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目的构建沉默DNMT1基因的重组体pshRNA-DNMT1,研究其体内外对胃癌AGS细胞增殖凋亡的影响。方法设计短发夹RNA(shRNA)的寡核苷酸片断,克隆到载体pTZU6+1中,构建重组体pshRNA-DNMT1,转染胃癌细胞株AGS,用Western印迹检测DNMT1蛋白质水平变化,用逆转录-聚合酶链反应(RT-PCR)法评估mRNA水平,四甲基偶氮唑盐比色法(MTT法)动态监测活细胞数,电镜和原位末端标记技术(TUNEL)观察其体外的促凋亡作用。构建裸鼠胃癌模型,经pshRNA-DNMT1处理后观察瘤体大小,绘制生长曲线;用苏木素伊红(HE)染色法、电镜和TUNEL检测细胞凋亡,用增殖细胞核抗原(PCNA)法评价细胞的增殖能力。结果成功构建重组质粒pshRNA-DNMT1后,进行序列分析得到确证。pshRNA-DNMT1转染AGS细胞后24h出现DNMT1蛋白质表达量减少,抑制率为28·24%,48h为68·54%,72h为81·47%,重组质粒pshRNA-DNMT1对胃癌AGS细胞中DNMT1基因的转录有着明显抑制作用,转染后24h抑制率为21·63%,48h为52·97%,72h为72·06%。转染后24、48和72h细胞数存活率为PBS组的79·49%、51·63%和39·16%。pshRNA-DNMT1能抑制肿瘤生长,呈一定的时间和剂量依赖性。结论重组质粒pshRNA-DNMT1能特异有效地抑制胃癌细胞株AGS内DNMT1基因的表达。在胃癌细胞株AGS的体内外实验中均能抑制细胞增殖,促进细胞凋亡。
Objective To construct recombinant pshRNA-DNMT1 silenced DNMT1 gene and study its effect on AGS cell proliferation and apoptosis in vitro and in vivo. Methods The oligonucleotide fragment of short hairpin RNA (shRNA) was designed and cloned into vector pTZU6 + 1. The recombinant pshRNA-DNMT1 was constructed and transfected into gastric cancer cell line AGS. The DNMT1 protein level was detected by Western blot. The mRNA levels were assessed by polymerase chain reaction (RT-PCR). The number of living cells was dynamically monitored by MTT colorimetry (MTT assay) Apoptosis. The gastric cancer model was established. The size of the tumor was observed after treatment with pshRNA-DNMT1, and the growth curve was drawn. The apoptosis was detected by hematoxylin and eosin (HE) staining, electron microscopy and TUNEL. Proliferative ability. Results The recombinant plasmid pshRNA-DNMT1 was successfully constructed and confirmed by sequence analysis. The expression of DNMT1 protein was down-regulated 24 h after pshRNA-DNMT1 was transfected into AGS cells, the inhibitory rate was 28.24%, 48.58% at 48h and 81.47% at 72h after transfection with pshRNA-DNMT1. The expression of DNMT1 gene in AGS cells The inhibition rate was 21.63% at 24h after transfection, 52.97% at 48h and 72.06% at 72h after transfection. The cell viability at 24, 48 and 72 h after transfection was 79.49%, 51.63% and 39.16% of the PBS group, respectively. pshRNA-DNMT1 can inhibit tumor growth, showing a certain time and dose-dependent. Conclusion The recombinant plasmid pshRNA-DNMT1 can specifically and effectively inhibit DNMT1 gene expression in gastric cancer cell line AGS. AGS in gastric cancer cell lines in vitro and in vivo experiments can inhibit cell proliferation and promote apoptosis.