Objective        To investigate the effects of ribonuclease inhibitor (RI) on the proliferation and apoptosis in mice melanoma B16 cells.        Methods        B16 cells was stably transfected with RI eukaryotic expression plasmid pIRES2-EGFP-RI

using the cells transfected with empty vector and untransfected cells as controls

positive clones were screened with G418. The transcription level of RI in B16 cells was determined by RT-PCR

while its protein level was determined by Western blotting and immunofluorescence assay. The cell proliferation was detected by MTT assay. Cell cycle and apoptosis were evaluated by flow cytometry (FCM)

TUNEL and Hochest33342 staining. The expression of apoptosis-related proteins and key molecules of ILK/PI3K/AKT signaling pathway were determined by Western blotting. B16-RI cells

B16 vector cells

and B16 cells were used to implant into the C57BL/6 mice to establish transplantation tumor model. The growth of tumor was observed

tumor microvessel density was surveyed by HE staining and CD31 immunofluorescence assay

and the expression of ILK/PI3K/AKT signaling pathway proteins in tumor tissues were further analyzed.        Results        The RI expression at mRNA and protein levels was significantly increased in B16 cells transfected with pIRES2-EGFP-RI plasmid comprised with the other 2 control cells. MTT assay indicated that the proliferation of B16-RI cells was notably decreased compared with those in B16 vector and B16 cells. Compared with B16 cells or B16 vector cells

the percentage of B16-RI cells at the S phase was increased

indicating more cells were arrested at S phase. A large number of apoptotic cells showed typical apoptotic morphological features

such as chromatin

nuclear fragmentation and bright blue fluorescence in B16-RI cells (P0.01). Western blotting indicated that the expression of ILK

p-Akt

p-GSK3β

Bcl-2 and β-catenin was distinctly decreased in B16-RI cells

while that of activated Caspase-3 and Bax was significantly increased compared with the control cells (P0.01). Animal experiment showed that the tumor weight and microvessel density in tumor tissue of B16-RI group were significantly reduced and the expression of ILK

p-Akt

p-GSK3β

and β-catenin were notably decreased simultaneously

when compared with the B16 and B16 vector cells groups. TUNEL found the apoptosis in the implanted tumor was consistent with that of the cells.        Conclusion        Over-expression of RI significantly inhibits the growth of mice melanoma B16 cells and induces cell apoptosis via regulating ILK/PI3K/AKT signaling pathway.