A Short-hairpin RNA Expression Vector-mediated Osteopontin RNA Interference Inhibits The Proliferation And Invasion Of Prostate Cancer PC-3 Cells

Objective:Substantial data have linked osteopontin (OPN), a secreted phosphoglycoprotein, with tumor progression and metastatic spread. OPN regulates cell migration and invasion in a variety of cancers and induces the activities of matrix metalloproteinase (MMP)-2 and MMP-9. It has been reported that OPN induce matrix metalloproteinase (MMP)-2 and MMP-9 activations through nuclear factor kappaB (NF-κB)-mediated signaling pathways. This study was to investigate the role of OPN in the proliferation and invasion of human prostate cancer PC-3 cells, and provide clues about the possible functions of IκB kinase (IKK) in OPN-induced MMP-2/9 activitions by nuclear factor kappaB (NF-KB)-mediated signaling pathways. Methods: Four sorts of OPN short-hairpin RNA (shRNA) recombinant plasmids were constructed and transfected into PC-3 cells. The most highly functional shRNA recombinant plasmid was selected by RT-PCR for further studies, then after the stablely transfected cell line were established, the different concentrations of IKK inhibitors were used to inhibit the activities of IKK-1 and IKK-2. The mRNA and protein expression levers of OPN, MMP-2 and MMP-9 were detected by real-time PCR and Western blot. MMP-2 and MMP-9 protein levels in the medium of the cells cultured by OPN were detected by ELISA. The different cell cycles, cell proliferation and invasion abilities of PC-3 cells were detected by flow cytometry, MTT and Transwell chamber assays, respectively. The tumor formation assay were used to observe the growth situation of PC-3 cells in vivo. Results:The most highly functional shRNA recombinant plasmid (PGPU6/GFP/Neo-OPN2) was selected by RT-PCR assay. It can obviously inhibit OPN expression in PC-3 cells and the stablely transfected cell line, PCs, PC/Vect and PC/OPN2 were established. Compared with untreated cells(PCs), the protein expression levers of OPN, MMP-2 and MMP-9 in PC-3 cells transfected with recombinant plasmids (PC/OPN2) were reduced by 55.22%,51.71% and 28.35%, respectively, thereby resulting in suppression of the proliferation, migration and invasion of PC/OPN2 cells. Moreover, the inhibition of IKK-2 inhibited the expressions of MMP-2 and MMP-9, while the inhibition of IKK-1 had no influence on the expressions of MMP-2 and MMP-9. Conclusions:A short hairpin RNA expression vector-mediated OPN gene silencing can not only inhibit OPN expression in PC-3 cells but also decrease the expression levels of MMP-2 and MMP-9. OPN shRNA recombinant plasmid can inhibit the malignant physiological behaviors of PC-3 cells and these processes are associated with the activities of MMP-2 and MMP-9. Moreover, IKK-2 may play a crucial role in the OPN-induced activations of MMP-2 and MMP-9 via NF-κB mediated signaling pathways.