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). The relative expression of Lgr5 mRNA measured by qPT-PCR was respectively 0.168±0.057 in the experimental group and 1.148±0.004 in the control group, with a statistically significant difference (t=28.778, P<0.05). (2) The capacity of self-renewal was detected by sphere forming assay. The results of sphere forming assay: the number of spheres was 29±6 in the experimental group and 410±10 in the control group, with a statistically significant difference (t=41.070, P<0.05). The results of colony formation assay: the numbers of colonies in the experimental group and control group were respectively 72±4 and 412±19, showing a statistically significant difference (t=31.433, P<0.05). The results of tumorigenesis: the volumes of tumors in the experimental group and control group were respectively (81±15)mm3 and (328±24)mm3 , with a statistically significant difference (t=11.304, P<0.05). (3) The effects of Lgr5 down-regulation on related genes, results of qRT-PCR detection: ① The mRNA relative expressions of Oct4, Sox2, Nanog and KLF4 (stem cells related genes) were 0.377±0.093, 0.662±0.104, 3.591±0.300, 0.425±0.091 in the experimental group and 1.957±0.026, 2.137±0.015, 5.831±0.165, 1.536±0.014 in the control group, with statistically significant differences (t=23.079, 22.261, 8.446, 19.186, P<0.05). ② The mRNA relative expressions of CD133, CD44 and ALDH (CSCs genes) were 1.490±0.155, 5.535±0.487, 1.640±0.039 in the experimental group and 2.488±0.061, 9.908±0.332, 5.718±0.292 in the control group, with statistically significant differences (t=8.170, 9.667, 27.849, P<0.05). ③ The mRNA relative expressions of Axin2, Wnt5a, Wnt3a, Fzd3, c-myc, VEGF, Ascl2 and claudin-1 genes in the Wnt/β-catenin pathway were respectively 1.592±0.267, 0.528±0.138, 2.153±0.078, 1.480±0.064, 0.248±0.128, 1.492±0.025, 0.658±0.095, 1.647±0.087 in the experimental group and 3.651±0.224, 2.570±0.093, 2.301±0.157, 1.636±0.058, 1.415±0.080, 2.610±0.159, 2.480±0.123, 3.432±0.273 in the control group. There were statistically significant differences in the mRNA relative expressions of Axin2, Wnt5a, c-myc, VEGF, Ascl2 and claudin-1 genes between the 2 groups (t=7.316, 15.332, 12.649, 12.320, 14.831, 9.063, P<0.05), and no statistically significant difference in the mRNA relative expressions of Wnt3a and Fzd3 between the 2 groups (t=2.887, 2.242, 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.
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