Effect of VDR gene silencing on proliferation and NF-κB activation of airway smooth muscle cells

Abstract

Objective: To investigate the effect of VDR gene silencing on proliferation of airway smooth muscle cells (ASMCs) and elucidate the role of NF-κB. Methods: A recombinant lentiviral vector specifically targeting VDR gene in rat was constructed by RNA interference. Rat ASMCs were divided into blank group, empty vector group and interference group. ASM cell line model stably silencing the VDR gene RNA expressing was selected by puromycin. Then MTT colorimetric assay and cell cycle analysis by flow cytometry were used to examine cell proliferation. The activation of nuclear factor-κB was determined by immunofluorescence double label method. Moreover, NF-κB-dependent transcription activity was tested through luciferase reporter gene assay. Western blotting was used for IκBα and phospho-IκBα protein levels and actinomycin D treatment was used to determine IκBα mRNA stability. All statistical analyses were performed using SPSS version 23.0 software. Differences between groups were analyzed using one-way ANOVA analysis. Multiple comparisons among groups were made by Student-Newman-Keuls test. Results: (1) As compared with those in the blank group and the empty vector group, the cell proliferation index (PI) and the percent of ASMCs at G2/M phase in the interference group were markedly increased (P<0.05), but their percent at G0/1 phase was decreased (P<0.05).(2) In the interference group, the nuclear translocation of NF-κB p65 in ASMCs was obviously induced. And its level of receptor gene NF-κB p65 (1.37±0.28) was significantly higher than that in the blank group (1.00±0.19,P=0.031) and in the empty vector group (0.96±0.18,P=0.027).(3) In the interference group, the IκBα protein level in ASMCs (0.13±0.04) was obviously less than that in the blank group (0.29±0.05, P=0.023) and in the empty vector group (0.32±0.07, P=0.014). Oppositely, the p-IκBα/IκBα level in the interference group (0.86±0.04) was much more than that in the blank control group (0.41±0.07, P=0.026) and in the empty vector group (0.37±0.05, P=0.017). (4) In the interference group, IκBα mRNA showed a shorter half-life, (171.31±9.67) min, compared to that in the blank group [(224.69±7.95) min,P=0.032] and in the empty vector group [(230.41±6.37) min,P=0.035]. Conclusion: VDR gene silencing could promote ASMC proliferation and the underlying mechanism may involve the activation of NF-κB signaling pathway.