6-磷酸果糖激酶-2／果糖双磷酸酶-2同工酶3基因对前列腺癌细胞增殖的影响

The impact of 6 - phosphofructo -2 - kinase/fructose -2,6 - bisphosphatase -3 gene on the prolif- eration of prostate cancer cells

目的观察6-磷酸果糖激酶-2/果糖双磷酸酶-2同工酶3（PFKFB3）在前列腺癌组织中的表达及其对PC3和DU145前列腺癌细胞增殖的影响。方法采用免疫组织化学染色（抗体浓度1∶50）方法，观察PFKFB3基因在前列腺癌组织及癌旁组织中的表达。实时荧光定量聚合酶链反应（FQ-PCR，40个循环，188 bp）、Western blot法[30 μl，10%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）]检测PFKFB3 mRNA和蛋白在前列腺癌细胞及前列腺上皮细胞中的表达。采用平板克隆形成实验（40 μmol/L H2O2）与细胞生长实验[20 μl噻唑蓝（MTT）]对比观察敲除PFKFB3基因前后前列腺癌细胞的生长。结果PFKFB3基因在前列腺癌组织中的表达（2.42±0.16）明显高于癌旁组织（1.38±0.10），差异有统计学意义（P＝0.031）；前列腺癌细胞中PFKFB3 mRNA和蛋白表达水平均高于前列腺上皮细胞，分别为2.0～2.6倍和2.5～4.5倍（P＝0.015）；敲除PFKFB3基因后，平板克隆形成实验结果显示细胞克隆形成率为敲除前的22%～35%（P＝0.018），细胞生长实验结果显示前列腺癌细胞的增殖在第4天受抑制，为对照组的28%～30%（P＝0.026）。结论PFKFB3基因在前列腺癌细胞和组织中高表达，PFKFB3基因对前列腺癌细胞的增殖有一定的促进作用。

To investigate the expression of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase-3 （PFKFB3） gene in the prostate cancer tissues and its impact on the proliferation of prostate cancer cells PC3 and DU145.Methods Immunohistochemical staining was used to detect the expression of PFKFB3 in the prostate cancer tissues and the adjacent tissues （antibody concentration 1∶50）. Real-time fluorescent quantitative polymerase chain reaction （FQ-PCR, 40 cycles, 188 bp） and Western blotting [30 μl, 10% sodium dodecyl sulfate polyacrylamide gel electropheresis （SDS-PAGE）] were applied to detect the mRNA and protein expression levels of PFKFB3 expression in the prostate cancer cells and prostate epithelial cells. After knocking out PFKFB3 gene, plate cloning （40 μmol/L H2O2） formation assay and cell growth assay [20 μl methyl thiazol tetrazolium （MTT）] were applied to measure the cell proliferation.ResultsThe expression of PFKFB3 in the prostate cancer tissues （2.42±0.16） was significantly higher than that in the adjacent tissues （1.38±0.10, P=0.031）. The mRNA and protein expression levels of PFKFB3 in prostate cancer cells were significantly higher than those in the prostate epithelial cells （P=0.015）, 2.0-2.6 times and 2.5-4.5 times respectively. When knocking out the PFKFB3 gene, cloning formation assay showed that the cloning formation rate decreased as 22%-35% compared to the control （P=0.018）. Cell growth assay showed that the proliferation of prostate cancer cells was inhibited as 28%-30% at day 4 （P=0.026）. ConclusionPFKFB3 gene is highly expressed in prostate cancer cells and prostate cancer tissues. PFKFB3 plays a promotional role in the prostate cancer cells.