MicroRNA-448 Suppresses the Proliferation, Migration, and Invasion of Glioma Cell Line U251 by Targeting B-cell Lymphoma-2
Glioma is one of the most common malignant tumors in the central nervous system, characterized by high aggressiveness and a median survival of less than two years post-diagnosis. B-cell lymphoma-2 (BCL-2), an important regulator in apoptosis signaling, is an adverse prognostic factor in glioma. Overexpression of BCL-2 inhibits cell apoptosis, a phenomenon observed in various cancer types. The intricate regulatory network of apoptosis has been increasingly understood, with BCL-2 inhibitors showing promise in clinical trials. Additionally, identifying upstream signaling mechanisms controlling BCL-2 expression and function offers further therapeutic targets. MicroRNAs (miRNAs), non-coding RNAs consisting of 18 to 25 nucleotides, regulate gene expression by binding to the 3′-untranslated region (UTR) of target mRNAs, leading to translational repression or mRNA cleavage. miRNAs are considered excellent biomarker candidates and potential therapeutic tools.
In this study, glioma tissues and peritumoral tissues were collected from patients who underwent glioma resection at the Third Affiliated Hospital of Soochow University between 2010 and 2015. The study was approved by the hospital’s Ethics Committee, and informed consent was obtained from all participants. Using miRDB, TargetScan, and miRNA databases, miRNAs targeting BCL-2 were predicted. Only miR-448 and miR-204-5p were predicted to target BCL-2 across all three databases. While miR-204-5p has been reported to inhibit glioma progression by targeting BCL-2, the role of miR-448 in glioma remains less understood. Recent findings indicate that miR-448 downregulates the cancer gene CTTN, inhibiting cell proliferation and promoting apoptosis in glioma cell lines. However, the expression of miR-448 in glioma tissues and its potential to target BCL-2 in glioma needed further investigation.
The expression levels of BCL-2 and miR-448 were measured in 20 glioma tissues, 20 peritumoral tissues, and two glioma cell lines (U251 and U373 MG Uppsala), as well as in the normal human astrocyte cell line (HEB) using quantitative real-time polymerase chain reaction (qRT-PCR). Results showed that BCL-2 expression was upregulated in glioma tissues and cell lines compared to normal tissues, whereas miR-448 was downregulated. A negative correlation between miR-448 and BCL-2 expression was observed in glioma tissues, suggesting that downregulation of miR-448 contributes to BCL-2 overexpression.
To validate the hypothesis that miR-448 targets BCL-2, the 3′-UTR of BCL-2 containing two putative miR-448 binding sites and mutant sites was cloned into the psi-check2 vector. BCL-2 coding sequences without the 3′-UTR were cloned into the pcDNA 3.1 vector. These constructs, along with miR-448 mimic or negative control, were transfected into Hela cells. Luciferase activities were analyzed using a dual-luciferase reporter system, with relative luciferase activity normalized to renilla luciferase activity. The results confirmed that miR-448 directly targets BCL-2 in glioma cells.
Further experiments involved transfecting the glioma cell line U251 with miR-448 mimic, either alone or in combination with pcDNA3.1-BCL-2, to evaluate cell proliferation, colony formation, and invasion ability. The findings revealed that miR-448 was downregulated in glioma tissues and cell lines, and its expression level negatively correlated with BCL-2 expression. Transfection with miR-448 mimic downregulated BCL-2 expression in U251 cells, suppressing cell proliferation, colony formation, and invasion. Overexpression of BCL-2 rescued the miR-448-mediated suppression of these cellular processes in U251 cells.
This study demonstrates that miR-448 directly targets BCL-2 and is downregulated in glioma tissues. miR-448 functions as a tumor suppressor miRNA by inhibiting BCL-2 expression, suggesting its potential as an effective biomarker and therapeutic strategy for glioma patients. The findings highlight the importance of miR-448 in regulating BCL-2 expression and its role in suppressing glioma cell proliferation, migration, and invasion.
The study’s results are supported by qRT-PCR data showing upregulated BCL-2 and downregulated miR-448 in glioma tissues and cell lines compared to normal tissues. The negative correlation between miR-448 and BCL-2 expression in glioma tissues further underscores the regulatory relationship between these molecules. The dual-luciferase reporter assay confirmed the direct targeting of BCL-2 by miR-448, and functional assays demonstrated the tumor-suppressive effects of miR-448 in U251 cells. Overexpression of BCL-2 reversed the inhibitory effects of miR-448 on cell proliferation, colony formation, and invasion, providing additional evidence of the miR-448/BCL-2 regulatory axis in glioma.
In conclusion, miR-448 serves as a critical regulator of BCL-2 expression in glioma, with its downregulation contributing to the malignant phenotype of glioma cells. The findings suggest that miR-448 could be a valuable biomarker and therapeutic target for glioma, offering new avenues for the development of miRNA-based therapies. Future studies should explore the clinical applications of miR-448 in glioma treatment and its potential synergies with existing therapeutic strategies.
doi.org/10.1097/CM9.0000000000000572
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