Astrocyte Elevated Gene-1 Serves as a Target of miR542 to Promote Glioblastoma Proliferation and Invasion
Glioma is the most common malignant neoplasm originating from the central nervous system, accounting for about 40% to 60% of intracranial tumors. Glioblastoma multiforme (GBM), which accounts for about half of gliomas, is a multigene-related disease caused by the abnormal regulation of gene networks that maintain stable cellular normality and differentiation. Despite comprehensive therapeutic strategies combining tumor resection with post-operative chemoradiotherapy, the high recurrence rate and unsatisfactory conventional chemotherapy remain significant challenges in GBM treatment.
Epithelial to mesenchymal transition (EMT) is a critical process in tumor invasion and metastasis, playing a vital role in carcinogenesis and cancer progression. Emerging evidence suggests that microRNAs (miRNAs) are closely associated with EMT by regulating targeted genes. MiR542 has been implicated in the EMT program and is associated with various cancers. However, the functions of miR542 and its underlying mechanisms in GBM remain largely unexplored. This study investigates the effect of astrocyte elevated gene-1 (AEG-1) on U251 cell aggressiveness, proliferation, apoptosis, and cell cycle, and explores the functional roles and mechanisms of miR542 in GBM.
The human GBM cell line U251 was used in this study. Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin, and streptomycin. MiRNA mimics and siRNA sequences were designed and synthesized, and transfection was performed using Lipofectamine RNAiMAX. Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8) assay, while cell migration and invasion were evaluated using Transwell assays. Cell cycle and apoptosis were analyzed using flow cytometry. Luciferase assays were conducted to confirm the interaction between miR542 and AEG-1. Western blot analysis and RT-PCR were used to measure protein and mRNA expression levels, respectively.
The study first investigated the effect of AEG-1 on U251 cell aggressiveness, proliferation, apoptosis, and cell cycle. siRNA targeting AEG-1 (si-AEG1) was transfected into U251 cells, and the results showed a significant suppression of AEG-1 expression at both mRNA and protein levels. AEG-1 deficient cells exhibited reduced aggressive abilities, as demonstrated by transwell assays. The proliferation of U251 cells was dramatically inhibited, with cells mostly arrested at the G1/S phase. Apoptosis was significantly promoted in AEG-1 deficient cells, suggesting that AEG-1 suppresses malignant phenotypes.
To identify the miRNAs involved in GBM, miRNA expression profiles were formed using deep sequencing technology from GBM samples. Three candidate miRNAs (miR128, miR520c, and miR542) were selected based on their differential expression. Functional investigations revealed that miR542 significantly reduced the migration and proliferation of U251 cells compared to the other miRNAs. The CCK-8 assay confirmed that all candidate miRNAs suppressed U251 cell proliferation, with miR542 showing the most pronounced effect.
The study then focused on the molecular mechanism of miR542. AEG-1 was predicted as a target gene of miR542 using bioinformatics tools. Luciferase assays confirmed that miR542 directly targets the 3’-UTR of AEG-1, significantly reducing luciferase activity in U251 cells. Mutation of the miR542 binding site in the AEG-1 3’-UTR abolished this effect, further confirming the interaction between miR542 and AEG-1.
The relationship between miR542 and EMT was also explored. U251 cells transfected with miR542 displayed lower expression of AEG-1 at both protein and mRNA levels. The expression of E-cadherin was upregulated, while Vimentin expression was downregulated in AEG-1 deficient cells, indicating that miR542 suppresses the EMT process by downregulating AEG-1.
The findings of this study demonstrate that miR542 inhibits the migration and proliferation of U251 cells and suppresses EMT by targeting AEG-1. MiR542 acts as a tumor suppressor in GBM, providing a new insight into U251 cell aggressiveness and proliferation. The identification of miR542 as a regulator of AEG-1 offers a potential therapeutic target for the treatment of glioma.
In conclusion, miR542 plays a crucial role in regulating the malignant phenotypes of GBM cells by targeting AEG-1 and suppressing the EMT process. This study highlights the potential of miR542 as a novel therapeutic target for GBM, offering new avenues for the development of effective treatment strategies.
doi.org/10.1097/CM9.0000000000001072
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