Abstract: Objective To observe the effect of oncolytic adenovirus carrying SPAG9 gene on the proliferation and invasion of prostate cancer PC-3 and DU145 cells. Methods 1.Construction of oncolytic adenovirus carrying SPAG9 gene (ZD55-SPAG9) and identification of adenovirus was tested by PCR. ZD55-SPAG9 were amplify in low passage HEK293 cells and purified by AAV Purification Miniprep Kit. The viral titer was determined by TCID50 method. 2. The transfection efficiency of adenovirus was detected with fluorescence microscopy compared with ZD55-EGFP. 3.The viability of PC-3 and DU145 cells was assessed by cell counting kit-8(CCK-8) assay.4.The cytotoxic effects of adenoviruses ZD55-SPAG9 to PC-3 and DU145 cells were detected by Crystal Violet Staining. 5. Cell invasion capacities of PC-3 and DU145 cells were detected by using transwell assay, respectively. 6.The expression of SPAG9 and invasion associated protein MMP-2, Vimentin and E-cadherin in PC-3 and DU145 cells were detected by Western Blot. Results The results of PCR assay showed that adenovirus ZD55-SPAG9 was constructed successfully. 2. After amplification and purification of adenovirus,the titer of ZD55-SPAG9 and ZD55-EGFP were 9.5×109pfu/ml and 9.0×109pfu/ml, respectively.The transfection efficiency ZD55-SPAG9 and ZD55-EGFP were highest at 48h. The cell viability decreased due to the killing effect of adenovirus with fluorescent protein expression decline after 72h. Moreover, the transfection efficiency of ZD55-SPAG9 was higher than ZD55-EGFP at the same time.CCK-8 assay showed that compared with the viability of PC-3 cells of ZD55-EGFP group (70.91±2.35)%, the viability of ZD55-SPAG9 group was (54.83±5.04)%, the difference with statistical significance. The viability of DU145 cells of ZD55-EGFP group and ZD55-SPAG9 group were respectively (75.45±4.32)% and (60.02±2.87)%, the difference with statistical significance. Crystal Violet Staining showed that ZD55-SPAG9 had the strongest cytotoxicity in PC-3 and DU145 cells compared with ZD55-EGFP. Transwell assay showed that the number of transmembrane PC-3 cells of ZD55-SPAG9 group was (58.40±3.20), compared with ZD55-EGFP group (74.80±7.60) and PBS group(121.60±12.20), the difference was statistically significant (P<0.05). The number of transmembrane DU145 cells of ZD55-SPAG9 group was (37.00±3.20), compared with ZD55-EGFP group (84.20±6.60) and PBS group(164.66±10.45), the difference was statistically significant (P<0.05). Western Blot showed that the SPAG9 expression of ZD55-SPAG9 group was lower than PBS and ZD55-EGFP group.Western Blot analysis also confirmed that ZD55-SPAG9 could increase the expression level of E-cadherin, concomitant with down-regulating the proteins expression of MMP-2 and Vimentin, their?difference?was significant(P<0.05). Conclusion Oncolytic adenovirus carrying SPAG9 gene could inhibit the growth and invasion effect in PC-3 and DU145 cells. We preliminarily explore the mechanism of ZD55-SPAG9 on the effect of cell invasion and the regulatory effect may related to EMT(epithelial-mesenchymal transition)