<i>ALKBH5</i> Suppresses Autophagy in Prostate Cancer Cells via Inhibiting m6A-Modification of <i>TSPAN1</i> mRNA
Xudong Zhao, Zhiqiang Zhao, Honglin Xie, Wen‐Jun Gao, Chunhua Ye
Abstract
Background: Activating autophagy promotes the invasion and progression of prostate cancer (PCa). Tetraspanin 1 (<i>TSPAN1</i>) has been found to promote autophagy flux and its up-regulation can enhance the migration of PCa cells. In addition, there is a binding relationship between <i>TSPAN1</i> and the N6-methyladenosine (m6A) demethylase AlkB homolog 5 (<i>ALKBH5</i>). Therefore, we wanted to know whether <i>ALKBH5</i> could affect autophagy by regulating <i>TSPAN1</i> expression, and thereby participate in PCa malignant progression. Methods: The expression of <i>ALKBH5</i> and <i>TSPAN1</i> in PCa was examined by quantitative real-time polymerase chain reaction (qRT-PCR), and the functional tests included cell counting kit-8 and 5-ethynyl-2′-deoxyuridine (EdU) staining assays. The expression of autophagy-related proteins was confirmed by western blot. Detection of the m6A level of <i>TSPAN1</i> was performed using methylated RNA immunoprecipitation sequencing (MeRIP)-qPCR. Results: <i>ALKBH5</i> was significantly downregulated in PCa cells (LNCaP, DU145 and PC3 cells; <i>p</i> < 0.001). Overexpression of <i>ALKBH5</i> inhibited cell viability and the number of EdU-positive cells (<i>p</i> < 0.01, <i>p</i> < 0.001), decreased the ratio of microtubule-associated protein light chain 3B (LC3B)-II/LC3B-I, and promoted P62 protein expression in LNCaP and DU145 cells (<i>p</i> < 0.001). The m6A level of <i>TSPAN1</i> was high in LNCaP and DU145 cells, but was inhibited by the overexpression of <i>ALKBH5</i> (<i>p</i> < 0.001). <i>TSPAN1</i> overexpression promoted cell viability (<i>p</i> < 0.001), increased EdU-positive cells and the LC3B-II/LC3B-I ratio (<i>p</i> < 0.001, <i>p</i> < 0.05), reduced P62 protein expression (<i>p</i> < 0.05, <i>p</i> < 0.001), and reversed the regulation of <i>ALKBH5</i> overexpression in LNCaP and DU145 cells (<i>p</i> < 0.01, <i>p</i> < 0.001). Conclusions: Promoting <i>ALKBH5</i> expression may inhibit PCa autophagy by reducing the m6A level of <i>TSPAN1</i>.