Objective      To construct the human ribonuclease inhibitor (RI) eukaryotic expression vector and determine the effect of RI over-expression on the invasion and migration in melanoma B16 cells.       Methods      Total RNA was extracted from human 7703 cells by Trizol reagents

the RI cDNA fragment was amplified by RT-PCR and the DNA fragment was inserted into the eukaryotic expression vector pIRES2-EGFP with DNA recombinant techniques. Then the vectors were transfected into B16 cells and positive clones were screened with G418. The efficiency of RI genes over-expression was determined by RT-PCR

Western blotting and immunocytochemical assay. B16 cells were observed for morphological changes by HE staining. Adhesion test

cell scratch test and Transwell chamber test were used for adhesion

migration and invasion abilities of the B16 cells. The expression of MMP-2

MMP-9

nm23-H1 and E-cadherin were detected by Western blotting. Lung metastasis nodule was observed by injecting the cells into c57BL/6 mice via tail vein.       Results      Enzyme digestion and DNA sequencing verified the constructed vectors was correct. The RI expression of mRNA and protein levels was significantly increased in B16 cells transfected with pIRES2-EGFP-RI vectors than in control cells. The cells transfected with pIRES2-EGFP-RI expression vectors spread well

with lower nuclear/cytoplasm ratio than controls

and their adhesion

migration and invasion abilities were decreased significantly

while the expression of MMP-2 and MMP-9 was obviously decreased but the nm23-H1 and E-cadherin significantly increased. The mice that were injected with the B16 cells transfected with pIRES2-EGFP-RI showed a smaller number of lung metastasis nodule than those in the control group.       Conclusion      The RI eukaryotic expression vector is successfully constructed. The vector improves RI expression

and decreases cell migration and invasion significantly in B16 cells.