ALKBH5 Suppresses Autophagy in Prostate Cancer Cells via Inhibiting m6A-Modification of TSPAN1 mRNA.

BACKGROUND: Activating autophagy promotes the invasion and progression of prostate cancer (PCa). Tetraspanin 1 ( TSPAN1 ) 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 TSPAN1 and the N6-methyladenosine (m6A) demethylase AlkB homolog 5 ( ALKBH5 ). Therefore, we wanted to know whether ALKBH5 could affect autophagy by regulating TSPAN1 expression, and thereby participate in PCa malignant progression.

METHODS: The expression of ALKBH5 and TSPAN1 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 TSPAN1 was performed using methylated RNA immunoprecipitation sequencing (MeRIP)-qPCR.

RESULTS: ALKBH5 was significantly downregulated in PCa cells (LNCaP, DU145 and PC3 cells; p < 0.001). Overexpression of ALKBH5 inhibited cell viability and the number of EdU-positive cells ( p < 0.01, p < 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 ( p < 0.001). The m6A level of TSPAN1 was high in LNCaP and DU145 cells, but was inhibited by the overexpression of ALKBH5 ( p < 0.001). TSPAN1 overexpression promoted cell viability ( p < 0.001), increased EdU-positive cells and the LC3B-II/LC3B-I ratio ( p < 0.001, p < 0.05), reduced P62 protein expression ( p < 0.05, p < 0.001), and reversed the regulation of ALKBH5 overexpression in LNCaP and DU145 cells ( p < 0.01, p < 0.001).

CONCLUSIONS: Promoting ALKBH5 expression may inhibit PCa autophagy by reducing the m6A level of TSPAN1 .