Effect of silencing sperm associated antigen 9 gene specially by RNA interference on human prostate carcinoma PC-3 cells

Abstract

Objective To investigate the effect of silencing sperm associated antigen 9 (SPAG9) gene specially by RNA interference on human prostate carcinoma PC-3 cells, and the possible mechanism. Methods The plasmid vector expressing SPAG9 short hairpin RNA (shRNA) was constructed and transfected into PC-3 cells by lipofectamine 2000. Cells were collected and assayed at 48 h after transfection. The SPAG9 mRNA and protein expression level was measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting, respectively. Cell proliferation was tested by cell counting kit-8 (CCK-8) assay. Cell apoptosis was detected by fluorescence-activated cell sorter (FACS). The invasion ability of cells was examined by Transwell assay. To explore its possible mechanism, the expression of matrix metalloproteinase (MMP)-2, vascular endothelial growth factor (VEGF), Vimentin and E-cadherin proteins was measured by Western blotting after SPAG9 gene knockdown for 48 h. Results The recombinant plasmid pGPU6-green fluorescent protein (GFP)-SPAG9 expressing SPAG9 shRNA had been constructed successfully. RT-PCR and Western blotting showed the expression level of SPAG9 mRNA and proteinin in pGPU6-GFP-SPAG9 group was 1.96±0.16 and 0.39±0.05 respectively, lower than empty plasmid group (3.23±0.18 and 1.13±0.04), blank control group (3.08±0.12 and 1.25±0.05), respectively, the differences were both statistically significant (P=0.000). The absorbance (A) value was 31.9±2.6 in pGPU6-GFP-SPAG9 group, weaken than that in empty plasmid group (47.8±2.8, P=0.023), blank control group (48.1±2.8, P=0.016), the differences were statistically significant. FACS analysis showed that the apoptosis rate was (12.12±0.69)% in pGPU6-GFP-SPAG9 group, more than empty plasmid group [(6.43±0.31)%, P=0.020], blank control group [(5.19±0.33)%, P=0.014], the differences were statistically significant. Western blotting showed the expression level of MMP-2, VEGF, and Vimentin in pGPU6-GFP-SPAG9 group was lower than in the control groups, however, the average expression level of E-cadherin in pGPU6-GFP-SPAG9 group was higher than in the control groups, the differences were all statistically significant (P=0.000). Conclusion Silencing SPAG9 gene can inhibit SPAG9 gene expression, suppress proliferation, induce cell apoptosis of PC-3 cells by decreasing MMP-2, VEGF and Vimentin expression level, and increasing E-cadherin expression level. Key words: Prostate cancer; Sperm associated antigen 9 gene; RNA interference