Effects of lentiviral short hairpin RNA silencing of Toll-like receptor 4 on the lens epithelial cell line HLEC
Abstract
The aim of this study was to observe the proliferation of, and cell-cycle changes in, the human lens epithelial cell line HLEC after Toll-like receptor 4 (TLR4) gene silencing. HLEC cells were transfected with four TLR4-short hairpin RNA (shRNA) lentiviral vectors or the control lentivirus (pGCL-GFP-shRP-1, -2, -3, -4, NC). TLR4 silencing was verified in these cells 96 h post-transfection using real-time polymerase chain reaction and western blot. We also observed the change in number of pGCL-GFP-shRP-4-transfected HLEC cells with silenced TLR4 (multiplicity of infection = 10). Cell proliferation was analyzed 48 h after transfection by a standard Cell Counting Kit-8 (CCK-8) assay, and the cell cycle changes were detected by flow cytometry. The number of cells with silenced TLR4 decreased with time. The decrease in TLR4 expression led to decelerated cell proliferation. Cells with silenced TLR4 (for 48 h) were arrested in the G1 phase; that is, the cell cycle was prolonged and cell division was decelerated. Lentivirus-mediated RNA interference effectively silenced TLR4 expression in HLEC cells, which decelerated their proliferation rate and extended the cell cycle.
The aim of this study was to observe the proliferation of, and cell-cycle changes in, the human lens epithelial cell line HLEC after Toll-like receptor 4 (TLR4) gene silencing. HLEC cells were transfected with four TLR4-short hairpin RNA (shRNA) lentiviral vectors or the control lentivirus (pGCL-GFP-shRP-1, -2, -3, -4, NC). TLR4 silencing was verified in these cells 96 h post-transfection using real-time polymerase chain reaction and western blot. We also observed the change in number of pGCL-GFP-shRP-4-transfected HLEC cells with silenced TLR4 (multiplicity of infection = 10). Cell proliferation was analyzed 48 h after transfection by a standard Cell Counting Kit-8 (CCK-8) assay, and the cell cycle changes were detected by flow cytometry. The number of cells with silenced TLR4 decreased with time. The decrease in TLR4 expression led to decelerated cell proliferation. Cells with silenced TLR4 (for 48 h) were arrested in the G1 phase; that is, the cell cycle was prolonged and cell division was decelerated. Lentivirus-mediated RNA interference effectively silenced TLR4 expression in HLEC cells, which decelerated their proliferation rate and extended the cell cycle.