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. 2019 Jul 29;9(1):10986.
doi: 10.1038/s41598-019-47360-9.

Altered Transcriptional Regulatory Proteins in Glioblastoma and YBX1 as a Potential Regulator of Tumor Invasion

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Free PMC article

Altered Transcriptional Regulatory Proteins in Glioblastoma and YBX1 as a Potential Regulator of Tumor Invasion

Manoj Kumar Gupta et al. Sci Rep. .
Free PMC article

Abstract

We have studied differentially regulated nuclear proteome of the clinical tissue specimens of glioblastoma (GBM, WHO Grade IV) and lower grades of gliomas (Grade II and III) using high resolution mass spectrometry- based quantitative proteomics approach. The results showed altered expression of many regulatory proteins from the nucleus such as DNA binding proteins, transcription and post transcriptional processing factors and also included enrichment of nuclear proteins that are targets of granzyme signaling - an immune surveillance pathway. Protein - protein interaction network analysis using integrated proteomics and transcriptomics data of transcription factors and proteins for cell invasion process (drawn from another GBM dataset) revealed YBX1, a ubiquitous RNA and DNA-binding protein and a transcription factor, as a key interactor of major cell invasion-associated proteins from GBM. To verify the regulatory link between them, the co-expression of YBX1 and six of the interacting proteins (EGFR, MAPK1, CD44, SOX2, TNC and MMP13) involved in cell invasion network was examined by immunohistochemistry on tissue micro arrays. Our analysis suggests YBX1 as a potential regulator of these key molecules involved in tumor invasion and thus as a promising target for development of new therapeutic strategies for GBM.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
(A) Workflow for the analysis of differentially expressed nuclear proteins of different grades of astrocytoma. (B) Nuclear preparation from the clinical specimens stained with DAPI and observed under fluorescent phase contrast microscope. (C) Sub cellular classification of differentially expressed proteins carried out using Human Protein Reference Database. The classification shows the enrichment of nuclear proteins in the preparation.
Figure 2
Figure 2
Ingenuity Pathway Analysis of differentially expressed nuclear proteins. The molecular functions and pathways were generated through the use of IPA (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis). Top five significant Canonical pathways (A) and Molecular and cellular functions (B) identified from analysis of non-redundant list of differentially expressed nuclear proteins (n = 244) from all grades combined and differentially expressed nuclear proteins grade-wise (Grade II- 162, Grade III-131 and Grade IV- 147 proteins).
Figure 3
Figure 3
Immunohistochemistry on tissue microarrays of different grades of astrocytomas (A) and MS/MS spectra of representative peptides (B) for six selected proteins - NUCKS1, SMARCA5, PARP1, PTBP1, HMGB2 and NFIB. IHC analysis of the proteins tested using commercially available tissue microarrays (US BioMax), confirmed overexpression of these proteins in multiple tumor specimens. The details of tissue microarrays used and IHC procedure are described in the Methods and the staining scoring details are shown in Supplementary Table S9. MS/MS spectra acquisition is described under Methods.
Figure 4
Figure 4
Protein-protein interaction network of proteins associated with transcription process. The interaction network was built with the transcription regulatory proteins including transcription factors and other DNA/RNA-binding proteins involved in transcription and post-transcription process, identified in GBM as shown in Supplementary Table 2. The network was constructed using STRING v10 web resource tool (https://string-db.org) as described in the Methods. Up or down regulation trend is represented by arrows.
Figure 5
Figure 5
Integration of transcription network proteins with their corresponding transcripts and miRNA regulators to construct 2 - Dimensional molecular map with regulatory cascades in relation to function. (A) Pipeline for mapping transcription network proteins and transcripts (as shown in Fig. 4) to their putative miRNA regulators in GBM. (B) Representative regulatory cascades mapped using the workflow. The Figure represents 2D map analysis of the key miRNAs and their targets identified in GBM with inverse relationship in their expression. Up or down regulation trend of all the entities is represented by arrows. A complete list of these cascades is provided in the Supplementary Table S10.
Figure 6
Figure 6
(A) Integrated protein-protein interaction network with transcription factors and proteins involved in the invasion process. The protein-protein interaction network was constructed with the transcription factors and invasion process entities, through the use of STRING v10 web resource tool (https://string-db.org). Two transcription factors YBX1 and PURA (circled with brown color) were captured in the interactions between the two - molecular functions. (B) Differential expression of YBX1 protein. This sub-panel showing the representative MS/MS spectra of YBX1 peptide with iTRAQ reporter ions and the peptide sequence identified in GBM as compared to the control (from the data from ref.). (C) Scatter plot showing expression of YBX1 mRNA in GBM and control sample using public domain data (TCGA mRNA expression data). Overexpression of YBX1 with 2.4 fold change was observed with p value < 0.001.
Figure 7
Figure 7
Immunohistochemistry on tissue microarrays of GBM for YBX1 and its interacting proteins - EGFR, MAPK1, CD44, TNC, MMP13 and SOX2. IHC analysis of the proteins tested using in-house prepared tissue microarrays, confirmed overexpression of YBX1 and its interacting proteins in multiple tumor specimens. The details of tissue microarrays used and IHC procedure are described in the Methods and the staining scoring details are shown in Supplementary Table S11.

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