Geng Haitao, Xiao Qian, Xu Dengyong, Hu Lifeng, Ding Kefeng
Department of Surgical Oncology, Zhejiang University School of Medicine, Hangzhou, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2012 Jan;32(1):66-70.
To construct a recombinant lentiviral vector that stably express hepatocyte nuclear factor 6 (HNF6) in colorectal cancer cell line and examine its effects on the invasive ability of SW620 cells.
The lentiviral vector pLeno-DCE-HA-HNF6 was constructed and transfected into 293T cells. The supernatant containing the lentivirus particles was harvested to determine the virus titer. A stable cell line was established by infecting SW620 cells with the lentivirus particles, and the transfection efficiency was examined by fluorescence microscopy and flow cytometry. The invasion ability of the transfected SW620-HNF6 cells was assessed by wound healing and transwell assays.
The recombinant lentiviral vector was correctly constructed and verified by sequencing. SW620-HNF6 cell line with stable HNF6 expression was established successfully, and the transfection efficiency reached 82.3%. Western blotting and quantitative PCR demonstrated significantly upregulated HNF6 expression in SW620-HNF6 cells, which showed obviously suppressed invasive ability in wound healing and transwell assays.
We have successfully established a colorectal cancer cell line SW620-HNF6 stably expressing HNF6, which shows a lowered migration activity in vitro.
构建在结肠癌细胞系中稳定表达肝细胞核因子6（HNF6）的重组慢病毒载体，并检测其对SW620细胞侵袭能力的影响。
构建慢病毒载体pLeno-DCE-HA-HNF6并转染至293T细胞。收集含有慢病毒颗粒的上清液以测定病毒滴度。用慢病毒颗粒感染SW620细胞建立稳定细胞系，通过荧光显微镜和流式细胞术检测转染效率。采用划痕愈合实验和Transwell实验评估转染后的SW620-HNF6细胞的侵袭能力。
经测序验证，重组慢病毒载体构建正确。成功建立了HNF6表达稳定的SW620-HNF6细胞系，转染效率达82.3%。蛋白质免疫印迹法和定量PCR结果显示，SW620-HNF6细胞中HNF6表达显著上调，在划痕愈合实验和Transwell实验中其侵袭能力明显受到抑制。
成功建立了稳定表达HNF6的结肠癌细胞系SW620-HNF6，其体外迁移活性降低。
