ABSTRACT
Aim To determine whether 8-bromo-7-me-thoxychrysin ( BrMC) inhibits in vitro carcinogenicity via up-regulating miR-519d expression and down-regu-lating Twist1 expression in liver cancer stem-like cells ( LCSLCs) derived from SMMC-7721 cell line. Meth-ods The second generation spheroids derived from SMMC-7721 cell line were obtained by sphere-forming assay and were considered as LCSLCs . Then LCSLCs were treated with various concentrations ( 1.0, 3.0, 10.0 μmol·L-1) of BrMC. The expression level of miR-519d was detected using real-time PCR. And in vitro carcinogenicity was investigated by sphere-forming assay and clone-forming assay in agar. The transcrip-tional activity and protein expression of Twist1 were an-alyzed using luciferase reporter assay and Western blot. Moreover, the molecular mechanism of BrMC was elucidated via miR-519 mimic transfection and Twist1 gene transduction, respectively. Results Compared with SMMC-7721 cells, miR-519d-3p was low-ex-pressed and Twist1 was over expressed in LCSLCs. And the sphere-forming ratio and the clone-forming ra-tio decreased by treatment with BrMC ( 1.0, 3.0, 10.0 μmol·L-1) in a dose-dependent manner. Fur-thermore, luciferase reporter assay demonstrated miR-519d could directly target the 3′ untranslated region of Twist1 mRNA and regulate protein expression. miR-519d mimic enhanced the effects of BrMC (3.0 μmol ·L-1) . However, Twist1 gene transduction effective-ly reversed the effects of BrMC ( 3.0 μmol·L-1) . Conclusion BrMC inhibits in vivo carcinogenicity via regulating miR-519/Twist1 signal axis in LCSLCs de-rived from SMMC-7721 cell line.
ABSTRACT
Objective: To examine whether and how Viticis Fructus total flavonoids (VFTF) inhibit the capacity of self-renewal in lung cancer stem cells (LCSCs) derived from human small cell lung cancer NCI-H446 cell line. Methods: NCI-H446 cell line was cultured in vitro. LCSCs were obtained and amplified by Magnetically activated cell sorting system (MACS) and suspended culture with serum-free conditioned medium. The capacity of self-renewal was detected by tumor sphere-forming assay. The protein expression levels of the self-renewal associated transcription factors, Bmi1 and p-Akt, were analyzed using Western blotting. Results: VFTF significantly suppressed the capacity of self-renewal in LCSCs, in a concentration-dependent manner (P < 0.05). The expression levels of Bmi1 and p-Akt were elevated in LCSCs, compared with parental cells. VFTF effectively down-regulated the expression levels of Bmi1 and p-Akt in LCSCs. In addition, LY294002, a PI3K specific inhibitor, enhanced the inhibition of VFTF on the capacity of self-renewal in LCSCs. Conclusion: VFTF could inhibit the self-renewal capacity of LCSCs derived from NCI-H446 cell line, which is associated with down-regulation of the expression of p-Akt and self-renewal associated transcription factors Bmi1.