Activation of Hedgehog pathway by circEEF2/miR-625-5p/TRPM2 axis promotes prostate cancer cell proliferation through mitochondrial stress.

Abstract

The purpose of this study was to identify the role played by circEEF2 (has-circ-0048559) in prostate cancer (PCa) development and to determine the potential mechanism involved. circEEF2, miR-625-5p, and the transient receptor potential M2 channel protein (TRPM2) were determined using RT-qPCR in PCa. Cell proliferation was determined by CCK-8 assay and colony formation assay, whereas migration and invasion were assessed by Transwell assay, and apoptosis was evaluated by flow cytometry after annexin V-FITC and propidium iodide staining. The interactions between circEEF2 and miRNAs were investigated through the Circular RNA Interactome database, and the downstream targets of miR-625-5p were forecasted using TargetScan. The interaction was confirmed using both the dual luciferase reporter gene assay and RNA pull-down assay. TRPM2, Hedgehog signaling pathway proteins (GLI1 and GLI2), ubiquinone oxidase subunit B8, and cytochrome C oxidase subunit IV (COX4) were analyzed by protein blotting. JC-1 fluorescence detection was applied for mitochondrial membrane potential changes, fluorescent probe assay for intracellular ROS levels, and immunofluorescence staining for γ-H2AX expression. The role of circEEF2 in PCa tumor growth was tested by xenograft experiments. CircEEF2 expression was upregulated in PCa (p<0.05). Cells of PCa were inhibited in proliferation, migration, invasion, and enhanced in apoptosis by depleting circEEF2 (p<0.05). circEEF2 directly targeted adsorbed miR-625-5p. TRPM2 bound to miR-625-5p. Upregulating TRPM2 likewise reversed the therapeutic effect of depleting circEEF2 on cancer development in PCa cells. circEEF2 activated the Hedgehog pathway through the miR-625-5p/TRPM2 axis, promotes mitochondrial stress, and promotes PCa development in vivo. circEEF2 upregulates mitochondrial stress to promote PCa by activating the Hedgehog pathway through the miR-625-5p/TRPM2 axis.