[Mechanism of G protein coupled receptor kinase interacting protein 1 RNA hairpin inhibiting osteoblasts migration].

OBJECTIVE

To study the function and mechanism of G protein coupled receptor kinase interacting protein 1 (GIT1) RNA hairpin (GIT1-RNAh) in osteoblast migration.

METHODS

The sixth passage osteoblasts were divided into 2 groups and were infected by GIT1-RNAh (experimental group) and green fluoresence protein RNA hairpin (GFP-RNAh) (control group) adenovirus for 12 hours respectively. Each group was further classfied into two groups according to with or without platelet-drived growth factor (PDGF) stimulation. The GIT1 expression and Paxillin distribution was analyzed by immunofluorescence staining. Paxillin phosphorylation was detected by Western Blot. The localization of Paxillin was determined by co-immunofluorescence staining after transfection with cyanine fluorescence protein tagged GIT1-RNAh (CFP-GIT1-RNAh) (experimental group) and GFP-RNAh (CFP-GFP-RNAh) (control group). The role of GIT1-RNAh (experimental group) and GFP-RNAh (control group) adenovirus in osteoblasts migration was determined by wound healing assay.

RESULTS

Immunofluorescence staining results showed that the GIT1-RNAh significantly inhibited endogenous GIT1 expression, interfered Paxillin distribution. Western Blot results showed that Paxillin phosporylation was obviously inhibited in osteoblasts infected with GIT1-RNAh adenovirus (P< 0.05). The wound healing assay results showed that GIT1-RNAh adenovirus significantly inhibited osteoblast migration induced by PDGF.

CONCLUSION

GIT1-RNAh inhibits osteoblasts migration by interfering paxillin distribution and decrease Paxillin phosphorylation.