NANOG-shRNA was transfected into CD133⁺ Eca-109 cells toobserve the changes of proliferation, and the effect of NANOG as gene therapyof esophageal cancer stem cells was evaluated.

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 CD133⁺ Eca-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.

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).

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.