| ObjectiveTo study over-expressing of RI on the EMT, invasion andmetastasis, then explore the mechanism underlying.Methods1.T24cells were transfected with pIRES2-EGFP andpIRES2-EGFP-RI plasmid respectively. Use G418to screen thepositive clone and named T24group(), T24vector group (transfectedwith pIRES2-EGFP plasmid) and T24-RI (transfectedwithpIRES2-EGFP-RI plasmid) group respectively. RT-PCR was usedto decte the expression of RI mRNA, immunofluorescence detectionand western blot used to exprolore the expression of protein RI.2.The change of morphology was detected by HE dying, cells’cytoskeleton was observated by conducted phalloidine expreriment.Flow cytometry examination was done to explore the cell cycle andMTT assay to the cell proliferation.To detect whether RI and ILKcolocalized, we conducted immunofluorescence laser scanning confocal detection, then we took immunofluorescence detection to detect theexpression level of protein p-Akt and p-GSK3β.3.Cell adhesion detection, wound healing assay and transwellassay was conducted to explore cell adhesion, immigration, invasionabilities respectivly.The expression of RI, ILK, the invasion releventprrotein MMP-2， MMP-9, the EMT relevent protein E-cadherin,N-cadherin, Vimentin, Snail, Slug, Twist and the cyclin-D1protins4.Cells of each group (T24, T24vector, T24-RI) were collectedand injected into the backs of the BALB/C nude mice,five weeks afterinjection, the mice were sacrificed and the tumors were removed andweighted. Immunofluorescence detection was done to explore the levelof p-Akt and p-GSK3β. The micorvessels of tumor and whether thecancer cell have metastasized to lung were detected by HE dying. Atthe same time, immunofluorescence assay of CD31was executed todetermine the influence of RI on new blood vessel formation.Immunohistochemical assays of key proteins were done in the animaltumor tissue (RI, ILK, MMP-2，MMP-9，E-cadherin, N-cadherin,Vimentin, Snail, Slug, Twist) or human bladder tissues (RI, ILK,E-cadherin, Vimentin, Snail, Slug, Twist). HE stain was done to detectthe change of micorvessels in human bladder tissues.Results1.The expression of RI mRNA and protein level was obviously increased.2.HE staining showed that up-regulating RI obviously changedcell morphology with an epithelial phenotype, smaller nucleuscompared with the other two control group cells, phalloidine-FITCstaining showed the cell cytoskeleton displayed over-expression of RIinduced a disorganized actin network, less actin fibers and epithelialmorphology. result of flow cytometry examination indicated that theproliferation of T24-RI cells was inhibited with G1phase, G2-M phasereducing and S phase arresting. RI has interaction with ILK in. Theexpression of ILK downstream targets p-Akt and p-Gsk3β decreased.3.Corresponding detections indicated that cell adhesion wasincreased (Ｐ＜0.01), migration and invasion were decreased(Ｐ＜0.01,Ｐ＜0.01). The results of western blot in vitro showed, theexpression of RI，E-cadherin proteins were increased in T24-RI groupcells than T24and T24vector group cells, while N-cadherin, MMP-2，MMP-9, Vimentin, Snail, Slug, Twist decreased and Cyclin-D1alsoobviuosly (Ｐ＜0.01, Ｐ＜0.01, Ｐ＜0.01, Ｐ＜0.01, Ｐ＜0.01, Ｐ＜0.01,Ｐ＜0.01, Ｐ＜0.05).4.After cells of each groups were injected into the backs of theBALB/C nude mice. The results showed that the T24-RI cells groupsignificantly inhibited the growth of bladder cancer compared with theother two control groups. Immunofluorescence assay of CD31and HE stain was executed. Results showed that T24-RI cell groups revealedaless microvessels in tumor tissue than two other control groups. Miceinjected with the T24-RI cell groups also showed a remarkablesuppression of the spontaneous lung metastasis, in which no metastasiswas observed in T24-RI cell groups. Results of immunohistochemicalassays reveled that the T24-RI groups displayed stronger positive signalof E-cadherin expressions and lower brown staining of MMP-2,MMP-9, N-cadherin, Vimentin, Cyclin-D1, Slug, Snail and Twistexpressions compared with the control groups in the animal tumortissue. Tumor with low metastasis capability and adjacent non-tumortissues showed stronger positive signal of E-cadherin and RIexpressions as well as weaker brown staining of ILK, Snail, Slug,Vimentin and Twist expressions compared with tumor with highmetastasis capability.ConclusionOur findings highlight that up-regulating ribonuclease inhibitorinhibited invasion and metastasis through epithelial-to-mesenchymaltransition and ILK signaling pathway in bladder cancer cells... |
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