CAJ | 학술논문

目的：探讨转录因子Ascl2对结肠癌细胞上皮间质转化（EM T ）相关的微小RNA （microRNA ）的影响。方法对结肠癌LS174T细胞进行 Ascl2的 RNA 干扰质粒转染，对照质粒 shRNA-control/EGFP和干扰质粒 shRNA-Ascl2/EGFP 转染LS174T细胞，经G418筛选建立了稳定转染的细胞系，并分别命名为shRNA-Ctr/LS174T和shRNA-Ascl2/LS174T细胞，实时荧光定量PCR（real-time PCR）和蛋白印迹（Western-blot）实验确定干扰效果后，采用 Transwell小室侵袭实验观察Ascl2干扰对细胞侵袭能力的影响，再进一步行microRNA芯片筛选与EM T相关的microRNA并行real-time PCR验证。结果干扰后细胞中Ascl2的mRNA和蛋白质表达均明显减少（P＜0．01）。Ascl2干扰后LS174T细胞的侵袭能力明显下降（P＜0．01）。microRNA芯片筛选出与 EM T 相关的 microRNA-200家族（包括 microRNA-200b、microRNA-200a、microRNA-429、microRNA-200c、mi-croRNA-141）在Ascl2干扰后均出现2倍以上的上调（P＜0．01）。结论 Ascl2可能通过转录调节microR-200家族，进而调控EM T从而影响结肠癌的浸润转移。
목적：탐토전록인자Ascl2대결장암세포상피간질전화（EM T ）상관적미소RNA （microRNA ）적영향。방법대결장암LS174T세포진행 Ascl2적 RNA 간우질립전염，대조질립 shRNA-control/EGFP화간우질립 shRNA-Ascl2/EGFP 전염LS174T세포，경G418사선건립료은정전염적세포계，병분별명명위shRNA-Ctr/LS174T화shRNA-Ascl2/LS174T세포，실시형광정량PCR（real-time PCR）화단백인적（Western-blot）실험학정간우효과후，채용 Transwell소실침습실험관찰Ascl2간우대세포침습능력적영향，재진일보행microRNA심편사선여EM T상관적microRNA병행real-time PCR험증。결과간우후세포중Ascl2적mRNA화단백질표체균명현감소（P＜0．01）。Ascl2간우후LS174T세포적침습능력명현하강（P＜0．01）。microRNA심편사선출여 EM T 상관적 microRNA-200가족（포괄 microRNA-200b、microRNA-200a、microRNA-429、microRNA-200c、mi-croRNA-141）재Ascl2간우후균출현2배이상적상조（P＜0．01）。결론 Ascl2가능통과전록조절microR-200가족，진이조공EM T종이영향결장암적침윤전이。
Objective To investigate the effects of transcription factor achaete scute-like 2(Ascl2)on epithelial-mesenchymal transition (EM T ) associated microRNAs .Methods Colon cancer LS174T cells were transfected with shRNA-Ascl2 vector and shRNA-control vector respectively ,then the transfected cells were selected with G 418 and stably transfected cell lines were estab-lished .Real-time PCR and Western-blot analysis were used to determine the interference effect .Transwell invasion experiment were used to observe the effects of Ascl2 RNA interference on cell invasion capability in vitro .MicroRNA chip analysis was used to de-tect the change of EMT-associated microRNA expression ,and real-time PCR experiment was used to validate the microarray re-sults .Results The mRNA and protein expressions were significantly reduced after Ascl2 interference (P<0 .01) .The numbers of invading cells were significantly decreased after Ascl2 interference (P<0 .01) .MicroRNA chip analysis found microRNA-200 fami-ly (including microRNA-200b ,microRNA-200a ,microRNA-429 ,microRNA-200c ,microRNA-141) was more than 2-fold upregula-tion after Ascl2 interference (P< 0 .01) .Conclusion Ascl2 regulates the invasion and metastasis of colon cancer cell ,possibly through transcription regulation of microRNA-200 family ,and then regulation of EM T .