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LncRNA FOXD3-AS1 Promotes Tumorigenesis of Glioma via Targeting miR-128-3p/SZRD1 Axis

Background The aim of the current study was to investigate the roles of LncRNA FOXD3-AS1 (FOXD3-AS1) in the glioma progression, and its underlying mechanism of competing endogenous RNA (ceRNA) network of FOXD3-AS1/miR-128-3p/SZRD1. Materials and Methods The FOXD3-AS1 expression and its prognostic relation were detected by bioinformatics tool. Next, glioma cell lines (HS683, U251, T98G, and SNB-19) were used to verify the FOXD3-AS1 expression. Furthermore, the roles of the FOXD3-AS1/miR-128-3p/SZRD1 axis on the glioma development in vitro and in vivo were examined. Results Bioinformatics analysis showed that FOXD3-AS1 was upregulated in the glioma and linked with poor prognosis. Consistently, FOXD3-AS1 level was overexpressed in the glioma cell lines (HS683 and U251). Subsequently, we verified that silencing of FOXD3-AS1 (si-FOXD3-AS1) restrained the cell proliferation, invasion, and tumor growth in vivo, and induced G0/G1 arrest, and promoted apoptosis. Further study also stated that FOXD3-AS1 interacted with miR-128-3p and SZRD1 was the target gene of miR-128-3p. Moreover, overexpression of miR-128-3p restrained the cell proliferation and metastasis of glioma, and reduced the SZRD1 level. Rescue assay illustrated that miR-128-3p inhibitor could reverse the suppressive impact of si-FOXD3-AS1 on the glioma progression. Similarly, SZRD1 overexpression could neutralize the influences of miR-128-3p mimic on glioma progression. Conclusion FOXD3-AS1 promoted the tumorigenesis of glioma, and exerted its function to modulate SZRD1 by targeting miR-128-3p.


Introduction
As a dominating primary intracranial tumor, glioma is featured by high aggression, poor prognosis, and high lethality. 1 On the basis of the grading criteria, glioma is classified as low-grade glioma (LGG, I-II) and high-grade glioma (HGG, III-IV). 2 Of these, patients with LGG have longer overall survival in contrast to HGG. 3 Glioblastomas (GBM), refers to the most aggressive type (grade IV), with a proportion of 65% in brain tumors. 4 Despite advances in therapeutic treatment, most patients achieve unsatisfactory efficacy and have poor prognosis, especially with GBM. 5 Like other tumors, the progression of glioma is a complex procedure involving changes of pathology, genes, and pathways. 6 Currently, more attention has been paid to the molecular mechanism of glioma progression. 7 However, there is no definite conclusion about pathogenesis of glioma. Thus, elucidating the molecular mechanism is an urgent need for glioma therapy.
Accumulating evidence has stated that LncRNAs exert crucial roles on the progression of various cancers including glioma. [8][9][10] Moreover, a previous study has reported that LncRNAs as competing endogenous RNAs (ceRNAs) by sponging miRNAs to directly target the mRNAs is the key mechanism for regulation of tumor growth and metastasis. 11 In terms of glioma, recent studies have verified this point. 12,13 Those findings suggested that the LncRNA as ceRNA may be the underlying mechanism to glioma development.
LncRNA FOXD3-AS1 (FOXD3-AS1) is highly expressed and may exert the crucial function on cancer progression through the ceRNA network, including lung cancer, malignant melanoma, and colon adenocarcinoma. [14][15][16] Consistently, FOXD3-AS1 is upregulated in glioma, which has been demonstrated in clinical specimen detection. 17 However, no reports of FOXD3-AS1 as ceRNA regulating glioma development are available. Microarray studies have been conducted to screen the aberrantly expressed miRNAs in glioma and found that miR-128-3p is observably declined. 18,19 Moreover, miR-128-3p is the target gene of FOXD3-AS1, which has been elucidated in cervical cancer. 20 Another study has also demonstrated that miR-128-3p suppresses the proliferation and metastasis of glioma cells via binding to 3ʹ-UTR of SZRD1, thereby influencing the glioma tumorigenicity. 21 Thus, we speculated that FOXD3-AS1 regulating miR-128-3p/SZRD1 axis may be an underlying mechanism in glioma progression. In the current study, we first verified that functioned as ceRNA to regulate the glioma progression. In addition, miR-128-3p/SZRD1 axis as a novel target of FOXD3-AS1 in regulation of glioma development may supply theoretical foundation for further study.

