摘要
目的目前有关5-脂氧合酶(5-LOX)与结肠癌干细胞的关系尚无明确结论。文中旨在探讨5-LOX抑制剂MK886对人结肠癌HT-29细胞株干性的影响。方法采用CCK-8法检测不同浓度MK886(12.5、25、50、75、100、200μmol/L)对体外培养的HT-29细胞生长增殖的抑制情况,并计算出半数抑制浓度(IC50);采用克隆形成和干细胞球形成实验观察MK886 IC50对细胞克隆形成和球形成能力的影响;采用实时定量PCR(real-time PCR)法检测MK886 IC50作用HT-29细胞24、48 h后干性标记物(CD133、Lgr5、Oct4和Ascl2)mRNA的表达变化;采用Western blot法检测MK886 IC50作用24、48 h及72 h后上述标记物蛋白质的表达情况。结果与对照组24、48 h细胞增殖抑制率比较(0%、0%),25μmol/L药物组[(20.46±1.14)%、(34.97±6.02)%]明显升高,50、75、100、200μmol/L药物组亦明显升高(P<0.05)。MK886组克隆形成能力、细胞球数量明显低于对照组[(10.60±1.71)%vs(44.67±3.21)%、(6.00±1.60)个vs(19.07±2.89)个],差异有统计学意义(P<0.05)。MK886组干预HT-29细胞后24、48 h CD133mRNA相对表达量[(0.72±0.10)、(0.39±0.07)]与0 h(1.66±0.33)比较显著降低,Lgr5、Oct4、Ascl2 mRNA亦显著降低(P<0.05)。结论 5-LOX抑制剂MK886可抑制HT-29细胞的增殖、克隆形成和球形成能力,可能与其下调干性标记物的表达水平,从而抑制结肠癌干细胞"干性"相关。
Objective It remains a controversy whether 5-lipoxygenase （5-LOX） is associated with colon cancer stem cells. This study was to investigate the effect of the 5-LOX inhibitor MK886 in maintaining the sternness of the human colon cancer cell line HT-29. Methods Using CCK-8 assay, we examined the inhibitory effects of different concentrations of MK886 （12.5, 25, 50, 15, 100, and 200 jjimol/L） on the colon cancer HT-29 cells cultured in vitro and calculated its half-inhibitory concentration （IC50） . Then, we de-tected the effects of MK886 IC50 on the clone- and sphere-forming a-bilities of the cells, determined the mRNA expressions of the sternness markers CD133, Lgr5, 0ct4 and Ascl2 by real-time PCR after 24 and 48 hours of MK886 IC50 intervention, and measured their protein ex-pressions by Western blotting after 24, 48 and 72 hours of MK886 IC50 intervention. Results The inhibition rates of MK886 on the HT-29 cells at 24 and 48 hours were significantly increased in a time-and dose-dependent manner （ [ 14.99±3.06] and [ 19.98±0.57] % at 12.5 jjimol/L, [20.46±1.14] and [ 34.97±6.02] % at 25 jjimol/L, [50.76±5.94] and [66.90±5.74]% at50 iJimol/L,[66.84±1.77] and [73.11±2.48]% at75 fjimol/L,[72.67±2.36] and [77.78土 3.30] % at 100 （jimol/L, [83.67±0.24] and [84.69±2.24] % at 200 （jimol/L） as compared with the blank control （0% and 0%） （P〈0.05）. The clone-forming rate and number of spheres formed were remarkably lower in the MK886 intervention than in the control group （ [ 10.60±1.71 ] vs [ 44.67±3.21 ] %, P〈0.05; 6.00±1.60ra 19.07±2.89, P〈0.05）. After 24 and 48 hours of MK886 interven- tion,the mRNA expression of CD133 in the HT-29 cells was markedly up-regulated in comparison with that at 0 hour （0.72±0.10 and 0.39±0.07 m 1.66±0.33, P〈0.05） , and so were those of Lgr5, Oct4 and Ascl2 （ P〈0.05） . Conclusion The 5-LOX inhibitor MK886 can inhibit the proliferation and clone- and sphere-forming abilities of human colon cancer HT-29 cells by down-regulating the expressions of the sternness markers and thus suppressing the sternness of the colon cancer stem cells.
出处
《医学研究生学报》
CAS
北大核心
2017年第9期907-911,共5页
Journal of Medical Postgraduates
基金
国家自然科学基金(81560467)
贵州省科学技术基金项目(黔科合J字LKZ[2013]18号)
贵州省高层次人才科研条件特助经费资助项目(TZJF-2011-32号)
遵义医学院博士科研启动基金(F-572)
