RESUMO
N6-methyladenosine (m6A) mRNA methylation has been considered a gene modulatory mechanism involved in disease progression and carcinogenesis. Herein, we aimed to explore the specific mechanism of m6A mRNA methylation in endometrial cancer. RT-qPCR was implemented to test the clinical correlation between m6A methylation and endometrial cancer. Bioinformatics analysis was performed to screen the genes related to endometrial cancer, and SRAMP was utilized for the prediction of m6A targets. Western blot assay and MeRIP-qPCR experiments were conducted to verify the effect of m6A methylation on the candidate genes and the signaling pathways involved in the occurrence of endometrial cancer. m6A-seq, RT-qPCR, and polysome profiling were used to confirm the mechanisms of m6A methylation in modulating related genes and pathways. The levels of m6A methylation, METTL3, and IGFBP4 were reduced in tumor tissues of patients with endometrial cancer, and SRAMP analysis confirmed that IGFBP4 and PAPPA had m6A methylation sites. Reduced m6A methylation promoted endometrial cancer cell progression and tumor formation in vivo. m6A methylation of RNA in endometrial cancer cells directly modulated IGFBP4 and PAPPA expression. m6A methylation regulated the PAPPA/IGFBP4 axis, thereby influencing endometrial cancer through the NF-κB and ERK signaling pathways. Knockdown of PAPPA or overexpression of IGFBP4 in endometrial cancer cells partially reduced disease progression caused by reduced m6A methylation. This research suggests that m6A mRNA methylation modulates the ERK/NF-κB/AKT signaling pathway through the PAPPA/IGFBP4 axis to induce cell proliferation and tumor formation in endometrial cancer. 1. METTL3 expressed modestly and m6A methylation of IGFBP4 and PAPPA mRNAs decreased in endometrial cancer; 2. YTHDF1-mediated IGFBP4 translation was reduced in HEC-1-A and AN3CA cells, and YTHDF2-mediated PAPPA mRNA degradation was blunted but its protein expression increased; 3. Increased PAPPA and reduced IGFBP4 activated IGF1-induced ERK, AKT, and NF-κB pathways by binding IGFR, thereby promoting cancer cell malignancy.
Assuntos
Neoplasias do Endométrio , NF-kappa B , Feminino , Humanos , Proliferação de Células/genética , Progressão da Doença , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Metilação , Metiltransferases/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/genéticaRESUMO
PURPOSE: The present work aimed to explore the aberrant expression of APEX1 in endometrial stromal cells (ESC) and the underlying mechanisms. METHODS: The levels of APEX1 and miR-24 in endometriosis tissues were tested by qRT-PCR and Western blot. After cell transfection, cells were correspondingly classified into pcDNA3.1-NC, sh-NC, mimic NC, inhibitor NC, pcDNA3.1-APEX1, sh-APEX1, miR-24 mimic, miR-24 inhibitor, sh-NC + inhibitor NC, inhibitor-NC + sh-APEX1, sh-NC + miR-24 inhibitor, pcDNA3.1-NC + mimic NC, mimic NC + pcDNA3.1-APEX1 and pcDNA3.1-NC + miR-24 mimic group. Besides, cell proliferation, apoptosis in addition to apoptosis-related proteins Bax, Bcl-2 and cleaved-casase-3 were analyzed by BrdU assay, flow cytometry (FCM) and Western blot assays, respectively. Additionally, RIP assay was conducted to determine the interaction between pri-miR-24 and miR-24. RESULTS: APEX1 and miR-24 were highly expressed in endometriosis tissues. Overexpression of APEX1 and miR-24 potentiates ESC proliferation and inhibits apoptosis, while those effects could be reversed by APEX1 and miR-24 silencing. Meanwhile, APEX1 and miR-24 could elevate ESC apoptosis-related proteins Bax and cleaved-caspase-3 and decrease Bcl-2 expression. Importantly, APEX1 was positively correlated with miR-24 expression. CONCLUSION: APEX1 promotes ESC proliferation and inhibits apoptosis by upregulating miR-24 expression.