Flow Cytometry Assay
For analysis of cell apoptosis, HS683 and U251 cells (1×10 6 cells/mL) were obtained after 48 h of transfection, and washed with cool phosphate buffered saline (PBS) three times. Cell apoptosis was detected by flow cytometer (NovoCyte, Agilent, Palo Alto, CA, USA) with annexin V-propidium iodide (PI) kit (Meilunbio, Dalian, China). To detect cell cycle, collected cells were fixed in the 75% ethanol. Having been washed, the cells were stained with PI, followed by analysis with flow cytometer (NovoCyte, Agilent, Palo Alto, CA, USA).

Transwell Assay
HS683 and U251 cells (1×10 5 cells/mL) were placed in a transwell chamber (Corning, Lowell, MA, USA), and the culture medium was in the lower chamber. Subsequently, the invasion and migration cells were fixed with 4% paraformaldehyde and then dyed with 0.1% crystal violet. The number of cells was quantified by microscope (CKX53, Olympus, Tokyo, Japan).

RNA Immunoprecipitation (RIP) Assay
RIP assay was conducted as previously described. 22 Briefly, transfected HS683 and U251 cells were lysed and incubated with magnetic beads conjugated with human anti-Argonaute2 (Ago2) antibody, negative control (anti-IgG) or input Control (Millipore) for six hours. Subsequently, immunoprecipitated RNA was isolated and detected by qRT-PCR.

Tumorigenesis Assay in vivo
A total of 72 nude mice (five weeks old) were obtained from Hangzhou Ziyuan Laboratory nimal Science and Technology Co. Ltd (Hangzhou, China). The mice were divided into six groups, which were inoculated with HS683 and U251 cells (1×10 6 cells/mL) transfected with control, sh-FOXD3-AS1, sh-NC; control, miR-128-3p mimic, and mimics-NC, respectively (12 mice per group). The nude mice in the control group were injected with glioma cells untreated. The tumor volume was assessed from one week to six weeks after tumor inoculation (once every week). Subsequently, mice were sacrificed by cervical dislocation and the tumor tissues were collected for the further experiments.

Bioinformatics Analysis
The FOXD3-AS1 expression profiles (GSE147352) in glioma patients were retrieved from the Gene Expression Omnibus (GEO) database. The dataset included 85 GBM samples, 18 LGG samples and 15 normal samples. Gene expression was estimated using fragments per kilobase of transcript per million mapped reads (FPKM) value and then Boxplot was visualized using the ggplot2 package in R Language. The differential expression between normal and tumor groups was analyzed by R package Deseq2 on basis of following criteria: fold change >2 and Padj <0.05. Samples were collected according to inclusion criteria as following: (1) patients were diagnosed as glioma; (2) data were available on expression of FOXD3-AS1; and (3) data of samples on FOXD3-AS1 expression had corresponding control group. Samples were excluded according to following exclusion criteria: (1) FOXD3-AS1 expression was not included; (2) control group was not included; and (3) cell samples were used.
The Cancer Genome Atlas (TCGA) data were applied to analyze the relationship between FOXD3-AS1 expression and patient's prognosis. 638 glioma patients with FOXD3-AS1 expression and survival information were included into current analysis. The survival analysis was performed using R language with "Survival" package and survival curves were plotted using "Survminer" package. Samples were collected according to inclusion criteria as following: (1)

Statistical Analysis
Data were presented as the means ± standard deviation (SD) and analyzed by SPSS 20.0 statistical software. The differences in the multiple groups were compared by oneway ANOVA followed by LSD test, respectively. P<0.05 was termed as statistically significant.

