蛋白激酶D3对前列腺癌细胞中基质金属蛋白酶7的负调控作用

Protein kinase D3 is involved in negative regulation of MMP-7 in prostate cancer cells

目的探讨蛋白激酶D3(PKD3)对前列腺癌细胞中基质金属蛋白酶7(MMP-7)的调控作用。方法应用佛波脂(PMA,100nmol/L)处理前列腺癌PC3细胞,Westernblotting和实时RT-PCR分别检测PKD3的激酶活性和MMP-7mRNA表达;将siRNA-PKD3瞬时转染PC3细胞以敲低PKD3的表达,同样用实时RT-PCR检测MMP-7mRNA的表达状况;在HEK293细胞中,分别过表达GFP-PKD3、siRNA-PKD3敲低PKD3的表达或先过表达GFP-PKD3再siRNA-PKD3敲低PKD3的表达,以上述同样的方法比较MMP-7mRNA相对表达量的变化。结果在PC-3细胞中,PMA诱导的PKD3的激活可明显降低MMP-7mRNA表达,反之,应用siRNA敲低PKD3的表达则明显提高MMP-7mRNA表达水平(P<0.01);同样,在HEK293细胞中,过表达PKD3可明显降低MMP-7的表达水平,而敲低PKD3可明显提高MMP-7的表达,且敲低PKD3可逆转PKD3过表达诱导的MMP-7下调。结论 PKD3可能通过负调控MMP-7的表达而介导前列腺癌的恶性进程。

Objective To explore the role of protein kinase D3 （PKD3） in the regulation of matrix metalloprteinases 7 （MMP-7） expression in prostate cancer cells. Methods PC-3 cells were either stimulated with 100 nmol/L PMA to activate PKD3 kinase activity, or transiently transfected with PKD3 siRNA, and the relative expression level of MMP-7 mRNA were analyzed by real-time PCR using 2~~method. MMP-7 mRNA levels were also analyzed and quantified in HEK293 cells with over-expression of wild-type PKD3, PKD3 knockdown （using PKD3 siRNA）, or over-expression of wild-type PKD3 followed by PKD3 knockdown. Results MMP-7 mRNA expression in PC3 cells was significantly decreased after PMA-induced PKD3 kinase activation. In contrast, PKD3 knockdown by siRNA transfection markedly increased MMP-7 mRNA level （P〈0.01）. MMP-7 mRNA level in HEK293 cells was significantly decreased by PKD3 over-expression, whereas obviously increased by PKD3 knockdown. Down-regulation of MMP-7 mRNA level in HEK293 induced by PKD3 over-expression was rescued by PKD3 knockdown. Conclusion PKD3 may contribute to the malignant progression of prostate cancer cells through negative regulation of MMP-7 expression.