TCGA dataset and Oncomine database MAGED2 mRNA and protein expression analysis in glioma
MAGED2 expression levels have been shown to be considerably greater in GBM tissues than in LGG tissues, according to the TCGA dataset (t = 8.21, P < 0.001, Fig. 1A). Furthermore, the clinical data and gene expression profiles of the 152 GBM patients were matched with those of the 510 LGG patients to determine if MAGED2 expression levels were related to patient survival, which was accomplished using Kaplan-Maier analysis and log-rank comparison. The results are shown in Fig. 1B, which show that a greater MAGED2 expression level is associated with a shorter OS in glioma patients (c2= 6.32, P < 0.010). These data suggested that a low MAGED2 expression level in glioma may be responsible for the patients' good OS.
Furthermore, by examining the ONCOMINE microarray datasets, researchers may detect variations in MAGED2 mRNA expression in distinct types of gliomas. The Beroukhim and Sun Brain dataset query results showed that MAGED2 mRNA expression in different types of glioma tissues, such as anaplastic astrocytoma (t=3.827, P < 0.001, Fig. 1C), glioblastoma (t=3.223, P<0.001, Fig. 1E), and oligodendroglioma (t=2.722, P=0.004, Fig. 1F), were all significantly increased when compared with normal brain tissue, with the exception for diffuse astrocytoma (t = 0.915, P=0.193, Fig. 1D). Moreover, the CGGA database results revealed that the expression of MAGED2 increased dramatically as the grade of glioma increased (Fig. 1G). Furthermore, the Gene Expression Profiling Interactive Analysis (GEPIA) database (http://gepia.cancer-pku.cn/) findings indicated that MAGED2 expression was considerably positive in LGG patients with (Fig. 1H), and it negatively correlated with MGMT expression (Fig. 1J). However, there is no significant correlation between MAGED2 expression and IDH1 in GBM patients, most likely because there are fewer individuals with IDH1 mutation (Fig. 1I). Moreover, there was no significant association between MAGED2 expression and MGMT expression (Fig. 1K).
The association of Glioma grading with Genotyping is demonstrated in Table 2. There was a significant association between glioma grading and IDH1/2 (P<0.05)、1p/19q (P<0.05), MGMT (P<0.05), Ki67 (P<0.01), MAGE-D2 (P<0.01). By contrast, glioma grading was not associated with TERT (P=0.156), EGFR (P=0.090) (Table 2).
In addition, utilizing the Human Protein Atlas (HPA) Database (http://www.proteinatlas.org), MAGED2 protein was found in 12 cases of glioma but not in normal tissue. According to the HPA database, the overall proportion of positive cells for MAGED2 protein was 100%. This included 33.33% with strong expression, 58.33% with moderate expression, and 0.08% with weak expression. The MAGED2 protein staining was primarily located in the cell cytoplasm and nucleus. These findings also revealed that MAGED2 may play a role in glioma formation, which may hold promise as a prognostic marker.
MAGED2 was overexpressed in human glioma tissues.
The expression levels of MAGED2 were analyzed in 98 glioma and 16 normal brain tissue specimens using RT-qPCR and Western blotting. There were significant differences between low or high-grade glioma compared with normal brain tissues for MAGED2 mRNA level (Fig. 2A, P<0.001). MAGED2 mRNA was considerably greater in low-grade glioma (P<0.001) and high-grade glioma (P<0.001) than in normal brain tissues, but there was no significant difference in MAGED2 mRNA between high-grade and low-grade gliomas (Fig. 2A). One-way ANOVA revealed significant differences between various kinds of glioma and normal brain tissues in terms of MAGED2 mRNA expression levels in each group (Fig. 2B, P<0.001). MAGED2 mRNA was highest in glioblastoma (P<0.001), and diffuse astrocytoma had the second highest value (P<0.001), followed by oligodendroglioma (P<0.001) and anaplastic astrocytoma (P<0.001), when compared with normal brain tissue. However, a post-hoc test determined that there was no significant effect of multiple comparisons (Fig.2B). High expression of MAGED2 was defined as a three-fold increase in the median value of MAGED2/GAPDH in normal brain tissues, as shown in Fig. 2C. The findings revealed that 57.14% of glioma tissues demonstrated high mRNA MAGED2 expression, whereas 0% of normal brain tissues did (P<0.001). When the percentage of high mRNA MAGED2 expression in low-grade (20.41%) and high-grade (36.73%) gliomas was compared to that of normal brain tissue, significant differences were found (P<0.001 and P<0.001, respectively); however, no significant differences were found between high-grade and low-grade glioma (P=0.82；Fig. 2C). Meanwhile, MAGED2 protein expression follows the same pattern as mRNA expression (p<0.001, Fig. 2D and E).
