抑制激肽释放酶10基因对结肠癌细胞增殖和凋亡及顺铂敏感性的影响

Inhibition of kallikrein 10 gene effecting on the proliferation and apoptosis of colon cancer cell and its chemosensitivity to cisplatin

目的通过小干扰RNA（siRNA）抑制激肽释放酶10（KLK10）基因在结肠癌细胞中的表达，观察其对结肠癌细胞增殖和凋亡及顺铂（DDP）敏感性的影响。 方法实时定量反转录聚合酶链反应（RT-qPCR）检测KLK10 mRNA在正常结肠细胞株NCM460和结肠癌细胞株HRT-S1、HCT116中的表达水平；利用Lipofectamine？ RNAi MAX转染试剂将KLK10特异性siRNA转染进入结肠癌细胞株HRT-S1、HCT116；采用噻唑蓝（MTT）、流式细胞术（FCM）法检测siRNA干扰KLK10基因对HRT-S1、HCT116细胞增殖和凋亡的影响；使用Western blot法检测细胞周期蛋白（Cyclin） D1和B细胞淋巴瘤/白血病-2（bcl-2）蛋白水平上的表达变化。结果KLK10mRNA在结肠癌细胞株HRT-S1和HCT116中表达较正常结肠细胞明显升高（P＝0.001）；siRNA干扰KLK10基因后，KLK10 mRNA表达水平在结肠癌细胞株HRT-S1和HCT116中明显降低（P＝0.002）。siRNA干扰KLK10基因后，Cyclin D1蛋白表达水平在HCT116细胞株中明显降低（P＝0.027），但在HRT-S1细胞株中差异无统计学意义（P＝0.081）；siRNA干扰KLK10基因后，bcl-2蛋白表达水平在HCT116和HRT-S1细胞株中均明显降低（P＝0.038、0.016）。siRNA抑制KLK10基因后，在HRT-S1和HCT116细胞株中，DDP的半数抑制浓度（IC50）值降低（P＝0.032和0.022），结肠癌细胞的增殖被抑制；DDP＋siRNA-KLK10组中HCT116和HRT-S1细胞凋亡率较DDP＋si-scramble组明显升高（P＝0.028、0.036）。结论抑制KLK10基因可以显著抑制结肠癌细胞增殖能力，能够促进肿瘤细胞凋亡，增强肿瘤细胞对DDP的敏感性；抑制KLK10基因可能通过抑制Cyclin D1和bcl-2的表达，从而抑制结肠癌细胞增殖，促进肿瘤细胞凋亡。

Objective To study the effect of KLK10 on the proliferation and apoptosis of colon cancer cell and its chemosensitivity to cisplatin （DDP） by small interfering RNA （siRNA） targeting KLK10.MethodsKLK10 specific siRNA was transfected into colon cancer cell lines HRT-S1 and HCT116 by Lipofectamine？ RNAi MAX transfection reagent. real-time quantitative reverse transcriptase-polymerase chain reaction （RT-qPCR） was used to detect the expression level of KLK10 mRNA in NCM460 and HRT-S1 and HCT116 colon cancer cell lines. The expression levels of Cyclin D1 and B cell lymphoma/leukemia-2 （bcl-2） protein were determined by Western blotting. Respectively, cell proliferation, apoptosis and half maximal inhibitory concentration （IC50） of cisplatin were detected by methyl thiazol tetrazolium （MTT） and flow cytometry （FCM） in vitro. ResultsCompared with those negative control groups, KLK10 mRNA level deceased in experimental group in HRT-S1 and HCT116 （P=0.002）; Cyclin D1 protein level deceased in HCT116 （P=0.027）; bcl-2 protein deceased in HRT-S1 and HCT116 （P=0.038 and 0.016）; The IC50 of cisplatin in experimental group was lower than that in the other groups in HRT-S1 and HCT116 （P=0.032 and 0.022）, the cells proliferation was suppressed. Apoptosis rate was significantly higher （P=0.028 and 0.036）.ConclusionKLK10 gene suppression can significantly inhibit colon cancer cell proliferation ability, can promote tumor cell apoptosis, enhance the sensitivity of tumor cells to DDP, by inhibitting the expression of Cyclin D1 and bcl-2; KLK10 gene is an important target in the treatment of colon cancer.