Effects and mechanisms of shRNA interfered with expression of leucine-rich repeat containing G-protein coupled receptor 5 on the malignant behaviors of colorectal cancer stem cells

Abstract

Objective To investigate the effects and mechanisms of shRNA interfered with expression of leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5) on the malignant behaviors of colorectal cancer stem cells (CSCs). Methods The experimental study was conducted. The CSCs expressing Lgr5+ were sorted by fluorescence activated cell sorting. Lgr5+ cells that were transfected with Lgr5-shRNA lentiviral vector and non-target shRNA lentiviral vector were respectively allocated into the experimental group and control group. The percentage of Lgr5+ cells was analyzed by flow cytometery. The relative expression of Lgr5 mRNA was detected by fluorescence quantitative real-time polymerase chain reaction (qRT-PCR). The capacity of self-renewal was detected by sphere forming assay. The tumorigenesis in vitro and in vivo were respectively measured by colony formation assay and xenografting experiment. The mRNA expressions of stem cells related genes (Oct4, Sox2, Nanog, KLF4), CSCs genes (CD133, CD44, ALDH) and Wnt/β-catenin pathway key genes (Axin2, Wnt5a, Wnt3a, Fzd3, c-myc, VEGF, Ascl2, claudin-1) were detected by qRT-PCR. Measurement data with normal distribution were represented as ±s. Comparison between groups was analyzed using the t test. Results (1) Transfection efficiency of shRNA lentiviral vector induced Lgr5 by flow cytometery was respectively 6.8%±1.0% in the experimental group and 92.7%±3.3% in the control group, with a statistically significant difference (t=43.148, P 0.05). Conclusion The malignant behaviors of colorectal CSCs are suppressed after shRNA lentivirus interfered with expression of Lrg5, and its mechanism is related to inhibiting activity of Wnt/β-catenin pathway. Key words: Colorectal neoplasms; Cancer stem cells; Self-renewal; Wnt/β-catenin pathway