IHC to examine the expression of the MAGED2 protein
IHC was used to examine MAGED2 protein expression in 98 glioma tissues (Table 3). The overall proportion of cells positive for MAGED2 protein was 79.59%, according to the findings. The MAGED2 protein was stained mostly in the cytoplasm and nucleus of the cells. The percentage of cells in low-grade glioma samples that expressed MAGED2-positive protein was 30.61%, whereas it was 48.97 % in high-grade gliomas (Table 3). According to the staining intensity, 57.14 % of patients had high MAGED2 protein expression (Figs. 3A and 3B), whereas 42.86 % had low MAGED2 protein expression (Fig. 3C). Furthermore, the presence of MAGED2 was essentially non-existent in 12 normal brain tissues that were stained (Fig. 3D). The positive control was a known glioma tissue segment with MAGED2-positive expression (Fig. 3E), while the negative control was omission of primary antibody (Fig. 3F).
Clinicopathological characteristics and MAGED2 protein expression.
Table 3 shows the relationship between MAGED2 protein expression and clinical variables. MAGED2 protein expression was shown to be associated with WHO grade (P<0.01), Ki-67 (P<0.01), IDH1/2 (P<0.05), MGMT (P<0.05). Conversely, MAGED2 protein expression was not associated with sex (P=0.765), age (P=0.814), tumor size (P=0.860), or extent of resection (P=0.554) (Table 3).
Prognostic impact of MAGED2 protein overexpression
The influence of MAGED2 expression and tumor categorization was analyzed using Kaplan-Meier survival analysis to identify the prognostic value for MAGED2. In the current investigation, 98 patients with glioma were followed up on and had complete clinical data. Patient follow-up lasted an average of 27.4 months (range, 19-72 months). In patients with glioma, a substantial positive correlation between MAGED2 protein expression, OS, and RFS times were discovered using clinical records. When compared to patients with low MAGED2 expression in malignant tissues, all patients with high MAGED2 expression in cancerous tissues had a substantially shorter median OS (50.00 vs. 224.21 months; P<0.001) and RFS (22.00 vs. 122.10 months, P=0.0031) times (Fig. 4A and B). Patients with high MAGED2 protein expression had significantly shorter median OS (100.00 vs. 246.00 months; P<0.001) and RFS (75.00 vs. 140.00 months; P=0.0013) times in low-grade gliomas (Fig. 4C and D); however, there was no significant difference in OS and RFS times in high-grade gliomas (P>0.05; Fig. 4E and F). Sex, age, tumor size (≥3.0 cm), extent of resection, WHO classification (High grade), IDH1 status (Mutant), Ki-67 expression (≥10%), and MAGED2 protein expression were used to categorize patients. Among these variables, an age of 39 years or older, WHO classification of High grade, IDH1 status (Mutant), Ki-67 expression ≥10% and high MAGED2 protein expression were all found to be important prognostic indicators in OS and RFS using univariate analysis. Patient survival was influenced by a variety of factors, necessitating the use of a multivariate analysis. The WHO classification, IDH1 status (Mutant), and MAGED2 protein expression were all found to have a substantial influence on OS and RFS in patients with glioma. Because there was a substantial positive relationship between high MAGED2 protein expression and glioma prognosis, it was assumed that MAGED2 protein expression may be identified as an independent prognostic factor for glioma patients (Table 4).
