Effects Of Ribounclease Inhibitor Up-regulated On Growth, Appototoc, Invasion And Metastasis Of Melanoma Cells And The Research On Its Molecular Mechanisms

Objective To construct the human ribonuclease inhibitor (RI) eukaryoticexpression vector and investigate the effect of RI overexpression on thegrowth,apoptotic, invasion and metastasis of murine melanoma cells.Methods Total RNA was extracted from QGY-7703cells by Trizolreagents,the RI cDNA fragment was amplified by RT-PCR and the DNAfragment was inserted into the eukaryotic expression vector pIRES2-EGFPwith DNA recombinant techniques.Then the vectors were transfected intoB16cells and positive clones were screened with G418.The efficiency ofRI genes overexpression was determined by RT-PCR,Western blot andimmunocytochemical method，these also were used in detecting ANG geneexpression when RI up-regulated.B16and B16-F10cells were observed formorphological changes by HE staining and cytoskeleton assays,anddetermined for adhesion ability by adhesion test,for metastasis ability by wound healing assay,and for invasion ability by transwell invasionassay.The expression of MMP-2, MMP-9, nm23-H1,E-cadherin,ILK, Bax,Bcl-2and Casepase3and so on were detected by western blot; Theinhibitory effect of RI up-regulated on cell proliferation was assessed byFlow Cytometry and MTT, The influence on cell apoptosis was assessed byHochest33342, Tunel Kit and Flow Cytometry; The B16/B16-F10，B16vector/B16-F10vector, B16-RI/B16-F10-RI were respectively injected intothe left flank or the vein of the socket of eye of syngeneic c57BL/6mice.Recording the time of the tumor emerged, and21days after injection, themice were killed, the tumors of animal growth mode and the lungs ofanimal pulmonary metastasis model were excised and weighed. The densityof tumor microvessel was detected by HE staining and CD31antibody ofimmunofluorescence；The expression of RI, MMP2, ILK, Snail, Slug,E-cadherin, pGSK-3β and so on were detectde by Immunohistochemistryand immunofluorescence.Results Enzyme digestion and DNA sequencing verified the constructedpIRES2-EGFP-RI vectors were correct. Compared with control cells,theRI expression of mRNA and protein significantly increased in B16orB16-F10cells by transfected with pIRES2-EGFP-RI plasmids; and theANG expression was decreased when RI expression up-regulated.the cellstransfected with pIRES2-EGFP-RI expression vectors spread well,indicated an unordered actin network，sparse actin fibers and epithelial morphology and the nuclearmass ratio was lower than controls,and theirproliferation,adhesion, metastasis and invasion abilities decreasedsignificantly,while the expression of MMP-2, MMP-9, Snail, Slug, Vimetin,N-cadherin, ILK, p-Akt, p-GSK3β， β-catenin and Bcl-2obviouslydecreased but the apoptosis and the expression of Bax, Casepase3,nm23-H1and E-cadherin significantly increased. The mouses that wereinjected with the B16/B16-F10cells transfected pIRES2-EGFP-RI showeda smaller tumors,a lower tumor microvessel density, a lighter lung weightand a fewer number of lung metastasis nodule and a lower incidence ratethan those in the control group,and the expressions of some proteins in vivowere consistent with the expressions in vitro.Conclusion The pIRES2-EGFP-RI eukaryotic expression vector issuccessfully constructed.The vector improves RI expression,and decreasescell growth, adherin, migration, and metastasis ability but increases cellsapoptosis significantly in vivo and vitro. Its molecular mechanisms arethat RI up-regulated inhibits the ability of invision and metastasis inmelanoma cells through regulating EMT and represses growth or improvecells apoptosis via regulating the ILK/PI3K/AKT signaling pathway.