LncRNA SNHG4 promotes prostate cancer cell survival and resistance to enzalutamide through a let-7a/RREB1 positive feedback loop and a ceRNA network

Background: Prostate Cancer threatens the health of men over sixty years old, and its incidence ranks first among all urinary tumors among men. Enzalutamide remains the first-line drug for castration-resistant prostate Cancer, however, tumors inevitably become resistant to enzalutamide. Hence, it is of great importance to investigate the mechanisms that induce enzalutamide resistance in prostate Cancer cells.

Methods: Bioinformatic analyzing approaches were used to identified the over-expressed genes in prostate Cancer tumor tissues from three GEO datasets. qRT-PCR, western blotting and immunochemistry/In situ hybridization staining assays were performed to assess the expression of SNHG4, RRM2, TK1, AURKA, EZH2 and RREB1. Cell cycle was measured by flow cytometry. CCK-8, plate colony formation and EdU assays were performed to assess the cell proliferation. Senescence-associated β-Gal assay was used to detect the cell senescence level. γ-H2AX staining assay was performed to assess the DNA damages of PCa cells. Luciferase reporter assay and RNA immunoprecipitation assay were performed to verify the RNA-RNA interactions. Chromatin immunoprecipitation assay was performed to assess the bindings between protein and genomic DNA.

Results: We found that RRM2 and NUSAP1 are highly expressed in PCa tumors and significantly correlated with poor clinical outcomes in PCa patients. Bioinformatic analysis as well as experimental validation suggested that SNHG4 regulates RRM2 expression via a let-7 miRNA-mediated ceRNA network. In addition, SNHG4 or RRM2 knockdown significantly induced cell cycle arrest and cell senescence, and inhibited DNA damage repair and cell proliferation, and the effects can be partially reversed by let-7a knockdown or RRM2 reoverexpression. In vitro and in vivo experiments showed that SNHG4 overexpression markedly enhanced cell resistance to enzalutamide. RREB1 was demonstrated to transcriptionally regulate SNHG4, and RREB1 was also validated to be a target of let-7a and thereby regulated by the SNHG4/let-7a feedback loop.

Conclusion: Our study uncovered a novel molecular mechanism of lncRNA SNHG4 in driving prostate Cancer progression and enzalutamide resistance, revealing the critical roles and therapeutic potential of RREB1, SNHG4, RRM2 and let-7 miRNAs in Anticancer therapy.

Enzalutamide resistance; Prostate cancer; RRM2; SNHG4; ceRNA.