| Objective: The simulated microgravity(SMG)regulates the proliferation of osteoblasts.However,the role of ras association domain family member 2(Rassf2)in the regulation of osteoblast proliferation by SMG remains unclear.The purpose of this study was to investigate the expression of Rassf2 in SMG environment and to investigate whether Rassf2 is involved in the effects of simulated microgravity on osteoblast proliferation.Method:(1)MEC3T3-E1 osteoblasts were cultured by Rotary cell culture system(RCCS)to simulate microgravity environment,and the growth of osteoblasts in SMG environment was observed by inverted phase contrast microscope.Western Blot was used to detect the changes in the expression levels of proliferation-related proteins(PCNA,Cyclin D1,Cyclin B1)and Ed U staining was used to detect the proliferation ability of osteoblasts.(2)48 h after osteoblasts were cultured with RCCS,the expression level of Rassf2 was detected by PCR and Western Blot.(3)The expression level of Rassf2 in osteoblasts was knocked down by transfection of small interfering RNA and up-regulated by transfection of overexpressed plasmid.The proliferative phenotype of osteoblasts was observed by Western Blot and Ed U staining.(4)Rassf2 was overexpressed in microgravity environment to restore the expression level of Rassf2 in osteoblasts,and to observe whether the inhibition of simulated microgravity on osteoblast proliferation was reversed.Results:(1)Compared with Normal gravity group(NG),the expressions of proliferation-related proteins PCNA,Cyclin D1 and Cyclin B1 in simulated microgravity group were significantly decreased(p < 0.05),and the proliferative ability of SMG-composed bone cells was also significantly decreased.(2)PCR and Western Blot results showed that the m RNA and protein expression of Rassf2 in MC3T3-E1 cells in SMG group was significantly lower than that in NG group(p <0.01).(3)Down-regulation of Rassf2 inhibited the proliferation of osteoblasts and the expression levels of PCNA,Cyclin D1 and Cyclin B1 proteins.In contrast,up-regulation of Rassf2 promotes osteoblast proliferation and protein expression levels of PCNA,Cyclin D1 and Cyclin B1.(4)Rescue experiment results showed that overexpression of Rassf2 in microgravity environment could partially eliminate the inhibitory effect of SMG on osteoblast proliferation(p < 0.05),and promote the expressions of PCNA,Cyclin D1 and Cyclin B1 proteins.Conclusion: Simulated microgravity environment can inhibit osteoblast proliferation by decreasing the expression of Rassf2 protein. |
