NGX6基因改变结肠癌细胞HT-29生物学特性及分子机制研究
首发时间:2012-02-16
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
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The Molecular Mechanism On the effect of the Human Colon Cancer Cells of the invasion and adhension By NGX6
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 expression of gene COX-2 was examined by RT-PCR analysis . Results NGX6 had inhibited proliferation and invasiveness of HT-29 colon cancer cells, suppressing extracellular matrix adhesion of HT- 29 cells, restoring gap junctional intercellular communication of HT-29, suppressing translocation of β-catenin from the nucleus and cytoplasm to the plasma membrane , inhibit the activity of Tcf4 trascript factor, down-express c-myc, cyclinD1 and COX-2. Conclusion: NGX6 inhibit the proliferation and invasiveness of HT-29 colon cancer cells by attenuating the WNT/-catenin signaling pathway.
Keywords: Colon carcinoma NGX6 gene β-catenin metastasis
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NGX6基因改变结肠癌细胞HT-29生物学特性及分子机制研究
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