Glioblastoma is the most frequent and lethal primary central nervous system tumor in adults, accounting for around 15% of intracranial neoplasms and 40–50% of all primary malignant brain tumors, with an annual incidence of 3–6 cases per 100,000 population. Despite maximum treatment, patients only have a median survival time of 15 months. Metformin is a biguanide drug utilized as the first-line medication in treating type 2 diabetes. Recently, researchers have noticed that metformin can contribute to antineoplastic activity. The objective of this study is to investigate the mechanism of metformin as a potential adjuvant treatment drug in glioblastoma. Glioblastoma cell lines U87MG, LNZ308, and LN229 were treated with metformin, and several cellular functions and metabolic states were evaluated. First, the proliferation capability was investigated using the MTS assay and BrdU assay, while cell apoptosis was evaluated using the annexin V assay. Next, a wound-healing assay and mesenchymal biomarkers (N-cadherin, vimentin, and Twist) were used to detect the cell migration ability and epithelial–mesenchymal transition (EMT) status of tumor cells. Gene set enrichment analysis (GSEA) was applied to the transcriptome of the metformin-treated glioblastoma cell line. Then, DCFH-DA and MitoSOX Red dyes were used to quantify reactive oxygen species (ROS) in the cytosol and mitochondria. JC-1 dye and Western blotting analysis were used to evaluate mitochondrial membrane potential and biogenesis. In addition, the combinatory effect of temozolomide (TMZ) with metformin treatment was assessed by combination index analysis. Metformin could decrease cell viability, proliferation, and migration, increase cell apoptosis, and disrupt EMT in all three glioblastoma cell lines. The GSEA study highlighted increased ROS and hypoxia in the metformin-treated glioblastoma cells. Metformin increased ROS production, impaired mitochondrial membrane potential, and reduced mitochondrial biogenesis. The combined treatment of metformin and TMZ had U87 as synergistic, LNZ308 as antagonistic, and LN229 as additive. Metformin alone or combined with TMZ could suppress mitochondrial transcription factor A, Twist, and O6-methylguanine-DNA methyltransferase (MGMT) proteins in TMZ-resistant LN229 cells. In conclusion, our study showed that metformin decreased metabolic activity, proliferation, migration, mitochondrial biogenesis, and mitochondrial membrane potential and increased apoptosis and ROS in some glioblastoma cells. The sensitivity of the TMZ-resistant glioblastoma cell line to metformin might be mediated via the suppression of mitochondrial biogenesis, EMT, and MGMT expression. Our work provides new insights into the choice of adjuvant agents in TMZ-resistant GBM therapy.
Introduction Glioblastoma (GBM) is the most common and lethal brain tumor. The current treatment is surgical removal combined with radiotherapy and chemotherapy, Temozolomide (TMZ). However, tumors tend to develop TMZ resistance which leads to therapeutic failure. Ancient ubiquitous protein 1 (AUP1) is a protein associated with lipid metabolism, which is widely expressed on the surface of ER and Lipid droplets, involved in the degradation of misfolded proteins through autophagy. It has recently been described as a prognostic marker in renal tumors. Here, we aim to use sophisticated bioinformatics and experimental validation to characterize the AUP1's role in glioma. Material and methods We collected the mRNA, proteomics, and Whole-Exon-Sequencing from The Cancer Genome Atlas (TCGA) for bioinformatics analyses. The analyses included the expression difference, Kaplan–Meier-survival, COX-survival, and correlation to the clinical factors (tumor mutation burden, microsatellite instability, and driven mutant genes). Next, we validated the AUP1 protein expression using immunohistochemical staining on the 78 clinical cases and correlated them with P53 and KI67. Then, we applied GSEA analyses to identify the altered signalings and set functional experiments (including Western Blot, qPCR, BrdU, migration, cell-cycle, and RNAseq) on cell lines when supplemented with small interfering RNA targeting the AUP1 gene (siAUP1) for further validation. We integrated the single-cell sequencing and CIBERSORT analyses at the Chinese Glioma Genome Atlas (CGGA) and Glioma Longitudinal