CAJ | 학술논문

目的:观察阻断己糖激酶-Ⅱ(HK-Ⅱ)基因表达对结肠癌LoVo细胞化疗敏感性的影响及其机制.方法:应用质粒介导的短发夹RNA(shRNA)真核表达法特异性阻断HK-Ⅱ基因表达.通过半定量逆转录-聚合酶链反应(RT-PCR)和Western印迹法分析RNA干扰效应.MTT法检测氟尿嘧啶(5-FU)和奥沙利铂(L-OHP)对LoVo细胞的半数抑制浓度(IC50);底物比色法测定半胱氨酸蛋白水解酶(caspase)-3活性;Western印迹法检测胸苷酸合成酶(TS)表达变化.结果:RNA干扰技术特异性下调结肠癌LoVo细胞HK-Ⅱ基因表达,mRNA和蛋白质水平的表达抑制率分别为72.1%和71.2%.阻断HK-Ⅱ基因表达后,结肠癌LoVo细胞5-FU和L-OHP的IC50值分别为(4.70±0.40)μg/ml 和(6.27±0.59)μg/ml,均明显低于正常培养细胞的(11.93±0.15)μg/ml和(9.77±0.60)μg/ml(P<0.01);与正常培养细胞相比,RNA干扰的LoVo细胞caspase-3 活性明显增高,TS蛋白表达下降.结论:阻断HK-Ⅱ基因表达可能通过活化caspase-3和降低TS表达的途径来增加结肠癌LoVo细胞化疗敏感性.
목적:관찰조단기당격매-Ⅱ(HK-Ⅱ)기인표체대결장암LoVo세포화료민감성적영향급기궤제.방법:응용질립개도적단발협RNA(shRNA)진핵표체법특이성조단HK-Ⅱ기인표체.통과반정량역전록-취합매련반응(RT-PCR)화Western인적법분석RNA간우효응.MTT법검측불뇨밀정(5-FU)화오사리박(L-OHP)대LoVo세포적반수억제농도(IC50);저물비색법측정반광안산단백수해매(caspase)-3활성;Western인적법검측흉감산합성매(TS)표체변화.결과:RNA간우기술특이성하조결장암LoVo세포HK-Ⅱ기인표체,mRNA화단백질수평적표체억제솔분별위72.1%화71.2%.조단HK-Ⅱ기인표체후,결장암LoVo세포5-FU화L-OHP적IC50치분별위(4.70±0.40)μg/ml 화(6.27±0.59)μg/ml,균명현저우정상배양세포적(11.93±0.15)μg/ml화(9.77±0.60)μg/ml(P<0.01);여정상배양세포상비,RNA간우적LoVo세포caspase-3 활성명현증고,TS단백표체하강.결론:조단HK-Ⅱ기인표체가능통과활화caspase-3화강저TS표체적도경래증가결장암LoVo세포화료민감성.
Objective:After knocking down the expression of hexokinase-Ⅱ(HK-Ⅱ) gene with RNA interference (RNAi) technique, chemotherapeutical effect and mechanism on colon cancer LoVo cell were investigated. Methods:In order to inhibit expression of HK-Ⅱ, a shRNA eukaryotic expression vector was constructed and transfected into LoVo cells. The mRNA and protein expression of HK-Ⅱ gene were assessed using semi-quantitive RT-PCR and Western blot analysis respectively. Then IC50 of LoVo cells by 5-FU and L-OHP were also assessed using MTT assay. The activation of caspase-3 was detected by substrate color reaction. Furthermore, the protein expression of thymidylate synthase (TS) was detected by Western blot analysis. Results:The expression of HK-Ⅱ gene was efficiently blocked by RNAi. The expression inhibition rates were 72.1% and 71.2% at mRNA and protein levels of LoVo cells respectively. The IC50 of 5-FU and L-OHP of the cells transfected with plasmids encoding HK-Ⅱ shRNA were (4.70±0.40)μg/ml and (6.27±0.59)μg/ml respectively, which were significantly lower than that of the normal cultured LoVo cells, (11.93±0.15)μg/ml and (9.77±0.60)μg/ml respectively (P<0.01). Compared with those of the normal cultured LoVo cells, the activation of caspase-3 were increased significantly and the protein expression of TS was decreased in the the cells transfected with plasmids encoding HK-Ⅱ shRNA plasmids respectively. Conclusion:Selective knock-down of the HK-Ⅱ gene expression can significantly enhance chemotherapeutical effect of LoVo cell via activation of caspase-3 and decreased expression of TS protein.