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 first 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 significant 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 infiltration 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 findings 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 significant, 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 paraffin-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 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).
Finally, analysis of western blot results showed that TXNDC12 protein levels were also elevated in human glioma cells U87MG, U251, T98, LN229, and A172 compared to human astrocytes (NHA) (Figure 2C, 2D). 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.
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2.3 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 efficiency on TXNDC12 in vitro. In the U251 cell line, the knockdown efficiency of si-TXNDC12-1 was 89.3% and that of si-TXNDC12-2 was 85% (Figure 3A). In the A172 cell line, the knockdown efficiency 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 efficiency 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).
2.4 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 fluorescence) 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 significantly 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).
2.5 Knockdown of TXNDC12 causes an increase in intracellular reactive oxygen species
Excessive production of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), superoxide radicals (O2-) 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 fluorescent superoxide probe (dihydroethidium, DHE) to study superoxide levels. si-TXNDC12-1 transfected A172 and U251 cells showed significantly higher fluorescence intensity than the control, which was approximately 2-fold elevated (Figure 5A, 5B). Similarly, the level of H2O2 in cells transfected with si-TXNDC12-1 was 1.8-fold higher than that in the control group, and H2O2 was significantly reduced after the application of hydrogen peroxide scavenger (N-acety1-L-cysteine,Figure 5C). The ROS fluorescence probe results showed that the green fluorescence 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.
2.6 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 significantly 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 significant apoptosis. There was less cell division in the tumor tissue (Figure 6D). In conclusion, these findings suggest that the downregulation of TXNDC12 inhibits the growth of GBM tumors in vivo.