CDC42沉默对结直肠癌细胞形态的影响

Effect of shRNA-mediated CDC42 knockdown on morphology of colorectal cancer cells in vitro

目的根据CDC42蛋白二级结构,设计CDC42 si RNA干扰片段,构建CDC42稳定干扰sh RNA载体。观察CDC42有效沉默对结直肠癌SW480细胞形态的影响。方法根据CDC42基因CDS区序列,设计4条si RNA片段。Western blot筛选最有效沉默片段,酶切插入真核表达载体,将构建的sh RNA质粒载体,瞬时转染结直肠癌SW480细胞,QPCR及Western blot验证CDC42沉默效率;观察转染前后,SW480细胞形态变化。结果 4条si RNA转染细胞后,验证结果显示si RNA-3沉默效率大于50%,沉默效果最好,将此片段插入真核表达载体,测序结果提示CDC42 sh RNA构建成功,转染SW480细胞后,CDC42基因m RNA及蛋白水平表达明显下降,差异有统计学意义;显微镜观察CDC42沉默后,SW480细胞周边伪足伸出减少,细胞变光滑,细胞整体体积明显减小。结论成功根据基因序列设计有效沉默片段,构建CDC42稳定沉默载体,证实CDC42沉默可以逆向改变结直肠癌细胞高侵袭性形态,为研究CDC42对结直肠癌的发生、发展提供分子工具。

Objective To test the effect of CDC42 （a member of Rho family of small GTPases） knockdown mediated by a CDC42 short-hairpin RNA （shRNA） on the morphology of colorectal cancer SW480 cells in vitro. Methods Four CDC42 siRNA fragments targeting CDC42 were designed and the most efficient siRNA for CDC42 knockdown was selected to construct the shRNA vector for transfection of colorectal cancer SW480 cells. The interference efficiency in the stably transfected cells （sw480. shCDC） was detected using real-time PCR and Western blotting, and the morphological changes of the transfected cells were observed. Results Western blotting result showed that siCDC42-3 was the most efficient fragment for CDC42 knockdown, which caused CDC42 knockdown by over 50%. DNA sequencing confirmed successful construction of the CDC42 shRNA vector. Transfection of the cells with the vector significantly reduced CDC42 expressions at both the mRNA and protein levels. The transfected cells exhibited reduced filopodia and cell size with smooth cell margins. Conclusion shRNA-mediated CDC42 knockdown can reduce the cytoskeleton dynamics of colorectal cancer cells to lower their invasiveness. This shRNA construct facilitates further study of the role of CDC42 in the tumorigenesis and progression of colorectal cancer.