Interplay Between Intergrin-Linked Kinase And Ribonuclease Inhibitor Regulates Epithelial-Mesenchymal Transition Through ILK Pathways In Bladder Cancer Cells

ObjectiveTo research the interplay between integrin-linked kinase(ILK) and ribonuclease inhibitor(RI) and investigate its effect on EMT and ILK signaling pathway in human bladder cancer cells. Methods1. To construct pEYFP-N1-ILK and pCMV-3×flag-ILK plasmid. Purchase lentiviral expression vector LV5-RNH1 homo and LV5 NC. Stably transfect vectors into EJ cells repectively, and the selected cells were named as EJ-RI, EJ-ILK, EJ-LV5, EJ-FLAG and EJ. The efficiency of overexpression of ILK and RI was detected by IF and Western blot.2. The interaction between RI and ILK in 293 and EJ cells was identified by glutathione-S-transferase(GST) pulldown assay and co-immunoprecipitation assay. Fluorescence microscope analysis was used to observe the co-location between RI and ILK in 293 cells and EJ cells. Using fluorescence resonance energy transfer(FRET) to further confirm that the two proteins possess a direct binding in EJ cells.3. CCK8 assay, flow cytometry assay and endothelial tube formation assay were conducted to analyze the interacton between ILK and RI and its impact on EJ cells.4. Through selection of stable cell lines, we detected the expression level of protein of EMT and ILK signaling pathway by Western blot.5. The stable–transfected EJ cell lines were injected subcutaneously into the flank of 4-week-old athymic nude mice. Tumors and lungs were harvested 30 days after injection. HE staining was done to observe the microvessels of tumors and metastasis of lungs. Immunofluorescence assay and IHC detection were conducted to explore the level of proteins related to EMT and signaling pathway. Results1. The expressing plamid pEYFP-N1-ILK and pCMV-3×flag-ILK were verified by restriction enzyme digestion and DNA sequencing. The results indicated that RI and ILK had stably high expressions in EJ cells respectively.2. Results indicate a directly physical interaction between ILK and RI in vitro and interaction between RI and ILK are present in the cellular content. A colocalization of ILK with RI was observed in the cytoplasm and near the cellular membrane.3. The proportion of EJ-RI cells in the S phase increased from 31 to 52.88 %. For EJ-ILK cells, around 64.99 % of the cells were in the G1 phase and 2.86 % in the G2 phase. Futhermore, data shows that ILK slightly increased the proportion of EJ cells in the G1 subphase while RI significantly decreased the proportion to 37.16 %. ILK significantly enhanced tube formation while RI inhibited the tube formation. The 450 nm absorbance is directly proportional to the number of living cells. The inhibitory rates of EJ-RI cells to EJ vector cells, and the promoting rates of EJ-ILK to EJ vector cells were 29.41% and 35.29% at 120 h respectively.4. The protein levels of p-Akt, p-GSK3β, p-PI3 K, p-PTEN, p-mTOR, β-catenin, MMP2, MMP9, N-cadherin, Vimentin, Twist, Snail and S100A4 were increased significantly in EJ-ILK cells whereas these protein levels decreased obviously in EJ-RI cells.5. In animal models, the rates of tumor formation were significantly lower in EJ-RI group compared with the other two control groups respectively. The results showed that the EJ-ILK cells group significantly promoted the growth of bladder cancer compared with the other control groups. Immunofluorescent analyses of CD31 and S100A4 were implemented to show that the EJ-ILK cells group displayed a high CD31 and S100A4 expression and apparent increase of angiogenesis in tumor tissue. Weaker ILK, p-Akt, p-GSK3β, p-PI3 K, p-PTEN, p-mTOR, β-catenin, MMP2, MMP9 and Vimentin expression were seen in tumor tissue of the EJ-RI groups. In human bladder cancer samples, ILK expression was negatively correlated with RI levels. ConclusionThe interaction between RI and ILK occurs in prokatyotic cells and mammalian cells. Up-regulating ILK could remarkably increase cell proliferation, regulate cell cycle and augment endothelial tube formation. Overexpression ILK significantly increased growth and metastasis of xenograft tumor as well as angiogenesis via epithelial-mesenchymal transition(EMT) and ILK signaling pathways; Whereas, RI has just the opposite effects to ILK. The interplay between ILK and RI regulating EMT via ILK/PI3K/AKT signaling pathways for bladder cancer.