Dickkopf-3过表达对结直肠癌LoVo细胞生物学行为的影响  

Effect of Dickkopf-3 overexpression on the biological behavior of LoVo cells in colorectal cancer

作　　者：邓润枢[1] 莫林耀[1] 何锡华[1] 陈建华[1] 吴志明[1] 郭裕良 

机构地区：[1]东莞市人民医院肿瘤外科,广东东莞523059

出　　处：《结直肠肛门外科》2016年第1期17-23,共7页Journal of Colorectal & Anal Surgery

摘　　要：目的采用基因过表达技术,过表达CRC细胞中DKK-3(Dickkopf-3)基因,运用迁移实验、侵袭实验等方法检测CRC细胞DKK-3基因过表达前后侵袭和转移能力的变化。方法使用Lipofectamine 2000试剂将针对目的基因DKK-3的过表达质粒p EGFP-N1-DKK-3-GFP转染入Lo Vo细胞。RT-PCR和Western Blotting分别用于检测质粒转染前后结直肠癌Lo Vo细胞DKK-3基因和蛋白表达水平的变化。通过WST-8,检测DKK-3过表达对结直肠癌Lo Vo细胞增殖能力的影响;通过FACSCalibur流式细胞仪和碘化丙啶(PI)染色,检测DKK-3过表达对结直肠癌Lo Vo细胞周期的影响;通过transwell小室,检测DKK-3过表达对结直肠癌Lo Vo细胞迁移和侵袭能力的影响。最后,通过Western Blotting检测相关蛋白caspase-3、caspase-9、Bax、Bcl-2、cytochrome c(Cyt-c)和β-catenin表达水平的变化。结果 p EGFP-N1-DKK-3-GFP介导的DKK-3过表达明显抑制了p EGFPN1-DKK-3组Lo Vo细胞的增殖(P<0.05),而未处理Lo Vo组和p EGFP-N1组细胞之间增殖速度无统计学差异(P>0.05)。p EGFP-N1-DKK-3组的穿膜细胞数明显少于未处理Lo Vo组(和p EGFP-N1组(P<0.05),结果显示DKK-3过表达可能抑制了Lo Vo细胞的侵袭能力。p EGFP-N1-DKK-3组的穿膜细胞数明显少于未处理Lo Vo组和p EGFP-N1组(P<0.05),结果显示DKK-3过表达可能抑制了Lo Vo细胞的迁移能力。同未处理Lo Vo组和p EGFP-N1组相比,p EGFP-N1-DKK-3组细胞G0/G1期细胞所占比例差异具有统计学意义(P<0.05)。DKK-3过表达同时也诱导了结直肠癌Lo Vo细胞的凋亡,表现为染色质凝聚和核碎片、Bax和Cyt-c蛋白表达上调、survivin和Bcl-2蛋白表达下调以及caspase-3和caspase-9的活化。此外,DKK-3过表达还减少了细胞溶质中β-catenin累积。结论 DKK-3过表达可能通过线粒体途径诱导CRC细胞的凋亡。另外,DKK-3可能参与了CRC的Wnt/β-catenin信号转导通路。Objective We used gene overexpression technology tooverexpress DKK-3 gene in CRC cells and to detect the changes of invasion and metastasis ability of CRC cells by using the method of migration assay and invasion assay.Methods： Overexpression of DKK-3 induced by p EGFP-N1-DKK-3-GFP plasmid in Lo Vo cells was performed using Lipofectamine 2000 reagent. Construction, sequencing and identification of the plasmid were completed by SHANGHAI GENECHEM CO., LTD. RT-PCR and Western Blotting were performed to determine the m RNA and protein expression levels of DKK-3, respectively. The cellular proliferation ability of transfected cells was measured via WST-8 assay. Cell cycle was analyzed on a FACSCalibur flow cytometer. Cell migration assay and tumour invasion assay were carried out by transwell chambers following the manufacturer＇s instructions. Caspase-3, caspase-9, Bax, Bcl-2, cytochrome c（Cyt-c） and β-catenin were measured by Western Blotting.Results：PEGFP-N1-DKK-3-GFP mediated overexpression of DKK-3 significantly inhibited the proliferation of Lo Vo cells（P〈0.05）, but there was no significant difference in the proliferation rate between Lo Vo and p EGFP-N1（P0.05）. The number of cells in the p EGFP-N1-DKK-3 group was significantly less than that in the untreated LoVo group（P〈0.05）, indicating thatthe expression of DKK-3 might inhibit the invasion of Lo Vo cells. The number of cells in the p EGFP-N1-DKK-3 group was significantly less than that in the untreated Lo Vo and p EGFP-N1groups（P〈0.05）, indicating that DKK-3 overexpression may inhibit the migration of Lo Vo cells. Compared with untreated Lo Vo group and p EGFP-N1 group, the difference of G0 / G1 phase cells in p EGFP-N1-DKK-3 group was statistically significant（P〈0.05）. The apoptosis of Lo Vo cells was also induced by DKK-3, which was manifested by caspase-9, Cyt-c, and protein expression, surviving, and Bax protein expression and activation of Bcl-2 and caspase-3. In addition, overexpression of DKK-3 also decreased the

关 键 词：DKK-3 过表达 凋亡 结直肠癌 侵袭 线粒体通路 

分 类 号：R735.34[医药卫生—肿瘤]