小分子干扰RNA对绒毛膜癌细胞人类白细胞抗原G 基因表达的影响

目的探讨小分子干扰RNA(siRNA)对绒毛膜癌(绒癌)细胞中人类白细胞抗原G(HLA-G) 基因表达的影响,了解HLA-G基因在绒癌发生、发展中的作用.方法设计并合成HLA-G siRNA,分为两组.实验组以不同浓度(分别为1.0、 2.5、 5.0 μg/L,下同)的HLA-G siRNA转染HLA-G基因高表达的绒癌细胞系JEG-3细胞,对照组以等量阴性对照siRNA转染JEG-3细胞.实时荧光定量RT-PCR技术检测转染后JEG-3细胞中HLA-G mRNA的表达,HLA-G mRNA的表达以PCR循环数阈值(Ct值)表示;蛋白印迹法测定JEG-3细胞中HLA-G蛋白的表达;显微镜观察并计数转染后存活的JEG-3细胞数.结果 HLA-G siRNA转染JEG-3细胞后,明显下调JEG-3细胞中HLA-G mRNA及蛋白的表达水平.实验组不同浓度的HLA-G siRNA转染后,JEG-3细胞的Ct值[分别为(20.67±0.02)、(21.37±0.03)、(21.43±0.02)个循环数],分别与对照组[分别为(20.33±0.01)、(20.37±0.02)、(20.40±0.03)个循环数]比较,差异有统计学意义(P<0.05).上述浓度转染后,实验组JEG-3细胞的HLA-G蛋白表达水平[分别为0.42±0.03、0.37±0.02、0.12±0.04],分别与对照组[分别为1.69±0.23、1.62±0.31、1.36±0.22]比较,差异有统计学意义(P<0.01).实验组JEG-3细胞生长较对照组明显受到抑制(P<0.05).结论 HLA-G siRNA可以下调HLA-G mRNA及蛋白的表达水平,抑制细胞生长,表明HLA-G基因在绒癌的发生、发展中具有重要作用.

Role of human leukocyte antigen-G small interference RNA in a choriocarcinoma cell line

OBJECTIVE: To study the role of human leukocyte antigen-G (HLA-G) in the carcinogenesis and development of choriocarcinoma. METHODS: We designed and synthesized a double strand small interference RNA (siRNA) of HLA-G, then transfected it into a HLA-G overexpressed choriocarcinoma cell line, JEG-3 by lipofectine; HLA-G mRNA level was detected by real time RT-PCR; HLA-G protein level was detected by western blot. The living cells of JEG-3 were counted under microscope after transfection by siRNA. RESULTS: Double strand siRNA of HLA-G effectively downregulated the mRNA level and the protein level of HLA-G. The mRNA levels by Ct value at different concentration (1.0, 2.5, 5.0 microg/L) were 20.67 +/- 0.02, 21.37 +/- 0.03, 21.43 +/- 0.02, respectively. They were significantly different compared with the control groups which were 20.33 +/- 0.01, 20.37 +/- 0.02, 20.40 +/- 0.03, respectively (P < 0.05). The protein levels were down regulated in different concentration compared with the control groups (0.42 +/- 0.03 vs 1.69 +/- 0.23, 0.37 +/- 0.02 vs 1.62 +/- 0.31, 0.12 +/- 0.04 vs 1.36 +/- 0.22; P < 0.01). Through downregulation of HLA-G expression, the growth of choriocarcinoma cells was inhibited. CONCLUSIONS: Small interference RNA of HLA-G can effectively down regulate HLA-G gene in mRNA and protein levels and inhibit tumor growth in choriocarcinoma. It shows that HLA-G plays an important role in the development of choriocarcinoma.