Effects of lamin A/C inhibition on proliferation and apoptosis of MC3T3-E1 cells exposed to stretch stimulus

Abstract

Objective To investigate the effect of lamin A/C inhibition using siRNA technology on proliferation and apoptosis of MC3T3-E1 cells exposed to the stretch stimulus. Methods Three chemically synthesized siRNAs targeting lamin A/C were transfected into MC3T3-E1 cell by lipofectamineTM 2000. The most efficient siRNA inhibiting lamin A/C mRNA and protein expression was identified by realtime polymerase chain reaction (PCR) and Western blotting respectively. After 72 h siRNA transfection,MC3T3-E1 cells were subsequently subjected to 2500 μe stretch for 6 h with four-point-bending system.DNA content was detected by Hoechst 33258 fluorochrome to evaluate cell proliferation. Flow cytometry was employed to investigate the changes in cell cycle and apoptosis of MC3T3-E1 cells. Results The siRNA-2 was identified to inhibit lamin A/C mRNA and protein expression significantly (P ＜ 0. 01 ) and utilized in the following experiment. DNA assay showed that stretch stress led to an increased DNA synthesis in a time-dependent manner in non-transfected cells, while the similar increase in DNA content was not displayed in siRNA transfected cells applied with the same mechanical stimulus (P ＜ 0. 01 ). Compared with non-transfected MC3T3-E1 cells, stretch stress increased the percentage of G0/G1 phase in transfected cells from 65. 19% to 85.82%, and decreased the percentage of S phase from 22. 57% to 11.37%. At the meantime, apoptosis rate was significantly increased from 11.49% to 19. 32% ( P ＜ 0. 01 ). Conclusion Inhibition of lamin A/C expression attenuates the mitogenic effects of MC3T3-E1 exposed to stretch stress,blocks more cells arrest in G0/G1 phase and increases cell apoptosis. Key words: RNA interference; Apoptosis; Proliferation; Stretch