MAGED2 knockdown in human glioma U251-MG cells using MAGED2 CRISPR
The human glioma U251-MG cell line was one of the most widely used glioma cell lines, with high tumorigenicity in vivo. The GFP-expressing MAGED2 CRISPR and Scramble CRISPR were created and transfected into U251-MG cells to better understand the role of MAGED2 in glioma. After lentiviral infection, almost 90% of cells displayed positive green fluorescence, as indicated in the data (Fig. 5A). qRT-PCR and Western blotting were used to determine the knockdown effectiveness. MAGED2 mRNA (t = 4.47, P = 0.0012, Fig. 5B) and protein (t = 10.58, P＜0.000, Fig. 5C) levels in U251-MG cells of the MAGED2 CRISPR group were significantly lower than those in the Scramble CRISPR group 72 hours after infection.
MAGED2 knockdown significantly reduced glioma U251-MG cell growth
CCK-8 and colony formation experiments were performed on glioma U251-MG cells transfected with MAGED2 CRISPR or Scramble CRISPR to better understand the role of MAGED2 in glioma cell proliferation. As shown in Fig. 6, MAGED2 knockdown significantly reduced the proliferation capacity and growth of U251-MG cells processed with MAGED2 CRISPR compared to the Scramble CRISPR group. Furthermore, the in vitro proliferation of the MAGED2 CRISPR-treated cells was significantly reduced at 72 hours (Scr CRISPR: 1.13±0.19 vs. MAGED2 CRISPR: 0.65±0.12, t=3.65, P=0.02), 96 hours (Scr CRISPR: 1.36±0.16 vs. MAGED2 CRISPR: 0.80±0.16, t=4.34, P=0.01), and 120 hours (Scr CRISPR: 1.41±0.17 vs. MAGED2 CRISPR: 0.86±0.12, t=4.62, P<0.01) (Fig. 6A). Similarly, inhibition of MAGED2 also resulted in a significant reduction in colony formation (t=5.22, P＜0.001, Fig. 6B). The effect of MAGED2 on proliferation following MAGED2 downregulation was also assessed using the EDU cell-image assay. The results of the EDU cell image test were consistent with the results of the CCK-8 and colony formation assays, indicating that the EdU-positive rates of U251-MG cells were lower in the MAGED2 CRISPR group than in the Scramble CRISPR group (t=5.47, P<0.0001, Fig. 6C). As a result of these three tests, it was concluded that knocking down MAGED2 inhibited the proliferation of glioma U251-MG cells.
Glioma U251-MG cells were arrested in G0/G1 phase and apoptosis was accelerated by MAGED2 suppression.
The cell cycle distribution demonstrated a direct relationship with cell proliferation. On this basis, flow cytometry was used to study the cell cycle distribution of U251-MG cells following MAGED2 downregulation. As depicted in Fig. 7A and B, inhibiting MAGED2 increased the number of cells entering the G0/G1 phase by 43.27% (t=19.13, P<0.0001) while decreasing the number of cells entering the S phase by 35.16% (t=30.18, P<0.0001). Furthermore, the impact of MAGED2 knockdown on apoptosis in glioma U251-MG cells was also investigated. The results showed that the percentage of apoptosis in U251-MG cells from the MAGED2 CRISPR group ((17.40 ± 2.44) % was significantly greater than the Scramble CRISPR group (10.40 ± 1.90) % (t=3.93, P<0.05, Fig. 7C and D). These findings demonstrated that MAGED2 knockdown effectively inhibited U251-MG cell growth by increasing the proportion of cells in the G0/G1 phase, lowering the percentage of cells in the S phase, and triggering apoptosis.
MAGED2 downregulation decreased cell growth through restoring CDKN1A
U251-MG cells were transfected with MAGED2 CRISPR or Scramble CRISPR to investigate the target of MAGED2 in glioma cells in vitro. Following that, CDKN1A mRNA and protein expression levels were determined by qRT-PCR and Western blotting, respectively. The results showed that when compared to Scramble CRISPR transfection, CDKN1A mRNA expression levels were clearly up-regulated following transfection with MAGED2 CRISPR (t=12.33, P<0.0001, Fig. 7E). Furthermore, CDKN1A protein expression was much greater in the MAGED2 CRISPR transfection group (t=11.52, P<0.0001, Fig. 7F).