沉默microRNA-20a对乳腺癌MCF7细胞表达NK细胞活化性受体配体MICA的研究

目的 采用寡核苷酸抑制剂特异性沉默乳腺癌MCF7细胞microRNA-20a的表达，检测MCF7细胞表面MICA分子的变化以及对于自然杀伤细胞（NK细胞）介导的杀伤活性的影响。方法 在脂质体介导下用 microRNA- 20a 抑制剂瞬时转染MCF7细胞，采用RT-PCR检测microRNA- 20a的表达量以明确抑制效果；采用免疫印迹法检测转染后MCF7细胞表达MICA分子的变化以及采用结晶紫实验检测MCF7细胞经过转染后以及在加入MICA封闭抗体前后对NK细胞介导的杀伤活性的变化。结果 与转染无关序列组等比较，经过microRNA-20a 抑制剂转染后MCF7细胞表达microRNA-20a下调，同时表达MICA蛋白分子上调；结晶紫法检测NK细胞对MCF7的杀伤活性，显示经过microRNA-20a 抑制剂转染后MCF7细胞的死亡率显著增加（P〈0.01），同时MICA分子的封闭性抗体能够逆转MCF7细胞升高的死亡率。结论 沉默microRNA-20a能上调MCF7细胞表达NK细胞活化性杀伤受体的配体MICA分子，并导致MCF7细胞在NK细胞介导的杀伤实验中死亡率显著上升。以microRNA- 20a为靶点的RNA干扰有望为增强肿瘤细胞的免疫原性从而上调NK细胞对其发挥杀伤作用提供新的靶点。

Study of microRNA-20a targeted silence on the expression of NK cell activation receptor ligand MICA on breast cancer MCF7 cells

Objective： To explore the effect of microRNA-20a targeted silence on the expression of MICA in breast cancer MCF7 cells and to investigate its effect on MCF7 cells sensitivity to natural killer （NK） cells cytotoxicity. Methods： MCF7 were transfected with microRNA-20a inhibitor using Lipofectamin2000,Real-time-PCR was applied to evaluate the transfection efficiency by detecting the expression of microRNA and Western-blotting was used to detect the expression of MICA. Then the susceptivity of MCF7 to the cytotoxicity of NK cells was assessed based on the crystal violet methods. Results： MicroRNA-20a targeted silencing resulted in the markedly decrease of microRNA-20a. The MCF7 cell up-regulated the expression of MICA and its susceptivity to the cytotoxicity of NK cells was increased significantly after the microRNA silence （P〈0.01）. Moreover, the up-regulated death rate of MCF7 was abolished by MICA antibody blocking peptide. Conclusions： MicroRNA-20a targeted silencing allows the increased expression of MICA, an important ligand for one of the activation receptors of NK cells. It implicates that downmodulation of microRNA-20a could serve as a target to increasing the susceptivity of breast cancer cells to the cytotoxicity of NKcells.