Loss of TXNDC12 Induces Oxidative Stress-mediated Autophagy in Glioblastoma Cell Lines

Redox homeostasis in glioblastoma cells is very important, but how it is maintained is still not well understood. Herein, we identied thioredoxin domain-containing protein 12 (TXNDC12) as a therapeutic target candidate which signicantly maintained redox homeostasis in GBM cells. Overexpression of TXNDC12 was associated with increased tumor grade and poor prognosis in patients with glioma (P < 0.001), according to the analysis of expression data from the public database Cancer Genome Atlas. TXNDC12 protein levels were signicantly elevated in glioblastoma compared to non-tumor brain tissue samples by immunohistochemistry and western blot analysis. Knockdown of TXNDC12 inhibited the proliferation of U251 and A172 in vitro. On day 21 post-implantation, tumor growth inhibition was 70% in A172 with stable expression of TXNDC12 shRNA in vivo (P < 0.001). Kaplan-Meier analysis of survival data showed that the overall survival of tumor-bearing animals increased from 20.3 days (control) to 28.7 days (knockout, P < 0.05). These data suggest that TXNDC12 is a key mediator in redox homeostasis both in vivo and in vitro. Thus, TXNDC12 is a potential treatment target of GBM. comprehensive analysis of expression and glioma of showed that patients with higher TXNDC12 expression levels had pathological malignancy, and prognosis. The results of cellular experiments indicated that TXNDC12 could promote the growth of glioma cells and TXNDC12 knockdown could signicantly inhibit the growth of glioma cells.


Introduction
Glioma is the most commonly diagnosed primary malignant brain tumor in adults, and 70% of them are high-grade gliomas (glioblastoma, GBM) 1 . In the clinical treatment of GBM, the median survival of patients is only 14 months despite the proven effectiveness of surgery combined with radiotherapy and chemotherpy 2 . Compared with other tumors, GBM is more sophisticated in treatment due to its high heterogeneity and high recurrence rate 3 . High redox in GBM cells is essential to maintain cell proliferation, division, and invasiveness, so it is important to explore the growth and proliferation mechanisms of The redox state of tumors changes with various intracellular physiological processes, for example, cells that are proliferating need to be in a reduced state more than differentiated cells; changes in the intracellular redox environment are important for either the cell cycle or apoptosis 4,5 . The intracellular redox state is mainly determined by intracellular redox coenzymes such as NAD(P)H-NAD(P) and reduced glutathione oxidized glutathione (GSH-GSSG) concentrations. Also, thioredoxin and its related proteins are disul de bond reductases that maintain the intracellular redox state [6][7][8] .
Thioredoxin domain-containing protein 12 (TXNDC12) is a protein with oxidase activity encoded by the TXNDC12 gene, which is a member of the thioredoxin superfamily 9,10 . TXNDC12 plays a key role in maintaining the intracellular reduction state, and its presence promotes the growth, migration, and invasion of hepatocellular carcinoma and gastric cancers 9 . However, information about the effect of TXNDC12 on GBM is unclear and awaits to be studied 11,12 .
To further reveal the role of TXNDC12 in the development of GBM, in this study, we use the Chinese Glioma Genome Atlas (CGGA) database, the brain tumor molecular database (Rembrandt), and The Cancer Genome Atlas (TCGA) to analyze the function of TXNDC12. The relationship between TXNDC12 and GBM grade and the patient prognosis was also investigated and analyzed by the gene knockdown technique. We found that TXNDC12 may promote the proliferation of GBM by facilitating the conversion of cells from the oxidized to the reduced state.

