Overexpression of NPRL2 is Linked to Patients with Bladder Cancer and Promotes Bladder Cancer Cells Progression by Regulating ENO1-SMAD2/3 Axis

Background: Nitrogen permease regulator-like 2 (NPRL2) acts as an effective gene for diverse tumor suppression and its expres-sion is up-regulated in castration-resistant prostate cancer (CRPC). However, potential mechanisms of NPRL2 in bladder cancer (BCa) progression remain to be discovered. To explore the function and molecular mechanism of NPRL2 in the development and progression of BCa, bioinformatics analysis and verification were performed. Methods: Extensive bioinformatic analysis explored NPRL2 expression associated with BCa using data from the Cancer Genome Atlas (TCGA) database. BCa cell lines (T24 and 5637 cells) were used to verify the function and molecular mechanism of NPRL2 in BCa, and they were divided into four groups: NC-CMV-NPRL2 (transfected with PDS237_pL-CMV-GFP vector), CMV-NPRL2 (transfected with PDS237_pL-CMV-GFP-TUSC4), NC-sh-NPRL2 (transfected with pl6.3-SHRNA-BSD vector), and sh-NPRL2 (transfected with pl6.3-SHRNA-GFP-NPRL2). The proliferation, invasion, migration, cell cycle and apoptosis of T24 and 5637 cells were further evaluated by Cell Counting Kit-8 (CCK-8), Transwell, flow cytometry and Western blot assays. Nude mice were used to establish xenotransplantation models, and three groups (control, sh1-NPRL2, sh2-NPRL2) were developed. The control group was subcutaneously injected with T24 cell suspension. The sh-NPRL2 groups were subcutaneously injected with two groups of T24 cells that had been stably transduced with NPRL2 knockout genes.Results: NPRL2 gene expression exhibited remarkable upregulation in BCa (p = 0.004). Overexpression of NPRL2 enhanced the proliferation and invasion of T24 and 5637 cells, while simultaneously inhibiting apoptosis. Furthermore, NPRL2 deficiency in the xenotransplantation model exhibited inhibition of tumorigenesis in vivo. Additionally, Immunoprecipitation and Shotgun Liquid Chromatography-Mass Spectrometry (LC-MS) analysis revealed a protein interaction between NPRL2 and alpha-enolase (ENO1) which was highly expressed in BCa. ENO1 had a noticeable effect on BCa stage and survival by promoting cell proliferation, epithelial-mesenchymal transition (EMT), small mothers against decapentaplegic (SMAD)2/3 phosphorylation, and ENO1's stability was enhanced by NPRL2.Conclusions: The present study reveals that NPRL2 is upregulated in BCa and can enhance SMAD2/3 phosphorylation by up-regulating ENO1. NPRL2 is a potential biomarker for BCa diagnosis and prognosis.