OBJECTIVE: Embryonic stem cells (ESCs) mainly originate from totipotent cells in early-stage of mammalian embryo and could proliferate in a manner of un-limitation. This study aimed to investigate roles of Axin2 in proliferation of ESCs and explore the associated mechanisms.
MATERIALS AND METHODS: Axis inhibition protein 2 (AXIN2) over-expression (LV5-AXIN2) and AXIN2 RNA interfere (LV3-AXIN2-RNAi) vectors were structured and transfected into H9 cells. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was used to evaluate cell proliferative activity. Flow cytometry analysis was employed to measure apoptosis of H9 cells. AXIN2, β-catenin, transcription factor 4 (TCF4), c-myc, c-jun and Cyclin D mRNA levels and protein expressions were determined using quantitative real-time PCR (qRT-PCR) and Western blotting assay.
RESULTS: LV5-AXIN2 and LV3-AXIN2-RNAi were successfully structured with higher transfecting efficacy. AXIN2 gene silencing remarkably increased proliferative activity and AXIN2 treatment significantly induced apoptosis of H9 cells, comparing with blank vector group (p<0.05). AXIN2 gene silencing significantly enhanced B-cell lymphoma-2 (Bcl-2) expression and remarkably inhibited cleaved caspase-3 expression comparing to that in blank vector group (p<0.05). AXIN2-RNAi treatment significantly enhanced and AXIN2 over-expression significantly reduced β-catenin and TCF4 expression, comparing to that in blank vector group (p<0.05). AXIN2 gene silence activated down-stream molecules of Wnt/β-catenin signaling pathway, including c-jun, c-myc, and Cyclin D1 (p<0.05).
CONCLUSIONS: AXIN2 gene silencing reduced apoptosis by regulating mitochondria-associated apoptosis signaling pathway and enhanced proliferation by modulating molecules in Wnt/β-catenin signaling pathway. Therefore, targeting of aberrant apoptosis and AXIN2 might be a novel clinical strategy to inhibit aging and enhance self-renewal of ESCs.Free PDF Download
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F. Fu, Q. Deng, R. Li, D. Wang, Q.-X. Yu, X. Yang, T.-Y. Lei, J. Han, M. Pan, L. Zhen, J. Li, F.-T. Li, Y.-L. Zhang, D.-Z. Li, C. Liao
AXIN2 gene silencing reduces apoptosis through regulating mitochondria-associated apoptosis signaling pathway and enhances proliferation of ESCs by modulating Wnt/β-catenin signaling pathway
Eur Rev Med Pharmacol Sci
Vol. 24 - N. 1