Results
2.1 TXNDC12 is overexpressed in human gliomas and is associated with a poor prognosis To investigate the role of TXNDC12 in human gliomagenesis, we rst analyzed the mRNA levels of this gene in human glioma samples using publicly available datasets from the TCGA database. TXNDC12 mRNA levels were elevated in low grade (WHO II; n = 226) and high-grade gliomas (WHO IV, n = 150; p < 0.0001) compared to non-neoplastic samples (n = 4, Figure 1A). TXNDC12 expression was markedly higher in oligodendrogliomas, mixed gliomas, astrocytomas, and GBM than in non-tumor tissues.
Rembrandt database results showed signi cant differences in TXNDC12 expression in WHO classes II, III, and IV, and the expression of TXNDC12 increased obviously with the increase of pathological grade ( Figure 1B). CGGA database also indicated that TXNDC12 mRNA levels were increased in high-grade gliomas ( Figure 1C).
Among all pathological types, GBM is the most malignant and has the worst prognosis. It has been demonstrated that the degree of in ltration of tumor microenvironmental cells, immune cells, and stromal cells may have an important impact on the prognosis of GBM, so we evaluated the differences in TXNDC12 expression between the classic, mesenchymal and anterior neurological types. The ndings from the CGGA database analysis suggested that TXNDC12 had the highest expression in the classic GBM, followed by the mesenchymal type, and the lowest expression was in the anterior neurological type ( Figure 1D). There was no apparent difference between the results of the Rembrandt database analysis and CGGA analysis, and the variation between pathological types was more evident ( Figure 1E). The TCGA database told us that the differences among classic, mesenchymal, and anterior neurological types were not statistically signi cant, but TXNDC12 expression was higher in the mesenchymal type and statistically different from the anterior neurological type ( Figure 1F).
To discuss the potential role of TXNDC12 expression in the survival of glioma patients, we generated Kaplan-Meier survival curves according to the CGGA database, Rembrandt database, and TCGA database. There was a noticeable difference between the high and low TXNDC12 expression groups, patients with a low expression of TXNDC12 had a longer survival time compared to the patients with a high level of TXNDC12 expression ( Figure 1G, Figure 1H, Figure 1I, p < 0.001).
2.2 Immunohistochemical staining and Western blot revealed increased expression of TXNDC12 in glioma samples and cell lines Immunohistochemical (IHC) staining of 60 para n-embedded clinical glioma specimens, including grade II (18 cases), grade III (18 cases), grade IV (18 cases), and non-tumor brain tissue specimens (6 cases), showed that TXNDC12 expression increased with tumor grade (Figure 2A, 2B). Also, we analyzed other Page 4/17 tumor-related factors, such as age, gender, tumor size, liquefactive necrosis, preoperative tumor edema, and tumor grade. The results showed that TXNDC12 expression was positively correlated with tumor grade and liquefactive necrosis, independent of age, gender, tumor size, and edema, suggesting that TXNDC12 may be a potential diagnostic marker for patients with glioma (Table 1, p < 0.001).
Taken together, these results suggest that TXNDC12 may play an important role in both glioma development and progression, and therefore TXNDC12 could be used as a new diagnostic protein marker.

Knockdown of TXNDC12 suppresses the viability of glioma cells
First, we designed and synthesized two independent small interfering RNAs (si-TXNDC12-1 and si-TXNDC12-2) and tested the knockdown e ciency on TXNDC12 in vitro. In the U251 cell line, the knockdown e ciency of si-TXNDC12-1 was 89.3% and that of si-TXNDC12-2 was 85% ( Figure 3A). In the A172 cell line, the knockdown e ciency of si-TXNDC12-1 was 88.3% and that of si-TXNDC12-2 was 79.3% ( Figure 3B). As the western blot results showed, the protein level of TXNDC12 was also visibly reduced after interfering with TXNDC12 mRNA expression, moreover, the e ciency of si-TXNDC12-1 was slightly higher than that of si-TXNDC12-2 ( Figure 3C, Figure 3D).
To verify the effect of TXNDC12, we performed cell viability assays on U251 and A172 cells with knockdown of TXNDC12. Compared with the control group, cell viability was greatly inhibited with the prolonged knockdown of TXNDC12. After 72h, the inhibition rate of cell activity was 39.5% for si-TXNDC12-1 and 24.5% for si-TXNDC12-2. The inhibitory effect of si-TXNDC12-1 was dramatically higher than that of si-TXNDC12-2 ( Figure 3E). Similar experimental results were shown in the A172 cell line, with 35.8% inhibition of cellular activity for si-TXNDC12-1 and 28.3% for si-TXNDC12-2 after 72 h ( Figure 3F).

