抑制NANOG表达对人食管鳞癌Eca-109干细胞增殖的影响

Influence of NANOG on the vitro proliferation of esophageal squamous cancer stem cells

目的采用NANOG短发夹RNA(shRNA)转染CD133+Eca-109食管鳞癌肿瘤干细胞,观察细胞增殖能力的变化,及NANOG对食管鳞癌干细胞的基因治疗效果。方法采用无血清培养基悬浮培养法分离食管鳞癌干细胞并通过RT-PCR和Western-Blot法检测分选效果。采用针对NANOG不同m RNA序列的两个shRNA分别转染食管鳞癌干细胞设为sh-N1组和sh-N2组,同时将转染不针对任何m RNA序列的质粒设为对照NC组。采用RTPCR和Western-Blot法检测细胞NANOG表达水平。采用CCK-8法检测细胞增殖能力。采用活死细胞染色检测细胞存活情况。采用无血清培养基悬浮培养并通过计数检测细胞成球能力。NANOG表达水平、CCK-8实验测得数据的比较采用单因素方差分析。结果 RT-PCR结果显示,正常培养的Eca-109食管鳞癌细胞CD133、CD44的表达水平1.03±0.02,1.02±0.02明显低于悬浮培养分离后肿瘤干细胞球中的表达10.12±0.19,9.21±0.26,(t=-79.952,-57.919;P均<0.01)。Western-Blot方法检测所得CD133、CD44表达结果与RT-PCR一致。shRNA转染食管鳞癌干细胞后,CCK-8实验结果显示,sh-N1组和sh-N2组细胞于24、48、96 h测得的A值分别为(0.33±0.02,0.52±0.04,0.61±0.04,0.81±0.03),(0.33±0.02,0.45±0.04,0.53±0.04,0.72±0.07),较对照NC组(0.9±0.01,1.41±0.01,2.31±0.02,3.12±0.07)下调(F=1121.33,525.73,1022.16,1198.29;P均<0.01),但并未出现细胞凋亡;在无血清培养条件下,细胞球计数结果显示,sh-N1组和sh-N2组(12±1,16±2)细胞形成肿瘤干细胞球的能力较NC组(80±3)降低(P<0.01)。结论 NANOG敲低对食管鳞癌干细胞的增殖具有明显的抑制效果,有望成为针对食管鳞癌的有效靶向性治疗手段。

Objective NANOG-shRNA was transfected into CD133＋ Eta-109 cells toobserve the changes of proliferation, and the effect of NANOG as gene therapyof esophageal cancer stem cells was evaluated. Methods CD133＋ Eca-109 cancer stem cells were sorted using serum- free suspension culture, and cell purity was evaluated using RT-PCR and Western blotting methods. shRNA were transfected into CD133Eta-109 in targeted different sequence in NANOG （sh-N1 group or sh-N2 group）. The blank control group （NC） was not targeted any sequence. The expression levels of NANOG were evaluated by RT-PCR and Western blotting methods. The cell proliferation was evaluated by CCK-8 method. The cell surviving was detected by Calcein-AM and PI staining. The capacity of tumor spheres formation was evaluated by serum-free suspension culture. Results The CD133 expression level of Eca-109 was significantly lower than the Eca-109 tumor spheres, and the difference was statistically significant （10.12±0.19,9.21 ±0.26, t = -79.952, -57.919 P 〈 0.01）. The results of Western blotting were in accordance with those of RT-PCR. After NANOG shRNA being transfected into Eca-109, CCK-8 assay showed that the A values of sh-N1 and sh-N2 groups （0.33± 0.02, 0.52 ± 0.04, 0.61 ± 0.04, 0.81± 0.03）, （0.33 ±0.02, 0.45 ± 0.04, 0.53 ± 0.04, 0.72±0.07）, lower than those in the NC group （0.9±0.01, 1.41±0.01, 2.31 ±0.02, 3.12±0.07）, and the difference was statistically significant （F=1121.33,525.73, 1022.16, 1198.29; P 〈 0.01）, but the cells were not dead. Serum-free suspension culture assay showed that the capacity of tumor spheres formation in sh-N1 and sh-N2 groups （12 ± 1, 16 ±2）, lower than those in the NC group （80_3）, and the difference was statistically significant （P 〈 0.01）. Conclusion Knockdown of NANOG has obviously inhibitory effect on the proliferation of CD133＋ Eca-109 esophageal squamous carcinoma stem cells, and is expected to be an effective targeted therapy for esophageal cancer.