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METTL3 promotes adriamycin resistance in MCF-7 breast cancer cells by accelerating pri-microRNA-221-3p maturation in a m6A-dependent manner.

ABSTRACT: Breast cancer (BC) is the most prevalent malignant neoplasm among women and is the fifth most common cause of cancer-associated death worldwide. Acquired chemoresistance driven by genetic and epigenetic alterations is a significant clinical challenge in treating BC. However, the mechanism of BC cell resistance to adriamycin (ADR) remains to be elucidated. In this study, we identified the methyltransferase-like 3/microRNA-221-3p/homeodomain-interacting protein kinase 2/Che-1 (METTL3/miR-221-3p/HIPK2/Che-1) axis as a novel signaling event that may be responsible for resistance of BC cells to ADR. A dual-luciferase reporter gene assay was employed to test the presence of miR-221-3p binding sites in the 3'UTR of HIPK2. Drug resistance was evaluated by immunoblotting multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP). Cultured ADR-resistant MCF-7 cells were assayed for their half maximal inhibitory concentration (IC50) values and apoptosis using an MTT assay and Annexin V-FITC/PI-labeled flow cytometry, and the cells were then xenografted into nude mice. METTL3 knockdown was shown to reduce the expression of miR-221-3p by reducing pri-miR-221-3p m6A mRNA methylation, thereby reducing the IC50 value of ADR-resistant MCF-7 cells, reducing the expression of MDR1 and BCRP, and inducing apoptosis. Mechanistically, miR-221-3p was demonstrated to negatively regulate HIPK2 and upregulate its direct target Che-1, thus leading to enhanced drug resistance in ADR-resistant MCF-7 cells. In vitro results were reproduced in nude mice xenografted with ADR-resistant MCF-7 cells. Our work elucidates an epigenetic mechanism of acquired chemoresistance in BC, in support of the METTL3/miR-221-3p/HIPK2/Che-1 axis as a therapeutic target for the improvement of chemotherapy.

SUBMITTER: Pan X

PROVIDER: S-EPMC8080609 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMETTL3 is known to be involved in all stages in the life cycle of RNA. It affects the tumor formation by the regulation the m6A modification in the mRNAs of critical oncogenes or tumor suppressors. In bladder cancer, METTL3 could promote the bladder cancer progression via AFF4/NF-κB/MYC signaling network by an m6A dependent manner. Recently, METTL3 was also found to affect the m6A modification in non-coding RNAs including miRNAs, lincRNAs and circRNAs. However, whether this mechanism is related to the proliferation of tumors induced by METTL3 is not reported yet.MethodsQuantitative real-time PCR, western blot and immunohistochemistry were used to detect the expression of METTL3 in bladder cancer. The survival analysis was adopted to explore the association between METTL3 expression and the prognosis of bladder cancer. Bladder cancer cells were stably transfected with lentivirus and cell proliferation and cell cycle, as well as tumorigenesis in nude mice were performed to assess the effect of METTL3 in bladder cancer. RNA immunoprecipitation (RIP), co-immunoprecipitations and RNA m6A dot blot assays were conducted to confirm that METTL3 interacted with the microprocessor protein DGCR8 and modulated the pri-miR221/222 process in an m6A-dependent manner. Luciferase reporter assay was employed to identify the direct binding sites of miR221/222 with PTEN. Colony formation assay and CCK8 assays were conducted to confirm the function of miR-221/222 in METTL3-induced cell growth in bladder cancer.ResultsWe confirmed the oncogenic role of METTL3 in bladder cancer by accelerating the maturation of pri-miR221/222, resulting in the reduction of PTEN, which ultimately leads to the proliferation of bladder cancer. Moreover, we found that METTL3 was significantly increased in bladder cancer and correlated with poor prognosis of bladder cancer patients.ConclusionsOur findings suggested that METTL3 may have an oncogenic role in bladder cancer through interacting with the microprocessor protein DGCR8 and positively modulating the pri-miR221/222 process in an m6A-dependent manner. To our knowledge, this is the first comprehensive study that METTL3 affected the tumor formation by the regulation the m6A modification in non-coding RNAs, which might provide fresh insights into bladder cancer therapy.

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METTL3 promotes adriamycin resistance in MCF-7 breast cancer cells by accelerating pri-microRNA-221-3p maturation in a m6A-dependent manner.

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