2024, Vol. 28 ›› Issue (32): 5177-5181

Effects of filament B knockdown on proliferation, migration and apoptosis of mouse MC3T3-E1 cells

Wang Xi1,2, Yu Li1, Jia Qiyu2, Huang Jinyong2, Liu Zebiao3, Zhang Jun2, Jiayidaer•Dilimulati2, Xie Zengru2, Ma Hairong1   

Abstract: BACKGROUND: Filamin B (FLNB) can crosslink the actin cytoskeleton into a dynamic structure that is essential for the directional movement of cells. It can regulate the proliferation, differentiation and apoptosis of chondrocytes. However, the effect of FLNB on osteoblast proliferation, migration and apoptosis has not been reported.
OBJECTIVE: To investigate the effect of FLNB on the proliferation, migration and apoptosis of MC3T3-E1 cells.
METHODS: The adenoviral vectors for knockdown of FLNB expression (sh-FLNB1, sh-FLNB2, sh-FLNB3) were constructed and infected with MC3T3-E1 cells. After screened by puromycin drug, the efficiency of FLNB knockdown was detected by western blot and RT-PCR. The MC3T3-E1 cell line with the best efficiency of FLNB knockdown was selected as the stable transient cell line of MC3T3-E1 for subsequent experiments. The cells were divided into blank group, mc3t3 group, sh-NC group (empty vector), and sh-FLNB group (sh-FLNB lentivirus). The blank group was cultured in cell-free α-MEM complete medium; the mc3t3 group was cultured in α-MEM complete medium alone; and the sh-NC and sh-FLNB groups were cultured with α-MEM medium containing 2.5 μg/mL puromycin. After 3 days of culture, cell counting kit-8 assay and cell scratch assay were used to detect the proliferation and migration ability of MC3T3-E1; flow cytometry was used to detect cell apoptosis; and RT-PCR was used to detect the expression of apoptosis-related genes.
RESULTS AND CONCLUSION: Western blot and RT-PCR results showed that the efficiency of FLNB knockdown was the best in the sh-FLNB3 (P < 0.000 1), which was used as a stable cell line for subsequent experiments. Cell counting kit-8 data showed that the proliferative ability of MC3T3 cells was significantly weakened after knockdown of FLNB (P < 0.05). Cell scratch assay results showed that the migration ability of MC3T3 cells was significantly decreased after knockdown of FLNB. Flow cytometry and RT-PCR results showed that the apoptotic rate of MC3T3-E1 cells increased after knockdown of FLNB, the expression of pro-apoptotic factor Bax increased significantly, and the expression of anti-apoptotic factor Bcl-2 decreased significantly (P < 0.05). To conclude, knockdown of FLNB can reduce the proliferation ability of MC3T3-E1 cells, decrease the migration ability of the cells, and increase cell apoptosis.

Key words: Filamin B, MC3T3-E1, proliferation, migration, apoptosis