NGX6基因改变结肠癌细胞HT-29生物学特性及分子机制研究.docVIP

 n NGX6 基因改变结肠癌细胞 HT-29 生物学 特性及分子机制研究# 郭勤，沈守荣，廖曼甜，连平，王晓艳** 5 10 15 20 25 30 35 40 （中南大学湘雅三医院消化内科，长沙 410013） 摘要：摘要目的 探讨 NGX6 基因对结肠癌细胞系 HT-29 体外侵袭转移的影响及产生的分子机 制。方法：以三组结肠癌细胞株 HT-29、pcDNA3.1(+)/HT-29、pcDNA3.1(+)/NGX6/HT-29 为 研究对象，采用 Transfilter Cell Invasion Assays、细胞外基质黏附实验和划痕标记染 料示踪实验探讨 NGX6 基因对结肠癌细胞系 HT-29 体外侵袭转移的影响。为了明确分子机制， 采用 RT-PCR、Western blotting、细胞免疫荧光实验分别从细胞水平、mRNA 水平和蛋白质 水平检测 NGX6 对 WNT 信号传导通路中的关键分子β-catenin 的影响, 通过荧光素酶报告基 因分析测定法检测 NGX6 对结肠癌 HT-29 细胞系中 Wnt 通路的关键分子 Tcf4 及下游靶基因 cyclinD1 的活性的影响, 通过 Western blot 方法检测 NGX6 对 Wnt 通路下游靶基因 c-myc、 cyclinD1 的表达的影响，运用 RT-PCR 方法检测 NGX6 对 Wnt 通路下游基因 COX-2 的影响。 结果：NGX6 基因抑制结肠癌细胞 HT-29 的侵袭转移， 能够恢复 HT-29 细胞的细胞间间隙连 接（GJIC）能力；其机制为 NGX6 基因抑制β-catenin 在细胞内总蛋白和核蛋白水平的表达， 抑制 Tcf4 启动子活性，从而抑制下游靶基因 cyclinD1 启动子活性，降低 c-myc、cyclinD1 蛋白表达水平以及 COX-2mRNA 表达水平。这说明 NGX6 很可能是 Wnt 信号通路中的负性调控 子，这很可能是 NGX6 抑制结肠癌增殖和转移的机制之一。结论：NGX6 基因通过抑制 WNT 信 号通路发挥抑制 HT-29 细胞的增殖和降低 HT-29 细胞的侵袭转移能力的作用。 关键词：结肠癌；NGX6 基因；β-catenin；侵袭转移 中图分类号：R735.35 The Molecular Mechanism On the effect of the Human Colon Cancer Cells of the invasion and adhension By NGX6 Guo Qin, Shen Shourong, Li Mantian, Lian Ping, Wang Xiaoyan (The Third Affiliated Hospital of Xiang Ya School of Medicine, Central South University, ChangSha 410013) Abstract: Object This study was designed to investigate NGX6 inhibit the growth and invasiveness of human colon cancer cells (HT- 29), and to elucidate the molecular mechanism of their action. Method Invasive behaviors of colorectal cancer cells were examined by cell adhesion, migration, and invasion assays. Gap junctional intercellular communication (GJIC) was assessed by the scrape-loading/dye transfer technique. The subcellular localization and expression of β-catenin protein was examined by immunofluorescence staining and Western blot analysis, respectively. The effect of NGX6 on Tcf4 was examined by performing luciferase reporter gene assays. Moreover,The expression of c-myc and cyclinD1 protein was examined by Western blot analysis . The e