OBJECTIVETo investigate the role of Gemin3 in cell proliferation and its regulation pathway.

METHODSUsing co-immunoprecipitation and GST pull-down assay to determine the domain of Gemin3 and p53 binding and interaction in vitro and in vivo. To check the effect of Gemin3 on p53 by luciferase reporter assay. Stable Gemin3 knock-down cell lines were generated by lentivirus-delivered small hairpin RNA then puromycin selection. Real-time PCR was used to confirm the effect of Gemin3 level on p53 and its downstream genes, and flow cytometry was used to analyze the effect of Gemin3 on apoptosis.

RESULTSThe C-terminal of Gemin3 interacted with the DNA binding domain of p53. The p53 reporter gene, PA3M-p53 and increasing amount of GFP-Gemin3 were co-transfected into Saos-2 cells. Gemin3 repressed p53 expression at transcription level. Real-time PCR indicated that the expression of p53, p21 and Bax in Gemin3 knock-down cells was higher than that in the control cells. Western blot showed Gemin3 knock-down cells had a higher p53 espression. Flow cytometric assay showed that knock-down Gemin3 expression led to an increased cell apoptosis.

CONCLUSIONGemin3 binds with p53 forming a complex and plays an anti-apoptotic role by repressing the p53 expression.

Apoptosis ; B-Lymphocytes ; cytology ; Cell Line, Tumor ; DEAD Box Protein 20 ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Genes, Reporter ; Humans ; Immunoprecipitation ; Lentivirus ; genetics ; Osteosarcoma ; genetics ; metabolism ; pathology ; Plasmids ; Protein Binding ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Transfection ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; bcl-2-Associated X Protein ; genetics ; metabolism