下调S100A8表达对卵巢癌细胞恶性生物学行为的影响及其机制

Effect of down-regulation of S100A8 expression on malignant biological behavior of ovarian cancer cells and its mechanism

目的探讨下调S100钙结合蛋白A8(S100A8)表达对卵巢癌细胞恶性生物学行为的影响及其机制。方法体外传代培养人卵巢浆液性乳头状囊腺癌细胞株SKOV3。取传3代、对数生长期、生长状态良好的SKOV3细胞,随机分为对照组、空转组、S100A8组,对照组不予转染,空转组转染NC-siRNA,S100A8组转染S100A8-siRNA,采用RT-PCR技术验证转染效率。收集各组转染后细胞,分别于继续培养24、48、72 h,采用MTT法检测细胞增殖活性;继续培养24 h,采用Annexin V-FITC/PI双染法检测细胞凋亡率,采用细胞划痕实验检测细胞迁移能力,采用RT-PCR技术检测磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(Akt)mRNA表达,采用Western blotting法检测PI3K、Akt蛋白表达。结果S100A8组S100A8 mRNA相对表达量显著低于对照组和空转组(P均<0.05),而对照组与空转组比较差异无统计学意义(P>0.05)。S100A8组继续培养24、48、72 h细胞增殖活性均显著低于对照组和空转组同期(P均<0.05),而对照组与空转组继续培养24、48、72 h比较差异均无统计学意义(P均>0.05);S100A8组继续培养72 h细胞增殖活性显著低于继续培养24、48 h(P均<0.05),而继续培养24 h与继续培养48 h比较差异无统计学意义(P均>0.05)。S100A8组细胞凋亡率显著高于对照组和空转组(P均<0.05),而对照组与空转组比较差异无统计学意义(P>0.05)。S100A8组划痕距离显著低于对照组和空转组(P均<0.01),而对照组与空转组比较差异无统计学意义(P>0.05)。S100A8组PI3K、Akt蛋白与mRNA相对表达量均显著低于对照组和空转组(P均<0.01),而对照组与空转组比较差异均无统计学意义(P均>0.05)。结论下调S100A8表达能够抑制卵巢癌细胞增殖和迁移并促进其凋亡,其机制可能与抑制PI3K/Akt信号通路激活有关。

Objective To investigate the effect of down-regulating S100 calcium binding protein A8(S100A8)expression on malignant biological behavior of ovarian cancer cells and its mechanism.Methods Human ovarian serous papillary cystadenocarcinoma cell line SKOV3 was subcultured in vitro.The SKOV3 cells in the third generations,in logarithmic growth period and with good growth condition were obtained.They were randomly divided into the control group,idling group,and S100A8 group.The cells in the control group were not transfected,the cells in the idling group were transfected with NC siRNA,and the cells in the S100A8 group were transfected with S100A8 siRNA,respectively.RT-PCR was used to verify the transfection efficiency.The transfected cells of each group were collected and cultured for 24,48 and 72 h,respectively.The cell proliferation activity was detected by MTT.We continued to culture them for 24 h,and used Annexin V-FITC/PI double staining method to detect the apoptosis rate.The cell migration ability was tested by cell scratch test.RTPCR was used to detect the mRNA expression levels of phosphatidylinositol 3-kinase(PI3K)and protein kinase B(Akt),and Western blotting was used to detect the protein expression levels of PI3K and Akt.Results The relative expression of S100A8 mRNA was significantly lower in the S100A8 group than in the control group and the idling group(both P<0.05),and there was no significant difference between the control group and the idling group(P>0.05).The proliferation activity of cells cultured for 24,48 and 72 h was significantly lower in the S100A8 group than in the control group and the idling group at the same time(all P<0.05),and there was no significant difference between the control group and the idling group at 24,48and 72 h(P>0.05).The proliferation activity of cells cultured for 72 h in the S100A8 group was significantly lower than that cultured for 24 and 48 h(both P<0.05),and there was no significant difference between the 24-hour culture and 48-hour culture(P>0.05).The apoptosis rate was significantly higher in the S100A8 group than in the control group and the idling group(both P<0.05),and there was no significant difference between the control group and the idling group(P>0.05).The scratch distance was significantly shorter in the S100A8 group than in the control group and the idling group(both P<0.01),but there was no significant difference between the control group and the idling group(P>0.05).The relative expression levels of PI3K,Akt protein and mRNA in the S100A8 group were significantly lower than those in the control group and the idling group(all P<0.01),but there were no significant differences between the control group and the idling group(all P>0.05).Conclusion Down-regulation of S100A8 expression can inhibit the proliferation and migration of ovarian cancer cells and promote their apoptosis,which may be related to inhibiting the activation of PI3K/Akt signaling pathway.