Knockdown of TXNDC12 Inhibits glioma cells proliferation
For further investigation of the mechanism associated with the inhibition of cell activity, we performed phenotypic experiments related to the knockdown of TXNDC12. As shown in the EdU experiments, the number of cells in the proliferation phase (red uorescence) was declined after the knockdown of TXNDC12 in U251 cells ( Figure 4A), and the number of proliferating cells decreased from 27.7% to 10.7% ( Figure 4B). Similarly, in the A172 cell line, cell proliferation was also found to be signi cantly inhibited after knockdown of TXNDC12, with the number of cell proliferation decreasing from 30.7% to 12.0% ( Figure 4C, 4D). The results of the clone formation assay showed that the number of clonal colonies decreased and the size of clonal colonies became smaller after knockdown of TXNDC12 ( Figure 4E), with a decrease in the total clonal area of about 54.5% in the U251 cell line and about 49.9% in the A172 cell line ( Figure 4F).

Knockdown of TXNDC12 causes an increase in intracellular reactive oxygen species
Excessive production of reactive oxygen species (ROS), such as hydrogen peroxide (H 2 O 2 ), superoxide radicals (O 2-) and highly cytotoxic hydroxyl radicals (-OH), as well as the decreased ability of cells to scavenge ROS are the main causes of intracellular oxidative stress. Since hydrogen peroxide is produced by superoxide, superoxide levels can indirectly respond to hydrogen peroxide levels. We used the uorescent superoxide probe (dihydroethidium, DHE) to study superoxide levels. si-TXNDC12-1 transfected A172 and U251 cells showed signi cantly higher uorescence intensity than the control, which was approximately 2-fold elevated ( Figure 5A, 5B). Similarly, the level of H 2 O 2 in cells transfected with si-TXNDC12-1 was 1.8-fold higher than that in the control group, and H 2 O 2 was signi cantly reduced after the application of hydrogen peroxide scavenger (N-acety1-L-cysteine Figure 5C). The ROS uorescence probe results showed that the green uorescence intensity of si-TXNDC12-1 transfected cells was approximately 1.85-fold higher than that of the control group ( Figure 5D, 5E). The above results suggest that TXNDC12 may play an important role in scavenging intracellular ROS to maintain the balance of intracellular redox reactions.

Knockdown of TXNDC12 suppresses the growth of GBM cells in vivo
To examine the effect of TXNDC12 on tumor growth in vivo in tumor-bearing nude mice, we implanted luciferase-expressing A172-sh-TXNDC12-1 and A172-NC cells into the brains of nude mice (10 mice per group). Tumor growth was continuously monitored over 21 days using a bioluminescence assay. By day 14, the bioluminescence values of the two groups of animals were signi cantly different ( Figure 6A). On day 21 post-implantation, the mean volume of A172-sh-TXNDC12-1 tumors was reduced by approximately 65% compared to the NC group (P < 0.01; Figure 6 B). Kaplan-Meier analysis showed that the overall survival of tumor-bearing animals increased from 20.3 days (control group) to 28.7 days (knockdown group, P < 0.05; Figure 6C). Histological examination revealed that A172-sh-TXNDC12-1 tumors were smaller than NC tumors, and A172-sh-TXNDC12-1 tumor cells underwent signi cant apoptosis. There was less cell division in the tumor tissue ( Figure 6D). In conclusion, these ndings suggest that the downregulation of TXNDC12 inhibits the growth of GBM tumors in vivo.

