Sialidase NEU1 suppresses progression of human bladder cancer cells by inhibiting fibronectin-integrin α5β1 interaction and Akt signaling pathway

Background Sialic acids are widely distributed in animal tissues, and aberrantly expressed in a variety of cancer types. High expression of sialic acid contributes to tumor aggressiveness by promoting cell proliferation, migration, angiogenesis, and metastasis. Sialidases are responsible for removal of sialic acids from glycoproteins and glycolipids. Methods N-glycomics of bladder cancer cells were detected by MALDI-TOF mass spectrometry. Sialic acid modification in bladder cancer tissue was determined by lectin blot. The down-regulation of NEU1 in bladder cancer cells was determined by high resolution liquid chromatography mass spectrometry (HR LC-MS). The effects of sialidase NEU1 expression on proliferation and apoptosis of human bladder cancer cells were examined by western blot, RT-PCR, confocal imaging and flow cytometry. Moreover, the function of sialic acids on fibronectin-integrin α5β1 interaction were assayed by immunoprecipitation and ELISA. The importance of NEU1 in tumor formation in vivo was performed using BALB/c-nu mice. Expression of NEU1 in primary human bladder cancer tissue samples was estimated using bladder cancer tissue microarray. Results (1) Downregulation of NEU1 was primarily responsible for aberrant expression of sialic acids in bladder cancer cells. (2) Decreased NEU1 expression was correlated with bladder cancer progression. (3) NEU1 overexpression enhanced apoptosis and reduced proliferation of bladder cancer cells. (4) NEU1 disrupted FN-integrin α5β1 interaction and deactivated the Akt signaling pathway. (5) NEU1 significantly suppressed in vivo tumor formation in BALB/c-nu mice. Conclusions Our data showed that NEU1 inhibited cancer cell proliferation, induced apoptosis, and suppressed tumor formation both in vitro and in vivo, by disrupting interaction of FN and integrin β1 and inhibiting the Akt signaling pathway. Our observations indicate that NEU1 is an important modulator of the malignant properties of bladder cancer cells, and is a potential therapeutic target for prognosis and treatment of bladder cancer. Video Abstract Graphical abstract


NEU1 gene silencing (siRNA)
Sequences of siRNA oligos for NEU1, synthesized by Invitrogen, were: HCV29 cells were cultured to 90% confluence in 12-well plates in growth medium without antibiotics, and transfected with siRNA oligos and Lipofectamine 2000. Transfection efficiency was determined by quantitative real-time RT-PCR and Western blotting.

Immunohistochemistry
Tissues were dewaxed, and antigen retrieval was performed using citrate buffer (0. Slides were counterstained with H&E.

Wound assay for motility
Cells were cultured in 6-well plates with complete medium and grown to 100% confluence.
Three separate wounds were scratched in each well using a 200 μL pipette tip. Wounds were washed with PBS and added with serum-free culture medium to prevent cell proliferation. Wounds were photographed at 0 and 24 h.
Adherent cells were fixed with 2% fresh paraformaldehyde for 10 min and stained with 0.1% crystal violet in 20% methanol for 10 min. Excess dye was washed off with PBS. Crystal violet in the cells was dissolved in 100 μL of 10% acetic acid, and absorbance was measured at 595 nm.

Determination of apoptosis by flow cytometry
Apoptotic cells were quantified using an Annexin V-FITC detection kit (Beyotime) according to the manufacturer's protocol. In brief, cells were collected and washed in cold PBS, resuspended in 1× binding buffer in the presence of Annexin V-FITC and PI, incubated for 20 min at RT in the dark, and subjected to flow cytometry (BD Biosciences; Franklin Lakes, NJ, USA).
Data were analyzed using the FlowJo software program (Tree Star; San Carlos, CA, USA).

Tissue microarray (TMA) analysis
Bladder cancer TMAs consisting of 44 cases of bladder carcinoma tissue with matched adjacent normal bladder tissue were from Shanghai Outdo Biotech Co. Nonspecific protein on slides was blocked by incubation with 5% BSA in PBS at 4 °C overnight. Specific antibodies were applied to slides and incubated for 3 h in the dark. Slides were scanned with a confocal scanner and photographed under fluorescence microscopy as above. Staining intensity of TMAs was evaluated and scored by automated quantitative imaging using the Image Pro Plus software program (Media Cybernetics; Carlsbad, CA, USA).

Co-immunoprecipitation (co-IP)
Cells were washed three times with ice-cold PBS and added with ice-cold weak RIPA

Extraction of detergent-soluble microdomain (DSM) and detergent-insoluble microdomain (DIM) fractions
DSM and DIM fractions were extracted as described previously [9]. In brief, cells were solution was added. The reaction was stopped by rinsing the slides with deionized water. Finally, testicular nuclei were counterstained with hematoxylin.

Cell cycle assay
Cells were harvested and suspended in 0.1% BSA/PBS. Cells were fixed with 70% EtOH and washed with PBS. RNAs were digested using RNase and DNAs were stained with PI in the dark.
Finally, cells were washed with PBS, resuspended, and subjected to FACS analysis. Data were analyzed using NovoExpress (ACEA, China) with Watson model.