Inhibitory effect of knocking down LOXL1 gene on elastin expression and cell biological behavior in HLECs

Objective  To investigate the effect of knocking down the lysyl oxidase-like protein 1 ( LOXL1) gene on the expression and aggregation of elastin in the human lens epithelial cells (HLECs), as well as its impact on the proliferation activity and migration ability of HLECs.

Methods  The human lens epithelial cell line HLE-B3 was cultured in vitro and divided into shLOXL1-1 group, shLOXL1-2 group, shLOXL1-3 group, and normal control group.The shLOXL1-1 group, shLOXL1-2 group, and shLOXL1-3 group cells were subjected to LOXL1 gene knockdown intervention using different sequences of lentiviral transfection methods, while the normal control group was subjected to meaningless sequence lentiviral transfection intervention.The relative expression level of LOXL1 mRNA in different groups was detected by real-time fluorescence quantitative PCR.The fluorescence intensity of elastin in HLE-B3 was determined by immunofluorescence.The expression of elastin in HLE-B3 was detected by Western blot.The content and aggregation degree of elastin in HLE-B3 was detected by electron microscopy scanning.The migration rate of HLE-B3 was detected by cell scratch assay.The proliferation activity of HLE-B3 was detected using the cell counting kit 8.

Results  After knocking down the LOXL1 gene, the relative expression levels of LOXL1 mRNA were lower in the shLOXL1-1, shLOXL1-2, and shLOXL1-3 groups than those in the normal control group, and the differences were statistically significant (all at P<0.001).The shLOXL1-3 group had the best knockdown effect, so the shLOXL1-3 group was selected for subsequent experiments and compared with the normal control group.After immunofluorescence staining, stable expression of elastin was observed in HLE-B3 cells.The average fluorescence intensity of elastin in the shLOXL1-3 group was 56.96±5.56, significantly lower than 80.52±4.78 in the normal control group ( t=5.572, P<0.001).The relative expression level of elastin in the shLOXL1-3 group was 0.807±0.002, significantly lower than 1.185±0.064 in the normal control group ( t=5.802, P<0.01).Under the electron microscope, the elastin density in the shLOXL1-3 group was lower than that in the normal control group, but its morphology, size, and aggregation degree did not show significant changes.At 24 and 48 hours after transfection, the relative migration rates of shLOXL1-3 group cells were 0.292±0.041 and 0.439±0.032, which were lower than 0.463±0.017 and 0.719±0.007 of normal control group, respectively, and the differences were statistically significant ( t=8.178, 2.611; both at P<0.05).After 24, 48, 72, and 96 hours of cultivation, the cell viability values of shLOXL1-3 group were lower than those of normal control group, and the differences were statistically significant ( t=2.555, 2.704, 6.695, 7.266; all at P<0.05).

Conclusions  Knocking down the LOXL1 gene in HLECs can cause a significant decrease in the expression level of elastin in the cells, as well as decreases in cell migration ability and proliferation activity.

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