FOXD3-AS1 Was Upregulated in Glioma and Correlated with Poor Prognosis
To investigate the function of FOXD3-AS1 in glioma, we downloaded the FOXD3-AS1 expression profiles (GSE147352) in glioma patients from GEO database. As displayed in Figure 1A and B, the FOXD3-AS1 expression in the GBM and LGG tissues (tumor group) was higher than that in normal tissues (normal group) (P<0.01) ( Figure 1A and B). Consistently, data from TCGA database showed that FOXD3-AS1 in the tumor group including GBM and LGG was higher than that in the normal group (P<0.01) ( Figure 1C). In addition, it also found that FOXD3-AS1 expression in glioma patients including GBM and LGG was negatively correlated with overall survival (P<0.01) ( Figure 1D). Similarly, FOXD3-AS1 expression in glioma cell lines (HS683 and U251) was higher than that of HEB ( Figure 1E) (P<0.01), indicating that HS683 and U251 cell lines can be used for further study.

Knockdown of FOXD3-AS1 Influenced Cell Cycle and Promoted Apoptosis of Glioma Cells via Modulating miR-128-3p
As shown in Figure 4A-D, the cell proportion at G0/G1 phase was prominently increased in the si-FOXD3-AS1 group, however miR-128-3p inhibitor reduced the cell proportion at G0/G1 phase (P<0.01). The cell proportion at different phases in si-FOXD3-AS1+miR-128-3p inhibitor group exhibited no obvious difference in contrast to control and si-NC groups (P>0.05). In the aspect of cell apoptosis, we discovered that apoptosis rate was notably increased in the si-FOXD3-AS1 group, whereas it was decreased in the miR-128-3p inhibitor group in contrast to control and si-NC groups (P<0.01). Moreover, miR-128-3p inhibitor can reverse the influences of si-FOXD3-AS1 on apoptosis rate (P<0.01) ( Figure 4E-H). Collectively, the aforementioned results concluded that si-FOXD3-AS1 influenced cell cycle and facilitated apoptosis of glioma cells via modulating miR-128-3p.

Silencing of FOXD3-AS1 Inhibited Tumorigenesis in vivo via Modulating miR-128-3p
To validate the effect of FOXD3-AS1 on glioma in vitro, xenograft tumor model was conducted. First, the  Figure 5A, FOXD3-AS1 expression was decreased while miR-128-3p level increased after transfected with sh-FOXD3-AS1 (P<0.01). Subsequently, we found that the tumor size and weight of the sh-FOXD3-AS1 group was remarkably lessened in contrast to control and sh-NC groups (P<0.05) ( Figure 5B-G).

MiR-128-3p Targeted the 3ʹ-UTR of SZRD1
As shown in Figure 6A, the 3ʹ-UTR of SZRD1 contains a targeting site for the region of miR-128-3p. Luciferase reporter assays showed that the relative luciferase activity of SZRD1-3ʹ-UTR-WT was remarkably decreased in overexpressed miR-128-3p group ( Figure 6B).

Overexpressed miR-128-3p Suppressed Proliferation and Invasion of Glioma Cells via Modulating SZRD1
MiR-128-3p mimic and OE-SZRD1 were co-transfected into HS683 and U251 cells, and it turned out that miR-128-3p expression was remarkably elevated in the miR-128-3p

Overexpression of miR-128-3p Inhibited Tumorigenesis in vivo via Modulating SZRD1
As shown in Figure 8A, qRT-PCR results validated transfection of miR-128-3p mimic (P<0.01). In vivo, it indicated that tumor size and weight of miR-128-3p mimic group was conspicuously decreased compared with control and mimics-NC groups (P<0.01) ( Figure 8B-G). Importantly, the schematic for the regulatory relationship among FOXD3-AS1, miR-128-3p, and SZRD1 in glioma was shown in Figure 9. Those finding proved that overexpression of miR-128-3p restrained tumorigenesis in vivo via modulating SZRD1.

