CAJ | 학술논문

目的：建立可条件性诱导PLK1-shRNA稳定表达的食管癌细胞株，为深入研究PLK1在食管癌发生发展中的作用及分子机制提供细胞模型。方法：合成PLK1-shRNA寡核苷酸，退火后连接到慢病毒表达载体pLKO-Tet-On并转化至感受态细胞Stbl3，利用菌落PCR和测序分析鉴定阳性重组子。用pLKO-shPLK1-Tet-On重组质粒和包装质粒共转染293T细胞，收集病毒上清，感染食管癌细胞KYSE510，利用嘌呤霉素筛选可诱导PLK1-shRNA稳定表达的细胞株。采用qRT-PCR和Western blot检测强力霉素(Dox)诱导PLK1-shRNA表达的效率。结果：菌落PCR和测序分析结果显示，pLKO-shPLK1-Tet-On重组质粒中PLK1-shRNA的序列及插入位点正确。包装、收获病毒并感染KYSE510细胞后，筛选获得了具有嘌呤霉素抗性的稳定细胞株KYSE510-shPLK1-Tet-On。qRT-PCR和Western blot的检测结果显示，0.1μg/mL Dox即可显著下调KYSE510-shPLK1-Tet-On细胞中PLK1的表达。结论：成功构建了诱导型PLK1-shRNA慢病毒表达载体，并筛选获得了可条件性诱导PLK1稳定敲降的食管癌细胞株，为进一步探讨PLK1异常表达与食管癌发生发展的关系提供了理想的细胞模型。
목적：건립가조건성유도PLK1-shRNA은정표체적식관암세포주，위심입연구PLK1재식관암발생발전중적작용급분자궤제제공세포모형。방법：합성PLK1-shRNA과핵감산，퇴화후련접도만병독표체재체pLKO-Tet-On병전화지감수태세포Stbl3，이용균락PCR화측서분석감정양성중조자。용pLKO-shPLK1-Tet-On중조질립화포장질립공전염293T세포，수집병독상청，감염식관암세포KYSE510，이용표령매소사선가유도PLK1-shRNA은정표체적세포주。채용qRT-PCR화Western blot검측강력매소(Dox)유도PLK1-shRNA표체적효솔。결과：균락PCR화측서분석결과현시，pLKO-shPLK1-Tet-On중조질립중PLK1-shRNA적서렬급삽입위점정학。포장、수획병독병감염KYSE510세포후，사선획득료구유표령매소항성적은정세포주KYSE510-shPLK1-Tet-On。qRT-PCR화Western blot적검측결과현시，0.1μg/mL Dox즉가현저하조KYSE510-shPLK1-Tet-On세포중PLK1적표체。결론：성공구건료유도형PLK1-shRNA만병독표체재체，병사선획득료가조건성유도PLK1은정고강적식관암세포주，위진일보탐토PLK1이상표체여식관암발생발전적관계제공료이상적세포모형。
OBJECTIVE: The aim of the study was to establish a suitable cell model for investigating the role and mechanism of PLK1 overexpression in esophageal cancer through inactivation of PLK1 expression employing lentiviral-mediated inducible shRNA expression system.METHODS:Chemically synthesized PLK1-shRNA oligonucleotides were annealed and ligated into the lentiviral vector pLKO-Tet-On. The ligation products were transformed into the competentE. coli Stbl3 cells. Colony PCR and sequencing analysis were used to identify the positive recombinants. The pLKO-shPLK1-Tet-On construct and packaging plasmids were co-transfected into 293T cells to produce the lentiviral particles. Esophageal cancer cells KYSE510 were infected with the viral supernatant and the stable cell strain was selected with puromycin. Doxcyclin-induced expression efficiency of PLK1-shRNA was determined by qRT-PCR and Western blotting. RESULTS:Colony PCR and sequencing analysis showed that PLK1-shRNA oligos were correctly inserted into the pLKO-Tet-On vector. The stable cell strain KYSE510-shPLK1-Tet-On was obtained through infecting the lentivirus expressing inducible PLK1-shRNA and then selected with puromycin. The results of qRT-PCR and Western blotting indicated that the expression of PLK1 in KYSE510-shPLK1-Tet-On cells could be markedly downregulated by 0.1μg/mL Dox.CONCLUSION:We successfully constructed a lentivirus-based inducible PLK1-shRNA expression vector and established an esophageal cancer cell line with stable expression of inducible PLK1-shRNA,providing an ideal cell model for further exploring the relationship between aberrant PLK1 expression and development and progression of esophageal cancer.