AnalySiS (GLASS) dataset to rationale the role of AUP1 in glioma. Results Firstly, the AUP1 is a prognostic marker, increased in the tumor component, and correlated with tumor grade in both transcriptomes and protein levels. Secondly, we found higher AUP1 associated with TP53 status, Tumor mutation burden, and increased proliferation. In the function validation, downregulated AUP1 expression merely impacted the U87MG cells' proliferation instead of altering the lipophagy activity. From the single-cell sequencing and CIBERSORT analyses at CGGA and GLASS data, we understood the AUP1 expression was affected by the tumor proliferation, stromal, and inflammation compositions, particularly the myeloid and T cells. In the longitudinal data, the AUP1 significantly dropped in the recurrent IDH wildtype astrocytoma, which might result from increased AUP1-cold components, including oligodendrocytes, endothelial cells, and pericytes. Conclusion According to the literature, AUP1 regulates lipophagy by stabilizing the ubiquitination of lipid droplets. However, we found no direct link between AUP1 suppression and altered autophagy activity in the functional validation. Instead, we noticed AUP1 expression associated with tumor proliferation and inflammatory status, contributed by myeloid cells and T cells. In addition, the TP53 mutations seem to play an important role here and initiate inflamed microenvironments. At the same time, EGFR amplification and Chromosome 7 gain combined 10 loss are associated with increased tumor growth related to AUP1 levels. This study taught us that AUP1 is a poorer predictive biomarker associated with tumor proliferation and could report inflamed status, potentially impacting the clinical application.
Introduction Gliomas, a type of brain neoplasm, are prevalent and often fatal. Molecular diagnostics have improved understanding, but treatment options are limited. This study investigates the role of INTS9 in processing small nuclear RNA (snRNA), which is crucial to generating mature messenger RNA (mRNA). We aim to employ advanced bioinformatics analyses with large-scale databases and conduct functional experiments to elucidate its potential role in glioma therapeutics. Materials and methods We collected genomic, proteomic, and Whole-Exon-Sequencing data from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) for bioinformatic analyses. Then, we validated INTS9 protein expression through immunohistochemistry and assessed its correlation with P53 and KI67 protein expression. Gene Set Enrichment Analysis (GSEA) was performed to identify altered signaling pathways, and functional experiments were conducted on three cell lines treated with siINTS9. Then, we also investigate the impacts of tumor heterogeneity on INTS9 expression by integrating single-cell sequencing, 12-cell state prediction, and CIBERSORT analyses. Finally, we also observed longitudinal changes in INTS9 using the Glioma Longitudinal Analysis (GLASS) dataset. Results Our findings showed increased INTS9 levels in tumor tissue compared to non-neoplastic components, correlating with high tumor grading and proliferation index. TP53 mutation was the most notable factor associated with upregulated INTS9, along with other potential contributors, such as combined chromosome 7 gain/10 loss, TERT promoter mutation, and increased Tumor Mutational Burden (TMB). In GSEA analyses, we also linked INTS9 with enhanced cell proliferation and inflammation signaling. Downregulating INTS9 impacted cellular proliferation and cell cycle regulation during the function validation. In the context of the 12 cell states, INTS9 correlated with tumor-stem and tumor-proliferative-stem cells. CIBERSORT analyses revealed increased INTS9 associated with increased macrophage M0 and M2 but depletion of monocytes. Longitudinally, we also noticed that the INTS9 expression declined during recurrence in IDH wildtype. Conclusion This study assessed the role of INTS9 protein in glioma development and its potential as a therapeutic target. Results indicated elevated INTS9 levels were linked to increased proliferation capacity, higher tumor grading, and poorer prognosis, potentially resulting from TP53 mutations. This research highlights the potential of INTS9 as a promising target for glioma treatment.
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