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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-GsiRNA,分为两组。实验组以不同浓度(分别为1.0、2.5、5.0μg/L,下同)的HLA-GsiRNA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-GmRNA的表达,HLA-GmRNA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-GsiRNA转染JEG-3细胞后,明显下调JEG-3细胞中HLA-GmRNA及蛋白的表达水平。实验组不同浓度的HLA-GsiRNA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-GsiRNA可以下调HLA-GmRNA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。
Objective To investigate the effect of small interfering RNA (siRNA) on human leukocyte antigen G (HLA-G) gene expression in choriocarcinoma (choriocarcinoma) cells and to understand the role of HLA-G gene in carcinogenesis and development of choriocarcinoma. Methods HLA-G siRNAs were designed and synthesized and divided into two groups. The experimental group were transfected with JEG-3 cells with high expression of HLA-G gene by HLA-G siRNA at different concentrations (1.0, 2.5 and 5.0 μg / L respectively), and the control group was treated with the same amount of negative control siRNA Transfection of JEG-3 cells. The expression of HLA-G mRNA in JEG-3 cells was detected by real-time fluorescent quantitative RT-PCR. The expression of HLA-G mRNA was expressed as the number of PCR cycles (Ct value). Western blotting was used to detect HLA-G The expression of JEG-3 cells was observed under a microscope and the number of surviving JEG-3 cells was counted. Results The expression of HLA-G mRNA and protein in JEG-3 cells was down-regulated by HLA-GsiRNA transfected JEG-3 cells. After transfection of HLA-GsiRNA with different concentrations in the experimental group, the Ct values of JEG-3 cells [(20.67 ± 0.02), (21.37 ± 0.03), (21.43 ± 0.02) cycles, respectively] (20.33 ± 0.01), (20.37 ± 0.02), (20.40 ± 0.03) cycles], the difference was statistically significant (P <0.05). After transfection, the expression of HLA-G protein in JEG-3 cells in the experimental group [0.42 ± 0.03,0.37 ± 0.02,0.12 ± 0.04], respectively, was significantly higher than that in the control group [1.69 ± 0.23,1.62 ± 0.31, 1.36 ± 0.22], the difference was statistically significant (P <0.01). The growth of JEG-3 cells in the experimental group was significantly inhibited compared with the control group (P <0.05). Conclusion HLA-G siRNA can downregulate the expression of HLA-G mRNA and protein and inhibit the cell growth, indicating that HLA-G gene plays an important role in the carcinogenesis and progression of choriocarcinoma.