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Pan Wang

 

Pan Wang

Affiliated Cancer Hospital of Chongqing University,
China

Abstract Title: miR-652-5p promoter hypermethylation enhances GBM malignancy

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Background To explore the molecular mechanism by which miR-652-5p promoter hypermethylation promotes glioblastoma maligancy in hypoxia. Methods KEGG analyses were performed on the upregulated differentially expressed miRNAs in HIF2α-ko cells. miRNA-652-5p promoter hypermethylation was verified by MSP and MeDIP. Mimicked or inhibited miRNA-652-5p expression and measured SDC1 and TGFβ2 levels in GBM cells. Cultured SDC1 and TGFβ2 knockdown cells under hypoxic conditions to measure CD63 and TGFβ2 levels in culture medium. PI3K-AKT signalling pathway component levels were measured after SDC1 and TGFβ2 knockdown, and cell proliferation, migration, invasion, fluc-positive viable cell, tumour weight and OS were analysed. Results miR-652-5p exhibited the largest log2FoldChange among significantly upregulated miRNAs between HIF2α-ko and control cells. The MSP and MeDIP demonstrated miR-652-5p promoter hypermethylation and a decrease in its methylation level after HIF2α knockout. SDC1, SDC1-Intra and TGFβ2 expression was increased after miR-652-5p inhibition. Live cell imaging and fluorescence staining showed that SDC1 regulated exosome production and TGFβ2 was secreted via exosomes. After SDC1 knockdown, the exosome number was decreased and TGFβ2 secretion was inhibited. BiFC and CoIP assays showed that secreted TGFβ2 bound to SDC1 or sSDC1 via HS chains and was linked to ERBB4 on recipient cells. After SDC1 and TGFβ2 knockdown, the levels of PI3K, PDK1, p-PDK1, AKT, p-AKT and mTOR were decreased and cell proliferation, migration, invasion, fluc-positive viable cells and tumour weights were inhibited but with longer OS. Conclusion In a hypoxic microenvironment, miR-652-5p promoter hypermethylation enhances glioblastoma malignancy through the SDC1/TGFβ2/pERBB4 complex via HIF2α-mediated regulation. Keywords miR-652-5p promoter hypermethylation; sSDC1, TGFβ2 and pERBB4 complex; HS chains of sSDC1; glioblastoma; autocrine and paracrine