干扰结肠癌上皮细胞Ascl2的表达致其向杯状细胞表型分化.docVIP

[通信作者] 汪荣泉，电话:(023E-mail: rongquanw@) 干扰结肠癌上皮细胞Ascl2的表达致其向杯状细胞表型分化 李晓桓，朱 蓉，田 音，汪荣泉 (400038 重庆，第三军医大学西南医院全军消化病研究所） [摘要] 目的 探讨转录因子Ascl2对结肠癌细胞分化方面的影响。方法 采用Ascl2分子干扰质粒和对照质粒对结肠癌HT-29和LS174T细胞进行转染，通过G418筛选建立稳定转染的细胞系，采用RT-PCR和Western blot方法检测干扰Ascl2分子表达对肠杯状细胞标志物Muc2和TFF3表达的影响。结果 成功构建了shRNA-Ascl2/EGFP质粒以及对照质粒shRNA-control/EGFP，经测序与预期相符，G418筛选获得shRNA-Ascl2/HT-29、shRNA-Ctr/HT-29、shRNA-Ascl2/LS174T和shRNA-Ctr/LS174T稳定转染细胞。 RT-PCR和Western blot检测证实干扰质粒能够有效的抑制HT-29和LS174T细胞内的Ascl2的表达（P0.01)。RT-PCR检测发现Muc2和TFF3 mRNA表达水平在Ascl2干扰后的HT-29细胞内均明显上调（P0.05）；Muc2 mRNA表达水平在Ascl2干扰后的LS174T细胞内明显上调，差异有统计学意义（P0.05），但是TFF3 mRNA表达水平在Ascl2干扰后的LS174T内升高不明显（P0.05）。Western blot检测发现Muc2蛋白质表达水平在Ascl2干扰后明显上调（P0.05）。结论 干扰结肠癌HT-29和LS174T细胞内Ascl2的表达导致其向杯状细胞的表型分化。 [关键词] Ascl2；结肠肿瘤；肿瘤细胞, 培养的；细胞分化；杯状细胞 [中图法分类号] R735.35 [文献标志码] A Ascl2 RNA interference in colon cancer cells leads to their differentiation towards goblet cells phenotype Li Xiaohuan, Zhu Rong, Tian Yin, Wang Rongquan(Gastroenterology Research Center, Southwest Hospital, Third Military Medical University, Chongqing 400038, China) [Abstract] Objective To investigate the regulation of transcription factor Ascl2 on colon cancer cells differentiation. Methods Colon cancer HT-29 and LS174T cells were transfected with shRNA-Ascl2 and shRNA-control plasmid, then transfected cells were selected with G418 and stably transfected cell lines were established. Real-time PCR and western-blot analysis were used to detect Muc2 and TFF3 mRNA and protein expression, for exploration on the effects of Ascl2 RNA interference on cell differentiation towards goblet cell phenotype. Results shRNA-Ascl2/EPGF plasmid and shRNA-control/EPGF plasmid were successfully constructed and confirmed by sequencing, after selection with G418, we obtained the stably transfected shRNA-Ascl2/HT29, shRNA-Ctr/HT-29, shRNA-Ascl2/LS174T and shRNA-Ctr/LS174T cells. Real-time PCR and Western blotting confirmed that Ascl2 interfere