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目的:应用反义RNA技术抑制人肝癌细胞SMMC-7721中POLD1基因的表达,探索采用该技术干预肝癌的可行性。方法:制备人POLD1基因的反义RNA表达质粒,同时建立空白对照组与阴性对照组,由此将实验分为阴性对照组(转染空载体的SMMC-7721细胞)、空白对照组(肝癌细胞SMMC-7721)和实验组(转染人POLD1基因反义RNA表达质粒的肝癌SMMC-7721细胞)3组。MTT实验分析细胞增殖变化,并在转染SMMC-7721细胞48h后,实时荧光定量PCR和蛋白质印迹法检测POLD1基因的表达。结果:MTT实验显示,与空白对照和阴性对照组相比,转染了反义RNA的实验组细胞增殖受到明显抑制。在转染后24～72h,实验组吸光度24h为0.306 7±0.015 4,48h为0.459 2±0.033 2,72h为0.567 1±0.061 1,与空白和阴性对照组相比,P值均<0.05。实时荧光定量PCR实验显示,实验组POLD1的mRNA表达明显下调为0.142 5±0.020 5,阴性对照组为1.017±0.188,空白对照组为1.00±0.00(F=26.5,P<0.05),说明POLD1基因表达受抑制。蛋白质印迹实验显示,POLD1基因蛋白水平(p125)变化趋势与mRNA相同,均为下调,其中实验组为0.222 3±0.009 7,阴性对照组为0.237 9±0.005 9,空白对照组为0.235 4±0.003 4(F=1 365.754,P值均<0.05),说明反义RNA可抑制p125蛋白表达,结论与基因水平趋势一致。结论:针对POLD1基因设计的反义RNA体外抑制了肝癌细胞SMMC-7721的增殖,这与POLD1在基因和蛋白水平的表达下调有关。
OBJECTIVE: To detect the expression of POLD1 gene in human hepatocellular carcinoma cell line SMMC-7721 using antisense RNA technique and to explore the feasibility of using this technique to interfere with hepatocellular carcinoma. Methods: The antisense RNA expression plasmid of human POLD1 gene was prepared, and blank control group and negative control group were established. The experiment was divided into negative control group (SMMC-7721 cells transfected with empty vector), blank control group SMMC-7721) and experimental group (SMMC-7721 cells transfected with human POLD1 antisense RNA expression plasmid). Cell proliferation was analyzed by MTT assay. The expression of POLD1 gene was detected by real-time fluorescence quantitative PCR and Western blot 48 h after SMMC-7721 cells were transfected. Results: MTT assay showed that compared with the blank control group and the negative control group, the proliferation of cells transfected with antisense RNA was significantly inhibited. At 24-72h after transfection, the absorbance of the experimental group was 0.306 7 ± 0.015, the value of 4,48h was 0.459 2 ± 0.033 2, and the value of 72h was 0.567 1 ± 0.061 1. The P values were all <0.05 compared with the blank and negative control groups. Real-time PCR showed that the mRNA expression of POLD1 in the experimental group was significantly decreased as 0.142 5 ± 0.020 5, in the negative control group was 1.017 ± 0.188 and in the blank control group was 1.00 ± 0.00 (F = 26.5, P <0.05) Expression is inhibited. Western blotting showed that the trend of P125 gene level was the same as that of mRNA, which was down-regulated in experimental group (0.222 3 ± 0.009 7), negative control group (0.237 9 ± 0.005 9) and blank control group (0.235 4 ± 0.003) 4 (F = 1 365.754, P <0.05), indicating that antisense RNA can inhibit the expression of p125 protein. The conclusion is consistent with the trend of gene level. CONCLUSION: Antisense RNA targeting POLD1 gene inhibits the proliferation of hepatocellular carcinoma SMMC-7721 in vitro, which is related to the down-regulation of POLD1 gene and protein.