MicroRNA-608 inhibits proliferation of bladder cancer via AKT/FOXO3a signaling pathway

Zhen Liang, Xiao Wang, Xin Xu, Bo Xie, Alin Ji, Shuai Meng, Shiqi Li, Yi Zhu, Jian Wu, Zhenghui Hu, Yiwei Lin, Xiangyi Zheng, Liping Xie, Ben Liu

Current evidence indicates that miR-608 is widely down-regulated in various malignant tumors including liver cancer, colon cancer, lung cancer and glioma, and acts as a tumor suppressor by inhibiting cell proliferation, invasion and migration or by promoting apoptosis. The specific biological function of miR-608 in bladder cancer is still unknown. qRT-PCR and Chromogenic in Situ Hybridization (CISH) was conducted to assess the expression of miR-608 in paired BCa tissues and adjacent non-tumor bladder urothelial tissues. Bisulfite sequencing PCR was used for DNA methylation analysis. CCK-8, colony formation and flow cytometry assays were performed, and a xenograft model was studied. Immunohistochemistry staining was performed with peroxidase and DAB. The target of miR-608 was validated with a dual-luciferase reporter assay, quantitative RT-PCR, and Western blotting. miR-608 is frequently down-regulated in human BCa tissues. The methylation status of CpG islands is involved in the regulation of miR-608 expression. Overexpression of miR-608 inhibits the proliferation and tumorigenesis of BCa cells in vitro and in vivo. Additionally, up-regulation of miR-608 in BCa cells induces G1-phase arrest through AKT/FOXO3a signaling. In contrast, down-regulation of miR-608 promotes proliferation and cell cycle progression in BCa cells. Moreover, the expression of FLOT1 was directly inhibited by miR-608, the down-regulation of FLOT1 induced by siFLOT1 could be significantly reversed by miR-608 inhibitor. Similarly, the up-regulation of FLOT1 by FLOT1 overexpression plasmid (pFLOT1) could also reverse the suppressed cell proliferation caused by miR-608. miR-608 is a potential tumor suppressor in BCa, and the restoration of miR-608 might be a promising therapeutic option for BCa.