Evaluation of ATG-5 and ATG-7 Gene Expression in the Autophagy Pathway by a Synthetic Circular MicroRNA Sponge Targeting miR-17 and miR-181 in the NALM-6 Cell Line
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Abstract
Introduction: MicroRNAs (miRNAs) can affect cellular processes by modulating the expression of different genes. Blocking specific miRNAs across all cancer types can reduce or prevent cancer cell activity. The present study aimed to investigate the inhibitory effects of MicroRNA sponges (miRSponge) on miR-17-5p and miR-181 in NALM-6 cells, a cellular model of acute lymphoblastic leukemia, and to evaluate their effects on the expression of the autophagy-related genes ATG-5 and ATG-7.
Materials and methods: The present study was conducted from 2021 to 2022 at Mashhad University of Medical Sciences, Iran, in collaboration with the Shiraz Paramedical Faculty, Iran, and focused on a miRSponge designed to target miR-17-5p and miR-181. The present study included four NALM-6 cell groups, including untreated cells as a control, miR-17-5p- and miR-181-sponge-transfected cells, and a vincristine-treated group. The NALM-6 cells were cultured and transfected with a miRSponge targeting miR-17-5p and miR-181. The expression levels of miR-17-5p, miR-181, ATG-5, and ATG-7 were quantified by real-time quantitative PCR, while apoptosis in cells from different study groups was assessed by flow cytometry using Annexin V/7-AAD staining. Cell viability following vincristine treatment was evaluated using MTT assay, and bioinformatics analyses were performed to investigate miRNA-target interactions.
Results: The current results indicated that the developed sponge significantly reduced the levels of these two oncogenic miRNAs in cells by binding to miR-17-5p and miR-181. Furthermore, the activity of ATG-5 and ATG-7, regulated by these two miRNAs, increased following the reduction in the expression of these miRNAs. Late-stage apoptosis was higher in miRSponge-treated cells than in vincristine-treated and control groups, although the difference was not statistically significant.
Conclusion: The present findings indicated that using a miRSponge significantly upregulated the ATG-5 and ATG-7 genes and enhanced autophagy and apoptosis in cancer cells. MicroRNA sponges reduced the effects of oncogenic miRNAs in acute lymphocytic leukemia cells.
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