Effect of leucine-rich repeat-containing G-protein coupled receptor 5 gene silencing on biological behaviors of colon cancer stem cells

Abstract

Objective To investigate the effect of leucine-rich repeat-containing G-protein coupled receptor 5 gene (LGR5) silencing on biological behaviors of colon cancer spheroid cells with stemness. Methods RNA interfering methods were used to knock down the LGR5 expression. Small interfering RNA (siRNA) targeting LGR5 and control siRNA were transfected into HT29 colon cancer spheroid cells by Lipofectamine RNAi MAX. Cells transfected with control siRNA or untransfected were used as negative control and blank control respectively. The expression of LGR5 mRNA and protein was detected by real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting. The proliferation of spheroid cells was detected by cell counting kit-8 (CCK-8) assay at 1st, 2nd, 3rd, and 4th day after transfection respectively. The self-renewal, invasion and cell cycle of spheroid cells were determined by sphere formation assay, Transwell assay and flow cytometry respectively at 48th h after transfection. Results At 48 h after transfection, the expression of LGR5 mRNA in experimental group (0.32±0.02) was remarkably lower than that in blank control group (1.00±0.16) and negative control group (0.99±0.12) (P=0.000). The expression of LGR5 protein in experimental group was also significantly down-regulated (P=0.000). CCK-8 assay showed that the absorbance (A) value in blank control group, negative control group and experimental group at 4th day was 0.540±0.010, 0.528±0.016 and 0.390±0.022, respectively (P=0.000). In addition, the experimental group (21.33±2.08) formed fewer secondary spheres than blank control group (72.33±3.06) and negative control group (70.67±2.52, P=0.000). Flow cytometry revealed that the cells in the experimental group were significantly arrested in G0/G1 phase (P=0.000). Furthermore, the number of cells passing through the Transwell membrane in experimental group (35.60±6.74) was remarkably decreased as compared with that in the blank control group (86.47±7.85) and negative control group (83.53±8.58, P=0.000). Conclusion LGR5 gene silencing markly inhibited the proliferation, self-renewal and invasion of HT29 spheroid cells, indicating that LGR5 may play an important role in manipulating the biological behaviors of colon cancer stem cells. Key words: Colon cancer; Cancer stem cells; Leucine-rich repeat-containing G-protein coupled receptor 5; Proliferation; Invasion