引用本文:张 璐1,杨雅莹1,黄子洋2,易永芬1.shRNA抑制高尔基体α-甘露糖苷酶Ⅱ基因的表达对胃癌细胞侵袭力的影响及机制[J].重庆医科大学学报,2014,38(3):310~317
shRNA抑制高尔基体α-甘露糖苷酶Ⅱ基因的表达对胃癌细胞侵袭力的影响及机制
Effect of Golgi α-mannosidase II expression inhibition by shRNA interference on invasive potential of human gastric cancer cell and its mechanism
DOI:
中文关键词:  高尔基体α-甘露糖苷酶Ⅱ  短发卡RNA  胃癌  侵袭
英文关键词:Golgi alpha-mannosidase Ⅱ  short hairpin RNA  gastric cancer  invasion
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作者单位
张 璐1,杨雅莹1,黄子洋2,易永芬1 1. 重庆医科大学基础医学院病理学教研室、分子医学与肿瘤研究中心重庆 4000162. 重庆医科大学附属第一医院老年科重庆 400016 
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中文摘要:
      目的:初步分析高尔基体α-甘露糖苷酶Ⅱ(Golgi alpha-mannosidase Ⅱ,GMⅡ)在低中不同分化人胃癌细胞系中的差异表达,以探讨GMⅡ在胃癌发生发展中的作用。方法:应用阳离子脂质体法将合成的靶向GMⅡ基因的pGPU6/GFP/Neo-GMⅡ-1406质粒载体,分别转染至低分化胃癌细胞MGC-803和中分化胃癌细胞SGC-7901中,然后荧光倒置显微镜观察并计算转染率,最后经过G418筛选出稳定转染的细胞株。应用RT-PCR和Western blot检测转染后GMⅡ和E-cadherin、α-catenin 的mRNA及蛋白表达变化情况,同时利用细胞划痕实验和Transwell体外侵袭实验来测定GMⅡ被沉默后两株细胞侵袭能力的变化。结果:GMⅡ短发卡RNA(short hairpin RNA,shRNA)转染胃癌MGC-803和SGC-7901细胞后GMⅡ的mRNA和蛋白表达明显抑制,而E-cadherin和α-catenin的2种因子检测结果明显升高(P<0.05);细胞划痕实验结果显示,转染重组质粒pGPU6/GFP/Neo-GMⅡ-1406组(GMⅡshRNA组)与对照组(空白对照组,阴性对照组)相比,GMⅡ被沉默后的胃癌细胞的侵袭能力降低(P<0.05);Transwell体外侵袭实验中GMⅡshRNA组胃癌细胞较空白对照组、阴性对照组细胞侵袭能力都明显降低,18 h穿过小室的细胞数分别为:(65±5)、(197±6)、(186±5),(72±4)、(178±4)、(184±5)个与对照组相比差别具有统计学意义(MGC-803:P=0.000,SGC-7901:P=0.000)。结论:GMⅡ基因沉默后的胃癌细胞的侵袭能力下降,可能与上调E-cadherin、α-catenin的表达有关。
英文摘要:
      Objective:To analyze the expression of Golgi alpha-mannosidase Ⅱ(GMⅡ) in different gastric cancer cell lines and to in-vestigate the role of GMⅡ in gastric cancer genesis. Methods:The pGPU6/GFP/Neo-GMⅡ-1406 gene was transfected into MGC-803 and SGC-7901 cells using cationic liposome assay. Rates of transfected positive cells were observed and calculated under invert microscope. Gene and protein expression of GMⅡ,E-cadherin and α-catenin were tested using RT-PCR and Western blot respec-tively. Invasive abilities of cancer cells were analyzed by cell scratch and Transwell assay. Results:Protein expression of GMⅡ was significantly inhibited and expression of E-cadherin and α-catenin in MGC-803 and SGC-7901 cells was up-regulated compared with those of untransfected cells(P<0.05). Cell scratch assay showed that the invasive abilities of cells transfected with GMⅡ gene were weaker than those of control group cells(P<0.05). Transwell invasive assay demonstrated that the invasive abilities of cells trans-fected with GMⅡ gene were weaker than those of shNC groups. Numbers of Transwell cells were 65±5,197±6,186±5,72±4,178±4,184±5 in three groups during 18 h,with statistical differences(P<0.05). Conclusion:GMⅡ gene silencing contributes to decreased invasive ability of gastric cancer cell lines,which is involved with up-regulation of E-cadherin and α-catenin expression.
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