on their interactions with other ncRNAs (Wu & Qian, 2019). miR-107 has been demonstrated as a tumor expressed at low levels in several human cancers such as colorectal cancer (Fu et al., 2019), cervical cancer Rui et al., 2018), and non-small cell lung cancer (Fan et al., 2020). However, studies have also suggested that miR-107 may confer chemoresistance to cancer cells (Liang et al., 2020) and promote growth and invasiveness of gastric cancer . This may be attributed to the different genes they regulated. In this study, we observed that miR-107 expression was reduced in glioma tissues and the acquired glioma cells. Artificial upregulation of miR-107 reduced proliferation, migration and invasion, and increased apoptosis of A172 and T98G cells. In concert with this, poor expression of miR-107 has been found in glioma in previous reports (Su & Song, 2018;Zhen et al., 2019). Restoration of miR-107 facilitated apoptosis, increased chemo sensitivity, and weakened proliferation, invasiveness, and angiogenesis of glioma cells (Chen et al., 2016;Su & Song, 2018;Wu et al., 2020).
In the study, a similar trend was reproduced in the in vivo experiments where upregulation of miR-107 in A172 cells significantly reduced the volume and weight of xenograft tumors.
E2F1 has been reported as an oncogene, and its downregulation by miRNAs has been suggested to reduce malignant behaviors, such as proliferation, invasiveness, and resistance to apoptosis of cancer cells (Han et al., 2020;Peng et al., 2020). This is also true for glioma, because upregulation of E2F1 has been correlated with proliferation, cell cycle progression, and carcinogenesis of glioma . In addition, previous studies have demonstrated that E2F1 can transcriptionally regulate multiple miR-NAs, such as elevating miR-17−92 cluster, miR-15/16, miR-203, and miR-449a/b levels, and reducing miR-30b and miR-1205 levels Ofir et al., 2011;Tan et al., 2014;Wang et al., 2015;Yang et al., 2009;Zhang et al., 2015). E2F1 belongs to the E2Fs family of transcriptional factors which regulate a multitude of genes involved in multiple key cellular processes. Here, we predicted the potential binding site between E2F1 and the promoter region of miR-107 and had the binding relationship validated using a ChIP-qPCR assay. As expected, high F I G U R E 7 A graphic abstract. E2F1 binds to the promoter region of miR-107 to suppress its transcription, which restores the expression of the miR-107 target gene CCND1, therefore promoting growth and metastasis of glioma cells expression of E2F1 was confirmed in the glioma tissues and cells, which showed an inverse trend with miR-107. Importantly, the proliferation, migration and invasion, and the resistance to apoptosis of cells reduced by miR-107 mimic were restored upon further overexpression of E2F1.
These results suggested that downregulation of miR-107 is possibly implicated in the oncogenic events mediated by E2F1 in glioma.
The bioinformatics analysis and luciferase assay confirmed CCND1 as a target transcript of miR-107. CCND1 is a crucial cell cycle regulatory protein whose expression and cellular localization is frequently transformed in tumor cells (Xie et al., 2017), and it is capable of inducing cell proliferation, invasion, and transformation in human malignancies (Lin, 2017). This is also true for glioma (Alqudah et al., 2013;Yamada et al., 2018). Here, we found CCND1 was highly expressed in glioma tissues and cells, and its expression was reduced by miR-107, but reduced by E2F1. Importantly, upregulation of CCND1 restored the proliferation, resistance to death, migration, and invasion of glioma cells and augmented the malignant growth of xenograft tumors. CCND1 is one of the important downstream targets of the Wnt/β-catenin signaling, a master regulator in carcinogenesis (Ghanavati et al., 2020;. Likewise, this signaling pathway is frequently activated during the pathogenesis of glioma (He et al., 2019). In the study by Xia et al. (2019), CCND1 has been reported to have elevated the nuclear translocation of β-catenin in cells. The authors suggested that CCND1 can accelerate the β-catenin of nuclear binding to the Nanog's promoter. As a transcriptional factor, Nanog possibly plays a role in the expression and nuclear accumulation of β-catenin. However, the specific mechanism remains to be further explored. Interestingly, miR-107 has been reported to suppress the activation of the Wnt/β-catenin signaling pathway to suppress proliferation and metastasis of cancer (Yao et al., 2021;Yu et al., 2018). Here, we confirmed that miR-107 reduces nuclear translocation of β-catenin, whereas the activity of the Wnt/β-catenin pathway was increased upon E2F1 or CCND1 overexpression.

CONCLUSION
In conclusion, by performing both cellular and animal experiments, we confirmed that the transcription factor E2P1 can induce transcriptional repression of miR-107 and block its inhibitory effect on CCND1, which leads to the malignant development of glioma with the involvement of the Wnt/β-catenin signaling pathway (Figure 7). This study confirmed the oncogenic roles of E2F1 and CCND1 and the antitumorigenic role of miR-107 in glioma. We hope these findings may offer a new understanding on the molecular mechanism involved in glioma pathogenesis.

ACKNOWLEDGMENT
We would like to thank the Key Scientific and Technological Research Projects of Jiangxi Provincial Education Department (No. 191404) for the funding support.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
All the data generated or analyzed during this study are included in this published article.

TRANSPARENT PEER REVIEW
The transparent peer review history for this article is available at https: //publons.com/publon/10.1002/brb3.2399