c-Ski癌基因对肿瘤相关成纤维细胞活化的影响

Effects of c-Ski oncogene on the activation of cancer-associated fibroblasts

摘要:

目的：研究癌基因c-Ski对乳腺癌相关成纤维细胞（cancer-associated fibroblasts，CAFs）活化的影响。方法：应用基因重组技术，将c-Ski基因克隆到载体pBABE-puro，转染至永生化的正常乳腺成纤维细胞（normal fibroblasts，NFs），嘌呤霉素药物筛选稳定转染细胞株；Western blot检测转染后c-Ski、α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）和成纤维细胞活化蛋白（fibroblast activation protein，FAP）的表达；通过MTT法、流式细胞术、细胞划痕实验及Transwell细胞侵袭实验研究转染前、后成纤维细胞增殖、迁移、侵袭能力的差异；收集细胞上清制备条件培养基，用Transwell细胞侵袭实验分析转染后成纤维细胞对MD-MB-231细胞侵袭能力的影响。结果：成功构建重组质粒pBABE-puro-Ski；Western blot检测结果显示，pBABE-puro-Ski转染组细胞中c-Ski、α-SMA和FAP表达明显高于空白对照组细胞（P=0.000，P=0.001，P=0.002）；MTT法结果显示转染组细胞增殖能力高于空白对照组细胞（P=0.011）；转染组细胞的S期含量（38.58±1.28）明显高于空载体组（25.05±0.52）和空白对照组（26.01±0.86），差异具有统计学意义（P=0.000）；细胞划痕实验及Transwell细胞侵袭实验结果表明转染组细胞的迁移、侵袭能力均高于空载体组和空白对照组细胞；转染组细胞条件培养基能明显促进MD-MB-231细胞的侵袭能力。结论：c-Ski癌基因表达上调能促进NFs活化为CAFs，并进一步促进乳腺癌细胞MD-MB-231的侵袭能力。

Abstract:

Objective：To research the effect of c-Ski oncogene on the activation of breast cancer-associated fibroblasts（CAFs）. Methods：The c-Ski gene was cloned into the vector pBABE-puro to construct the expression vector pBABE-puro-Ski then was transfected into normal fibroblasts（NFs）；the stable transfected fibroblasts were selected by puromycin. Western blot was used to ana－lyze the expression of c-Ski，α-smooth muscle actin（α-SMA） and fibroblast activation protein（FAP） after the transfection. MTT as－say，flow cytometry，wound-healing experiment and Transwell assay were used to study the differences of cell proliferation，migration and invasion before and after the transfection. Conditioned media were collected and used to study the effect of fibroblasts on the in－vasion of MDA-MB-231 cells by Transwell assay. Results：The recombinant plasmid pBABE-puro-Ski was successfully constructed. Western blot showed that the expressions of c-Ski，α-SMA and FAP were higher in cells transfected with pBABE-puro-Ski than in those untransfected cells（P=0.000，P=0.001，P=0.002）. MTT showed that the proliferation of pBABE-puro-Ski transfected cells was higher than that of untransfected cells（P=0.011）. FCM showed that the S phase of pBABE-puro-Ski transfected cells（38.58±1.28） was significantly higher than that of empty vector transfected（25.05±0.52） or untransfected cells（26.01±0.86）（P=0.000）. Wound-healing experiment and Transwell assay revealed enhanced migration and invasion of the pBABE-puro-Ski transfected cells com－pared with those of the other two groups. Transwell assays showed that pBABE-puro-Ski transfected cells could promote the invasion of MDA-MB-231 cells. Conclusion：Overexpression of c-Ski could stimulate the activation of breast CAFs and activated CAFs further promote the invasion of MDA-MB-231 cells.