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目的探讨增强子结合蛋白α(C/EBPα)在结肠癌转移中的分子调控机制,为防治结肠癌及其转移提供研发方向和临床思路。方法比较Ad C/EBPα感染后在正常的人结肠黏膜上皮细胞株、结肠癌细胞株SW620和LOVO中C/EBPα及相关基因、蛋白的表达差异,用CCK-8法检测SW620和LOVO细胞生长增殖情况,以流式细胞仪检测细胞的凋亡情况;体外分离人正常结肠上皮细胞和结肠癌细胞株SW620和LOVO以观察其体外存活率;构建实验性结肠癌和肝转移的小鼠模型,部分小鼠分别经瘤内或尾静脉注射导入Ad C/EBPα接受治疗,以进一步明确C/EBPα在体内对实验性结肠癌和肝转移小鼠的作用机制。结果 Ad C/EBPα以MOI400感染结肠癌细胞株SW620和LOVO 4 d后,Westernblot检测K-ras基因表达上调,C/EBPαmRNA和ANXA1蛋白表达明显上调;免疫组化法也显示结肠癌细胞中的C/EBPαmRNA和蛋白表达明显上调,定位在细胞核内。CCK-8法检测SW620和LOVO细胞生长增殖的结果显示,Ad C/EBPα感染7 d后出现明显的生长抑制。Ad C/EBPα感染结肠癌细胞株SW620和LOVO 5 d后,流式细胞仪检测结果表明细胞株凋亡率差异无统计学意义。而体外分离细胞的实验结果表明,Ad C/EBPα感染7 d后,结肠癌SW620和LOVO细胞的存活率明显降低。经C/EBPα治疗实验性结肠癌或肝转移小鼠,发现其能显著抑制瘤体重量和体积,且采用Real-TimeRT-PCR法检测发现经C/EBPα治疗的小鼠其K-ras基因和ANXA1蛋白表达明显低于未经治疗组,提示C/EBPα对结肠癌及其肝转移的作用与Raf/MEK/ERK/MAPK细胞信号通路的激活有关系,从分子水平揭示了C/EBPα的作用机制。结论本研究提示C/EBPα治疗结肠癌转移的分子机制为激活Ras/Raf/MEK/ERK/MAPK细胞信号通路,下调ANXA1表达和K-ras基因,从而抑制肿瘤细胞的增殖、生长和远端转移。
Objective To investigate the molecular regulation mechanism of enhancer binding protein α (C / EBPα) in colon cancer metastasis and to provide research directions and clinical ideas for the prevention and treatment of colon cancer and its metastasis. Methods The expression of C / EBPα and its related genes and proteins in normal human colon mucosal epithelial cell line, colon cancer cell line SW620 and LOVO after Ad C / EBPα infection were compared. The growth and proliferation of SW620 and LOVO cells were detected by CCK-8 , The apoptosis of cells was detected by flow cytometry; human normal colon epithelial cells and colon cancer cell lines SW620 and LOVO were isolated to observe the in vitro survival rate; a mouse model of experimental colon cancer and liver metastasis was constructed, part of which Mice were injected with Ad C / EBPα by intratumoral or tail vein injection respectively to further clarify the mechanism of action of C / EBPα in vivo on experimental colon cancer and liver metastasis mice. Results The expression of K-ras gene and C / EBPαmRNA and ANXA1 protein were upregulated in AdC / EBPα-infected colon cancer cell lines SW620 and LOVO for 4 d by Western blot, and C / EBPαmRNA and protein expression was significantly upregulated, located in the nucleus. The results of CCK-8 assay for the proliferation of SW620 and LOVO cells showed that AdC / EBPα showed obvious growth inhibition after 7 days. The results of flow cytometry showed that there was no significant difference in apoptotic rate between AdC / EBPα-infected colon cancer cell lines SW620 and LOVO for 5 days. The experimental results of cells isolated in vitro showed that the survival rate of SW620 and LOVO cells was significantly decreased after 7 days of Ad C / EBPα infection. C / EBPα treatment of experimental colon cancer or liver metastasis mice, was found to significantly inhibit the tumor weight and volume, and using Real-TimeRT-PCR assay found that C / EBPα-treated mice K-ras gene and ANXA1 protein expression was significantly lower than the untreated group, suggesting that C / EBPα on colon cancer and liver metastasis and Raf / MEK / ERK / MAPK cell signaling pathway activation has revealed the molecular level of the role of C / EBPα mechanism. Conclusions This study suggests that the molecular mechanism of C / EBPα in the treatment of colon cancer metastasis is activation of Ras / Raf / MEK / ERK / MAPK signaling pathway and down-regulation of ANXA1 expression and K-ras gene, thereby inhibiting the proliferation, growth and distant metastasis of tumor cells .