Discussion
Despite breakthroughs in basic glioma research over the past decades, the median patient survival is less than 15 months [13][14][15] . The development of chemotherapeutic drugs for glioma has been slow, mainly due to the lack of ability of most drugs to be delivered across the blood-brain barrier and the fact that increasing drug doses can have signi cant side effects on patients' somatic functions, while the application of chemotherapeutic drugs can also enhance drug resistance in glioma 2,16,17 . Therefore, exploring the mechanisms related to the biological behavior of glioma cells and nding new targets for glioma therapy are expected to provide new directions for the treatment of glioma.
The TXNDC12 gene encodes a member protein of the thioredoxin superfamily 12,18 . The main feature of this family member is that it has a thioredoxin-folding active motif that catalyzes the formation and isomerization of disul de bonds 19,20 . What's more, This protein may play a role in the defense against ROS 9, 21 . In this study, a comprehensive analysis of TXNDC12 expression levels in databases and clinical glioma samples as well as the prognosis of patients showed that patients with higher TXNDC12 expression levels had higher pathological grade, higher malignancy, and worse prognosis. The results of cellular experiments indicated that TXNDC12 could promote the growth of glioma cells and TXNDC12 knockdown could signi cantly inhibit the growth of glioma cells.
The role of ROS in tumor therapy has received increasing attention, and various drugs that cause oxidative stress in cells by increasing ROS levels are more frequently used in clinical practice 22 , but there is no uniform understanding of the role of oxidative stress in tumor therapy, especially there is no quantitative study on whether different levels of ROS in the body play an anti-cancer or pro-cancer role 23,24 . Many drugs are used to treat tumors through oxidation, which can be broadly classi ed into two categories: those that increase intracellular ROS levels and induce tumor cell death, and those that inhibit the intracellular antioxidant enzyme system [25][26][27] . This study provides a theoretical basis for the treatment of glioma with drugs that inhibit intracellular antioxidant enzyme systems.
In GBM cells, the oxidative and antioxidant systems are maintained in a relative balance, and an increase in pro-oxidant levels or a decrease in antioxidant capacity can lead to an increase in ROS levels in vivo, resulting in a series of changes [28][29][30][31] . In this study, we further studied the mechanism of the role of TXNDC12 in regulating the proliferation process of glioma cells. TXNDC12 plays an important role in maintaining this balance. When TXNCD12 expression is inhibited, the ROS content in glioma cells is signi cantly increased, leading to apoptosis. In summary, this study demonstrated that TXNDC12 expression was signi cantly elevated in glioma patients and positively correlated with glioma disease grade, which means that patients with high TXNDC12 expression have a poorer prognosis. TXNDC12 not only affected the prognosis of glioma patients but also was associated with the promotion of glioma cell proliferation. This study provides more grounds for considering TXNDC12 as a therapeutic target for glioma and lays a theoretical foundation for the development and clinical application of TXNDC12related drugs. Furthermore this study provides a theoretical basis for the inhibition of intracellular antioxidant enzyme system drugs for the treatment of glioma and lays the foundation for the next development of chemotherapeutic drugs and targeted drugs.

Pathological Samples
A total of 60 patients who underwent cerebral lesion resection in the Department of Neurosurgery, Haiyang People's Hospital from January 2017 to January 2020 were selected, including 18 patients with pathological diagnosis of WHO grade II, 18 patients with pathological diagnosis of WHO grade III, 18 patients with pathological diagnosis of WHO grade IV, and 6 normal brain tissue samples from patients who underwent decompression due to cerebral hemorrhage or traumatic brain injury and had postoperative pathology con rmed as normal brain tissue. All patients had signed the informed consent forms, which were approved by the Ethics Committee of Haiyang People's Hospital.

