重组质粒pcDNA3.0-hugl-1转染结直肠癌细胞后生物学效应的观察

Biological effects of recombinant pcDNA3.0-hugl-1 plasmid on colorectal cancer cell line LS174

目的:观察外源基因hugl-1转染对结直肠癌LS174细胞生物学行为的影响,探讨hugl-1基因与结直肠肿瘤的相关性。方法:构建重组质粒pcDNA3.0-hugl-1,转染至结直肠癌细胞株LS174细胞中,RT-PCR、Western印迹检测转染后hugl-1 mRNA及蛋白的表达;通过软琼脂集落形成试验、划痕修复试验、细胞黏附试验及Transwell细胞侵袭试验等进一步对转染后LS174细胞增殖、黏附、运动和侵袭能力进行分析,并与空质粒载体及未转染LS174细胞作对照。结果:成功构建重组质粒pcDNA3.0-hugl-1;RT-PCR、Western印迹检测结果显示,重组体pcDNA3.0-hugl-1转染细胞株中hugl-1 mRNA及蛋白表达明显高于空载体转染及未转染组细胞(P<0.05)。重组体转染组细胞克隆形成率(32.23%)与未转染及空载体转染组细胞相比(35.76%、33.91%)无明显改变;重组体转染组LS174细胞克隆的迁移细胞数(82.14±7.62)明显低于空载体组(135.61±3.74)及未转染组细胞(142.37±6.12,P<0.05);转染后120min,重组体转染组LS174细胞克隆黏附力明显高于空载体组及未转染组(P<0.05);重组体转染组穿膜细胞数(63.7±8.0)明显少于空载体组及未转染组(158.3±16.5、156.3±13.0,P<0.05)。结论:人hugl-1基因表达上调能降低结直肠癌细胞迁移运动和侵袭能力,增加细胞黏附能力,但对肿瘤细胞增殖能力无明显影响;其表达降低可使肿瘤细胞播散。

Objective： To observe the biological behaviors of colorectal cancer LS174 cells before and after pcDNA3. 0-hugl-1 transfection, so as to investigate the association of hugl-1 with colorectal cancer. Methods： The eukaryotic expression vector pcDNA3.0- hugl-1 was constructed and transfected into LS174 cells. RT-PCR and Western blotting methods were used to analyze the expression of hugl-1 mRNA and protein in LS174 cells before and after transfection. Soft agar colony formation assay, wound-healing experiment, adhesion assay and Matrigel invasion assays were used to study the effects of hugl-1 expression on the proliferation, adhesion, movement and invasion in LS174 cells. Results： The recombinant plasmid pcDNA3.0-hugl-1 was successfully constructed. RT-PCR and Western blotting showed that the hugl-1 expression was higher in cells transfected with pcDNA3.0-hugl-1 than in those un-transfected or empty vector-transfected cells （P〈0.05）. The colony forming rates showed no significant difference between the 3 groups （35.76%,33.91% and 32.23% ）. Wound healing assays showed a significant reduction in the migrated cells in pcDNA3.0-hugl-1 transfected cell clones （82.14±7.62） compared with un-transfected cell lines（142.37 ± 6. 12） and empty vector transfected cell line （ 135. 61 ± 3.74, P〈 0.05）. Attachment assays revealed enhanced adhesion of the pcDNA3. 0-hugl-1-transfected cell clones compared with the other 2 groups. Matrigel invasion assay showed a significant reduction in the invasive ability of the pcDNA3.0-hugl-1-transfected cell （63.7± 8.0） in comparison to the un-transfected cells （156. 3 ± 13. 0） and pcDNA（-） cells （158. 3 ±16. 5, P〈0. 05）. Conclusion： Overexpression of hugl-1 eukaryotic expression vector can decrease migratory and invasive ability of LS174 cells, but has no influence on cell proliferation.