Discussion
To our knowledge, dysregulation of LncRNAs frequently occurs in the various cancers, which is deemed as a master regulator for disease progression. 23 Here, our study showed that FOXD3-AS1 was upregulated in glioma and correlated with poor prognosis using bioinformatic analysis. In addition, FOXD3-AS1 level in the glioma cell lines was consistent with bioinformatic analysis. Furthermore, knockdown of FOXD3-AS1 suppressed the tumorigenesis of glioma via targeting miR-128-3p/SZRD1 axis, as evidenced by cell viability, invasive ability, apoptosis rate, cell cycle, and tumorigenesis in vivo.
FOXD3-AS1, is a 963-bp lncRNA, which is located in the chromosome 1p31.3 upstream of FOXD3 promoter, 24 indicating that its function is closely associated with adjacent protein-coding transcripts. 25 Beyond that, FOXD3-AS1 acts as independent prognostic indicator for prediction of neuroblastoma progression, which has been proved. 24   Similarly, dysregulation of FOXD3-AS1 expression is observed in numerous cancers. 14,26,27 In terms of glioma, Chen et al 17 have clarified that si-FOXD3-AS1 inhibited the tumor development as demonstrated by cell proliferation, migration, and invasion. Consistently, our study found that FOXD3-AS1 was highly expressed in glioma, and si-FOXD3-AS1 restrained the tumor growth in vivo and in vitro. In addition, glioma patients with overexpressed FOXD3-AS1 had lower overall survival, indicating FOXD3-AS1 was closely associated with poor prognosis. Furthermore, Wang et al 28 have demonstrated that FOXD3-AS1 may be functioning as ceRNA to exert the pivotal role in the regulation of cancer progression. To date, knowledge about FOXD3-AS1 ceRNA network involved in glioma progression remains vague.
MiR-128-3p was initially discovered in GBM, 29 which attracts more attention at present because it acts as a tumor suppressor and early diagnostic indicator. 30,31 A similar study has verified that miR-128-3p can increase the sensitivity of chemotherapy of colorectal cancer. 32 In the gliomas, Bendahou et al 33 have revealed that patients with high miR-128-3p expression have longer overall survival than low miR-128-3p using bioinformatic analysis. A similar study has demonstrated that miR-128 may be effective therapy for disruption of tumor-relevant phenotypes and tumor ingrowth. 34 Furthermore, a previous study has reported that miR-128-3p restrained the cell proliferation and accelerated apoptosis. 18 Aforementioned findings were aligned with our results. Our study showed that overexpression of miR-128-3p restrained proliferation and tumorigenesis in vivo, and accelerated apoptosis of glioma cells. On the contrary, inhibition of miR-128-3p accelerated the glioma development. In regard to the interaction between miRNAs and LncRNAs, previous studies have been reported. For example, a previous study reported by Fu et al 22 has uncovered that miR-128-3p recuses the regulation of LncRNA PVT1 on glioma tumorigenesis. Another study also showed that inhibition of miR-128-3p eliminates the influence of si-LINC00346 on metastasis of glioma. 21 However, the relationship between miR-128-3p and FOXD3-AS1 in glioma has not been reported. Accordingly, our study firstly disclosed that miR-128-3p inhibitor reversed the effects of si-FOXD3-AS1 on glioma progression. Taken together, FOXD3-AS1 may be involved in the modulation of glioma development via sponging miR-128-3p.
In the current study, we also found SZRD1 is the targeted gene of miR-128-3p. SZRD1, a highly conserved protein, is first found in cervical cancer, which functions as a tumor suppressor. 35 Conversely, SZRD1 exerts the push roles on tumor development in the oligodendrogliomas, lymphoma, and glioma, 21,36,37 which are in accordance with our results that overexpression of SZRD1 facilitated the tumorigenesis. Moreover, our study displayed that miR-128-3p mimic reduced the SZRD1 expression, whereas overexpression of SZRD1 reversed the function of miR-128-3p mimic on inhibition of glioma development, indicating that miR-128-3p was bound with SZRD1. This results were in line with a previous study, which revealed the interaction between 9046 miR-128-3p and SZRD1. 21 The aforementioned findings manifested that miR-128-3p modulated the proliferation, and invasion of glioma cells via targeting SZRD1.
There were some limitations in the current study. First, the overall survival of mice among different groups was not examined in this study. In future, we will explore the effects of compounds on survival of mice including sh-FOXD3-AS1 and miR-128-3p. In addition, whether overexpression of FOXD3-AS1 influences on behaviors of glioma cell lines is unclear. Importantly, comparison of the growth rate and invasion among the glioma cell lines will be a potential research direction.

Ethics Approval and Informed Consent
The study was approved by the ethical committee of Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University (No. 2021-468), and all experiments conformed to Guide for the Care and Use of Laboratory Animals.

Author Contributions
All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding
The study was funded by Major Science and Technology Innovation Project of Shandong Province (2020SFXGFY03-2).