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. 2022 Aug 11:13:912039.
doi: 10.3389/fneur.2022.912039. eCollection 2022.

Chromobox 7/8 serve as independent indicators for glioblastoma via promoting proliferation and invasion of glioma cells

Zong-Qing Zheng  1 , Gui-Qiang Yuan  1   2 , Na-Ling Kang  3 , Qian-Qian Nie  1 , Guo-Guo Zhang  1 , Zhong Wang  1
Affiliations

Chromobox 7/8 serve as independent indicators for glioblastoma via promoting proliferation and invasion of glioma cells

Zong-Qing Zheng et al. Front Neurol. .

Abstract

Background: The chromobox family, a critical component of epigenetic regulators, participates in the tumorigenesis and progression of many malignancies. However, the roles of the CBX family members (CBXs) in glioblastoma (GBM) remain unclear.

Methods: The mRNA expression of CBXs was analyzed in tissues and cell lines by Oncomine and Cancer Cell Line Encyclopedia (CCLE). The differential expression of CBXs at the mRNA level was explored in The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) databases with the "beeswarm" R package. The protein expression of CBXs in GBM was further examined on Human Protein Atlas (HPA). The correlations between CBXs and IDH mutation and between CBXs and GBM subtypes were investigated in the TCGA portal and CGGA database with the "survminer" R package. The alteration of CBXs and their prognostic value were further determined via the cBioPortal and CGGA database with the "survival" R package. The univariate and multivariate analyses were performed to screen out the independent prognostic roles of CBXs in the CGGA database. Cytoscape was used to visualize the functions and related pathways of CBXs in GBM. U251 and U87 glioma cells with gene intervention were used to validate the role of CBX7/8 in tumor proliferation and invasion. Proliferation/invasion-related markers were conducted by Western blot and immunostaining.

Results: CBXs presented significantly differential expressions in pan-cancers. CBX2/3/5/8 were upregulated, whereas CBX6/7 were downregulated at mRNA level in GBM of TCGA and CGGA databases. Similarly, high expression of CBX2/3/5 and low expression of CBX6/8 were further confirmed at the protein level in the HPA. CBX2/6/7 were positively correlated with IDH mutation and CBX1/2/4/5/8 were closely related to GBM subtypes. CBX7 and CBX8 presented the independent prognostic factors for GBM patient survival. GO and KEGG analyses indicated that CBXs were closely related to the histone H3-K36, PcG protein complex, ATPase, and Wnt pathway. The overexpression of CBX7 and underexpression of CBX8 significantly inhibited the proliferation and invasion of glioma cells in vivo and in vitro.

Conclusion: Our results suggested that CBX7 and CBX8 served as independent prognostic indicators that promoted the proliferation and invasion of glioma cells, providing a promising strategy for diagnosing and treating GBM.

Keywords: chromobox (CBX); glioblastoma (GBM); invasion; multi-omics; prognosis; proliferation.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
mRNA expression of CBX family in different cancers from tissues to cell lines. (A) Transcriptional expression of CBXs in 20 different types of cancers tissues (ONCOMINE). The difference in transcriptional expression was analyzed by Student's t-test. The cut-off of p-value was below 0.05. Fold changes should be beyond 1.5, and gene ranks should be in the top 10%. The yellow box emphasized the Brain and CNS cancers. (B) The difference of each CBX member at mRNA level in cell lines (CCLE) of 33 various cancers. Statistical comparisons were performed using one-way ANOVA and post hoc least significant difference tests for multiple comparisons. The dashed line represented the mean of the expression in the above 33 cancers.
Figure 2
Figure 2
The differential expressions of CBX family at mRNA level in TCGA and CGGA. (A-H) The mRNA expression difference of CBXs in GBM was analyzed in The Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA) databases. UALCAN provided 156 GBM samples for analysis (ns, no significance; *p < 0.05, **p < 0.01, ***p < 0.001). 218 GBM samples from CGGA were downloaded and analyzed via “survminer” R package. The p-value was set at 0.05.
Figure 3
Figure 3
Representative immunohistochemistry images of CBX family in GBM and normal brain tissues from Human Protein Atlas (HPA). (A–H) The protein expression of each tissue was marked under the image. The scale bar represented 4 μm, and the image in the black square represented a 4x zoom. The antibody used in the staining was labeled below.
Figure 4
Figure 4
IDH mutation and molecular GBM subtypes were correlated with the mRNA expression of CBX family. (A) The difference in the mRNA expression of CBX family between IDH mutation (Red) and wildtype (Green) groups was explored in CGGA database by “survminer” R package. The p-value was set at 0.05. (B) Molecular subtypes in GBM, including Classical, Proneural, Neural, Classical, and Mesenchymal subtypes, were explored in differential mRNA expressions of CBX family by TCGA portal. (*p < 0.05, **p < 0.01, ***p < 0.001).
Figure 5
Figure 5
The prognostic value of CBX family in GBM patients. (A) The genetic mutation of CBX members in GBM patients was analyzed on cBioPortal (TCGA, Firehose Legacy). The z-scores of mRNA expression relative to diploid samples were set at 1.5. Except for the gray region (No alteration), the other colors represented different genetic mutations in these patients. (B) The prognostic value of alterations in CBX genes was analyzed by Kaplan-Meier analysis on cBioPortal. (C) The correlation between mRNA expression and GBM patients' survival was explored by Kaplan-Meier analysis in CGGA data. The red dash represented high mRNA expression, and the blue meant low expression. (D) The univariate Cox survival analysis was conducted to screen out the significant single factors that contributed to the survival of GBM patients. The p-value threshold was 0.5. (E–G) Further multivariate Cox survival analysis was used to confirm the independent prognostic values of the above significant single factors. The above Kaplan-Meier, univariate, and multivariate survival analyses using R software with the “survival” and “survminer” packages.
Figure 6
Figure 6
The potential function prediction and validation of CBX family in proliferation and invasion of glioma cells. The top 50*8 genes associated with CBX members in GBM were screened out by GEPIA and used to conduct the GO and KEGG analyses by Cytoscape. (A) Biological process (BP), (B) Cellular components (CC), (C) Molecular functions (MF), and (D) the KEGG pathways influenced by CBX family. The red box emphasized the functions that had the most count and significant difference in these analyses. (E) Clinical validation of CBX7 and CBX8 through immunochemistry staining in tumor-adjacent normal and GBM tissues. The black scale bar represented 4 μm. N = 3. (*p < 0.05). (F,G) Western blots were performed to confirm the effectiveness of lentivirus transfection in U87 and U251 glioma cells. β-tubulin was set as the loading control. N = 3. (*p < 0.05). (H) Ki-67 immunofluorescent staining was performed to evaluate the effect of CBX7 and CBX8 on the proliferation capacity in glioma cells. The white scale bar represented 20 μm. (I) The orthotopic U87 glioma model in nude mice measured the proliferation of glioma cells with intervention on CBX7 and CBX8. The subcutaneous tumor was photographed and measured at 0 h and 28 days after injection using the formula: volume (mm3) = length*width*height. N = 3. (J,K) Western blots were performed to assess the invasion-related markers by detecting the MM2 and MMP9. β-Tubulin was set as the loading control, N = 3, (*p < 0.05). (L) The wound-healing assay explored the invasion abilities of U87 glioma cells with different interventions on CBX7 and CBX8 compared to the control. The gap was measured at 0 h and 24 h after the scratch. The white scale bar represented 4 μm. (*p < 0.05, **p < 0.01, ***p < 0.001).
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