Cells and Reagents
Glioma cell lines U251 and A172 were purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Immunohistochemical secondary antibody SP reagent (SP-9001), goat anti-rabbit secondary antibody (ZB-2301), and DAB color development reagent (Zli-9032) were purchased from Beijing Zhongsun Jinqiao Biotechnology Co. Ltd. SiRNA was purchased from Shanghai Jima Pharmaceutical Technology Co. CCK-8 kit was ordered from Dongren Chemical Technology (Shanghai) Co. Cell proliferation imaging kit (EdU method) was purchased from Guangzhou Ribo Biotechnology Co. Crystalline violet was purchased from Shanghai Biyuntian Biotechnology Co.

Bioinformatics Analysis
Chinese Glioma Genome Atlas (CGGA), American Brain Tumor Molecular Database (Rembrandt), The cancer genome atlas (TCGA) were used to investigate TXNDC12 expression and prognostic differences, and the TCGA database was further analyzed for TXNDC12-related signaling pathways.

Cell Culture and Transfection
Glioma cells were cultured in DMEM medium containing 10% fetal bovine serum and placed in a constant temperature incubator at 37℃ with 5% CO2 for routine passaging. Cells were digested and passaged with 0.05% trypsin solution. Well-grown U251 and A172 cells were selected to make cell suspensions for cell counting and then passed to six-well plates. When the cells grew to 60%, 1 mL of fresh medium was replaced in each well and the blank control group and experimental group were set up simultaneously. The cells were transfected according to the lipofectamine 2000 transfection kit.

Western Blotting
After transfection for 48 h, the cells were collected, added with an appropriate amount of RIPA lysis solution, lysed on ice for 30 min, centrifuged at 12000 r/min for 5 min at 4℃, and then the supernatant (protein) was placed in a centrifuge tube and stored at -20℃ in a refrigerator. After the protein concentration was detected by the BCA method, 5× protein loading buffer was added to it, and the protein was denatured by boiling water bath for 10 min. For each group, about 40 µg samples were separated by electrophoresis on SDS-PAGE gels and transferred to PVDF membranes at 4℃. 5% skimmed milk powder was used for blocking and primary antibodies (1:1000 dilution of β-actin and TXNDC12 antibodies) were added separately overnight at 4℃. After washing with TBST for 3×5 min, the secondary antibody coupled with horseradish peroxidase (1:5000) was added and incubated at room temperature for 1 h. After TBST washing, ECL was added dropwise, developed, and nally, the grayscale values of each band were analyzed by Image J software.

Cell Viability and Proliferation Assays
Cell viability was assessed using the CCK-8 assay. U251 and A172 cells in good condition were inoculated in 96-well plates at 3000 cells/100 µL, and the cells were transfected 24 h later. Three replicates were set up for each group, and four 96-well plates were inoculated and incubated at 37℃ in a 5% CO 2 incubator. At 24h and 48h, 96-well plates were taken out separately and 10 µL of CCK-8 was added to each well. After incubation for 60 min at 37℃, the absorbance at 450 nm was measured using the enzyme-labeled instrument. Proliferation was assessed using the EdU incorporation assay. U251 and A172 cells in good cell status were inoculated in 24-well plates at 10,000 cells/500 µL, and the cells were transfected after 24 h. Two replicates were set up for each group and incubated at 37℃ in a 5% CO 2 incubator. After 48h incubation, xation and staining were performed according to the instructions of the EdU kit, and uorescence microscopy was applied to take pictures.

Statistical Analysis
SPSS 22.0 software was applied for statistical analysis. All measures were tested for normal distribution using the Shapiro-Wilk (S-W) method. Compliance with normal distribution was expressed as x±s. Oneway ANOVA was used for the comparison of means between groups, and LSD or SNK methods were used for two-way multiple comparisons. p < 0.05 was considered a statistically signi cant difference (twotailed).      Representative images of hematoxylin and eosin-stained sections from brains of orthotopic A172-sh-TXNDC12-1 or A172-NC tumor-bearing nude mice. Scale bar, 100 µm.