STGC3基因表达下调对NP69细胞系生长增殖的影响

发布时间：2018-03-20 15:25

本文选题：鼻咽癌　切入点：NP69细胞系　出处：《南华大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的:设计靶向STGC3基因的shRNA(miR30-based shRNA),构建针对STGC3基因的特异性shRNA真核表达载体,将pRNAT-U6-shRNA-STGC3瞬时转染永生化人鼻咽上皮细胞系NP69,观察STGC3基因表达降低对NP69细胞系的影响,分析STGC3基因在细胞生长增殖中的作用。方法:运用生物信息学方法分析与筛选STGC3基因的干扰靶点,根据miRNA30框架结构,设计靶向STGC3基因的shRNA,构建pRNAT-U6-shRNA-STGC3真核表达载体,用Lipofectamine 2000将重组pRNAT-U6-shRNA-STGC3表达载体瞬时转染NP69细胞系,通过荧光显微镜下观察GFP表达,确定转染效率,以RT-PCR,检测STGC3基因mRNA表达水平,评估miR30-based shRNA对STGC3基因的干扰效果,扩大培养转染细胞,进行细胞克隆形成实验,观察pRNAT-U6-shRNA-STGC3- NP69细胞克隆形成能力,使用流式细胞仪,分析pRNAT-U6-shRNA-STGC3- NP69细胞周期的改变和凋亡情况。结果构建载体,经PCR、双酶切鉴定和测序鉴定,确定成功构建pRNAT-U6-shRNA-STGC3真核表达载体。将pRNAT-U6空质粒和pRNAT-U6-shRNA-STGC3重组质粒分别转染NP69细胞系,转染36小时后,荧光显微镜下观察NP69、pRNAT-U6-NP69和pRNAT-U6-shRNA-STGC3-NP69三组细胞的GFP表达,pRNAT-U6-NP69和pRNAT-U6-shRNA-STGC3-NP69两组细胞可见绿色荧光,而NP69细胞则无绿色荧光,结果表明空质粒和重组质粒均成功转入NP69细胞系; RT-PCR检测结果证实pRNAT-U6-shRNA-STGC3-NP69细胞中STGC3基因mRNA的表达水平明显低于NP69和pRNAT-U6-NP69细胞,结果表明miR30-based shRNA成功地干扰了STGC3在NP69细胞系中的表达。细胞克隆形成实验结果显示,pRNAT-U6-shRNA-STGC3-NP69较对照组克隆形成能力增强(p0.05);流式细胞仪检测,pRNAT-U6-shRNA-STGC3-NP69组G_2期和S期的细胞增加,生长增殖速度增快,凋亡减少,与对照比较差异具有显著性意义(p㩳0.05)。结论 1.成功构建了pRNAT-U6-shRNA-STGC3真核表达载体。 2. miRNA30-based shRNA可下调STGC3基因在NP69细胞系中的表达。 3. STGC3表达下调,使NP69细胞系生长增殖速度加快,其机制可能与影响细胞周期和减少细胞凋亡有关。
[Abstract]:Aim: to design shRNA(miR30-based shRNAs targeting STGC3 gene, construct a specific shRNA eukaryotic expression vector for STGC3 gene, and transfect pRNAT-U6-shRNA-STGC3 into immortalized human nasopharynx epithelial cell line NP69, and observe the effect of decreased STGC3 gene expression on NP69 cell line. To analyze the role of STGC3 gene in cell growth and proliferation. Methods: the interference targets of STGC3 gene were analyzed and screened by bioinformatics. According to the miRNA30 frame structure, the pRNAT-U6-shRNA-STGC3 eukaryotic expression vector was constructed by designing the shRNAs targeting STGC3 gene. The recombinant pRNAT-U6-shRNA-STGC3 expression vector was transiently transfected into NP69 cell line by Lipofectamine 2000. The expression of GFP was observed under fluorescence microscope, the transfection efficiency was determined, the expression level of mRNA of STGC3 gene was detected by RT-PCR, the interference effect of miR30-based shRNA on STGC3 gene was evaluated, the transfected cells were expanded, and the cell clone formation experiment was carried out. The clone forming ability of pRNAT-U6-shRNA-STGC3- NP69 cells was observed. The cell cycle changes and apoptosis of pRNAT-U6-shRNA-STGC3- NP69 cells were analyzed by flow cytometry. Results the eukaryotic expression vector of pRNAT-U6-shRNA-STGC3 was successfully constructed by PCR, restriction endonuclease digestion and sequencing. The empty pRNAT-U6 plasmid and the pRNAT-U6-shRNA-STGC3 recombinant plasmid were transfected into NP69 cell line for 36 hours, respectively. The expression of GFP in three groups of NP69-pRNAT-U6-NP69 and pRNAT-U6-shRNA-STGC3-NP69 cells was observed under fluorescence microscope. Green fluorescence was observed in the cells of pRNAT-U6-NP69 and pRNAT-U6-shRNA-STGC3-NP69, but no green fluorescence was observed in NP69 cells. The results showed that both empty plasmid and recombinant plasmid were successfully transferred into NP69 cell line, and the expression level of STGC3 gene mRNA in pRNAT-U6-shRNA-STGC3-NP69 cells was significantly lower than that in NP69 and pRNAT-U6-NP69 cells. The results showed that miR30-based shRNA successfully interfered with the expression of STGC3 in NP69 cell line. The results of cell clone formation assay showed that pRNAT-U6-shRNA-STGC3-NP69 enhanced the ability of clone formation compared with the control group (p0.05G), and flow cytometry was used to detect the increase of pRNAT-U6-shRNA-STGC3-NP69 cells in G2 and S phases. The rate of growth and proliferation increased and the apoptosis decreased, and the difference was significant compared with the control. 0.05. Conclusion. 1. The eukaryotic expression vector of pRNAT-U6-shRNA-STGC3 was successfully constructed. 2. MiRNA30-based shRNA down-regulated the expression of STGC3 gene in NP69 cell line. 3. The down-regulation of STGC3 expression speeds up the growth and proliferation of NP69 cells, which may be related to the influence of cell cycle and the reduction of apoptosis.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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