S100P shRNA慢病毒载体的构建及其对胃癌MGC-803细胞生物学行为的影响-东南大学学报医学版

S100P shRNA慢病毒载体的构建及其对胃癌MGC-803细胞生物学行为的影响

单位：东南大学附属中大医院普外科, 南京江苏 210009

分类号：R735.2

出版年·卷·期（页码）：2015·34·第一期（65-70）

摘要：

目的:构建针对S100P基因的shRNA慢病毒载体,并在胃癌MGC-803细胞上鉴定沉默效率,观察其对细胞生物学行为的影响。方法:筛选的S100P基因特异性siRNA靶点,合成短发卡结构shRNA,并与GV115-GFP慢病毒载体重组形成shRNA表达载体,与pHelper 1.0和pHelper 2.0质粒共转染293T细胞,产生慢病毒。用病毒感染MGC-803细胞,RT-PCR和Western blot检测靶基因的沉默效率、克隆形成情况、细胞周期变化和凋亡实验观察靶基因沉默对MGC-803的影响。结果:构建的重组shRNA慢病毒载体,经293T细胞包装获得病毒颗粒,和阴性对照相比,慢病毒感染组S100P mRNA表达下降了83.4%,蛋白表达也明显受到抑制。S100P沉默后MGC-803的克隆形成能力明显减弱,细胞周期发生S期阻滞,细胞凋亡明显增加。结论:成功地构建了S100P基因shRNA慢病毒表达载体,该载体能够在MGC-803细胞中沉默S100P基因的表达,导致胃癌细胞克隆形成能力降低,细胞周期阻滞,诱导细胞凋亡,表明S100P基因有可能具有影响胃癌发生发展的作用。

Objective: To construct a shRNA lentiviral vector targeting S100P gene, and detect the efficiency of gene silence and its biological role on MGC-803 cells. Methods:Specific targets sequence of S100P were designed and synthesized short hairpin RNA, then were recombined into shRNA expression vector GV115-GFP. 293T cells were cotransfected with reconbined lentiviral vector(sh-S100P), pHelper 1.0 and pHelper 2.0 to produce shRNA lentiviral particles. MGC-803 cells were tansfected with lentivirus, the silencing efficiency of targeting sequence was detected by RT-PCR and Western blot, the biological function of S100P gene silence on MGC-803 cells was evaluated by colony formation assay, cell cycle and apoptosis analysis. Results: We successfully constructed the shRNA lentiviral expression vector and obtained the virus by packaging the 293T cell. The expression level of S100P mRNA in MGC-803 cells transfected with identificated shRNA lentiviral particles was decreased by 83.4%.The expression of protein was suppressed notably. Downregulating S100P expression suppressed the cell colony formation, blocked cell cycle and promoted the apoptosis in MGC-803 gastric cancer cell. Conclusion: The successfully constructed recombinant vector can silence S100P expression in MGC-803 cells and suppress colony formation, induce cell cycle arrest and apoptosis, which indicates S100P may be involved in the progress of gastric cancer cells.

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