Abstract: Background and purpose: Protein interactions dominate the life processes, including cell signal transduction, transmembrane transport, DNA synthesis and transcriptional regulation, playing important and complex roles in life activities. Our previous study demonstrated the direct combination of ribonuclease inhibitor (RI) and integrin-linked kinase (ILK) in vitro and in vivo using GST pull down and Co-IP. The purpose of this study was to investigate the role of interplay between RI and ILK on ILK/AKT/mTOR pathway and bladder carcinoma growth in vitro and in vivo. Methods: Immunofluorescence staining was performed to analyze the co-localization of RI and ILK in bladder cancer EJ cells. Fluorescence resonance energy transfer (FRET) was applied to demonstrate the interaction between RI and ILK. Subsequently, the stable EJ cell lines with overexpression of RI or ILK were generated respectively. The protein levels of RI, ILK, and the molecules related to ILK/AKT/mTOR pathway were determined using Western blot. And the cell viability and cell cycle were analyzed by cell counting kit-8 (CCK-8) and flow cytometry assays respectively. In addition, the model of bladder cancer xenograft in nude mouse was constructed, and the effects of overexpression of RI or ILK on xenograft growth were observed. The expressions of RI, ILK and the proteins related to ILK/AKT/mTOR pathway in xenograft tissues were examined using immunohistochemistry and immunofluorescence. Results: The co-localization and interaction of RI and ILK were observed and verified in EJ cells. Overexpression of RI suppressed cell proliferation capacity (P<0.05), led to the blockage of S phase (P<0.05), repressed bladder cancer xenograft growth (P<0.05), and inhibited ILK/AKT/mTOR pathway in vitro and in vivo (P<0.05). However, overexpression of ILK promoted cell proliferation and xenograft growth (P<0.05), and overactivated ILK/AKT/mTOR pathway in vitro and in vivo (P<0.05). Conclusion: RI interacts with ILK, inhibits ILK/AKT/mTOR pathway and suppresses bladder cancer growth in vitro and in vivo.

Key words: Ribonuclease inhibitor, Integrin-linked kinase, Bladder cancer, Proliferation