Abstract: Objective To investigate the effects of ribonuclease inhibitor expression on EMT and metastasis of mouse melanoma B16-F10 cells. Methods RI eukaryotic expression plasmid pIRES2-EGFP-RI was constructed, then transfected into B16-F10 cells, RI expression was identified by RT-PCR, Western blot and immunofluorescence assay. Cell morphology was observed by HE staining and phase contrast microscope. The cytoskeleton change was detected with FITC phalloidin staining under laser scanning confocal microscope. The adhesion assay, scratch assay and Transwell assay were used to detect the cell adhesion, migration and invasion ability. The expressions of metastasis and EMT related proteins were determined by Western blot. Various B16-F10 cells were respectively injected into the vein of the eye socket of c57/BL mice to establish pulmonary metastasis animal model. Three weeks after injection, the mice were sacrificed, lungs were removed and weighed. And then the number of metastasis nodules was counted under a dissecting microscope. The lung tissue sections were stained with HE staining, and the expressions of metastasis and EMT related proteins were detected by immunohistochemistry.Results The results showed that up-regulation of RI inhibited migration and invasion as well as changed cell morphology, adhesion and rearranged cytoskeleton in vitro. In addition, the data indicated that over-expression RI induced up-regulation of E-cadherin, accompanied with decreased expressions of proteins associated with EMT such as N-cadherin, snail, slug, vimentin and twist both in vitro and in vivo. Furthermore, RI restrained matrix metalloproteinase MMP-2 and MMP -9 secretions in B16-F10 melanoma cells. Finally, the data showed that RI suppressed invasiveness and metastasis by the experimental metastasis models of melanoma with lighter lung weight, a fewer metastasis nodules and a lower incidence rate, with respect to the control groups. Conclusion Up-regulating ribonuclease inhibitor could significantly inhibit EMT, invasion and metastasis of B16-F10 cell. These results suggest that RI could be a therapeutic target protein for melanoma.

Key words: ribonuclease inhibitor, B16-F10 cells, EMT, metastasis