S100A6 siRNA对卵巢癌细胞生物学行为的影响

Influence of S100A6 siRNA on biological features of human ovarian cancer cell

目的探讨S100A6蛋白表达对卵巢癌细胞生物学行为的影响。方法以卵巢癌细胞A2780为研究对象，利用RNA干扰技术特异性降低S100A6表达水平后，分别用实时定量PCR和Western blot检测S100A6 siRNA对S100A6表达水平的抑制效率。用流式细胞术检测S100A6 siRNA转染前后A2780细胞的细胞周期变化，用Transwell试验观察S100A6siRNA转染前后侵袭能力的变化，用MTT法检测S100A6 siRNA对A2780细胞顺铂敏感性的影响。结果S100A6siRNA转染组细胞中S100A6 mRNA和蛋白表达水平与S100A6 siRNA呈浓度、时间依赖性明显下调，抑制效率可达63．75％～80．68％。在与空白对照组和阴性对照siRNA组相比，S100A6 siRNA出现了G0／G1期比例升高，S期比例降低，差异有统计学意义（X^2=6．211，P=0．045）。Transwell试验表明，6．25、12．5nmol／LS100A6 siRNA分别转染48h后，A2780细胞芽膜细胞数目分别为78±7．51和27±8．24，与阴性对照组（91±5．78）相比，穿膜细胞显著减少，差异有统计学意义（X^2=3．898，P=0.048）。MTT法测定S100A6 siRNA转染后A2780细胞对顺铂敏感性的影响结果显示，S100A6 siRNA引起顺铂对A2780细胞的抑制率增加（X^2=9．609，P=0．022），而空白对照组和阴性对照siRNA组之间无明显差异。对顺铂半数致死剂量（IC50）的计算结果显示，分别转染6．25、12．5nmol／LS100A6 siRNA的A2780细胞对顺铂IC50值分别为13．42μmol／L和11．32μmoL／L，与空白对照组和阴性对照siRNA组相比均显著降低，差异有统计学意义（X^2=5．566，P=0．018）。结论S100A6 siRNA转染卵巢癌A2780细胞后，引起A2780细胞GO／G1期阻滞，侵袭能力降低，同时对顺铂的敏感性有所增强，可能是卵巢癌靶向治疗的候选基因。

Objective To investigate the influence of S100A6 protein on biological features of human ovarian cancer cell line A2780. Methods Human ovarian cancer A2780 cell line was transfeeted with small interfering RNA （siRNA） to reduce the expression of S100A6 specifically, and real time PCR （RT-PCR） and Western blot were used to detect the suppression efficacy of S100A6 siRNA on S100A6 expression. The cell cycle was evaluated by flow eytometry （FCM） , and the invasion ability was determined by Transwell assay. The sensitivity to eisplatin was determined with MTT assay. Results The levels of S100A6 mRNA and protein were greatly reduced in a dose and time dependent manner. S100A6 siRNA could effectively suppress S100A6 expression, with the inhibiting rate of 63.75%-80.68%. The cell cycle assayed by using flow eytometry （FCM） analysis showed that S100A6 siRNA also resulted in accumulation in GO/G1 phase and decline in S phase in A2780 cells （X^2 =6.211 ,P =0. 045）. The number of penetrating cells in 6.25 and 12.5nmol/L S100A6 siRNA transfected groups decreased to 78 ± 7.51 and 27 ± 8.24, respectively. Compared with the negative control group （91 ± 5.78） , the number reduced significantly （X^2 = 3. 898,P = 0. 048）. MTT assay indicated that the inhibition rate of A2780 caused by cisplatin increased （X^2 = 9. 609, P = 0.022 ） , but there were no significant differences from blank control group and negative control group. Pretreatment with 6.25 anti 12.5nmol/L S100A6 siRNA, the 50% inhibitory concentration （IC50） value of cisplatin in A2780 cells was 13. 42μmol/L and 11. 32μmol/L, respectively, which were significantly decreased compared with blank control group and negative control group （X^2 = 5. 566, P = 0. 018）. Conclusion Transfeeting A2780 eel1 line with S100A6 siRNA can induce G0/G1 arrest. Inhibiting S100A6 expression can reduce the invasion ability and improve sensitivity to cisplatin in ovarian cancer cells. It may be a new promising approach in targeting therapy of ovarian cancer.