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[Screening and identification of effective small interfering RNAs against bone morphogenetic protein-9]
1Department of Biochemistry and Molecular Biology, Chongqing Medical University, Chongqing 400016, China. yangbi1981@yahoo.com
Objective:
To screen specific small interfering RNA (siRNA) targeting mouse BMP9 gene and identify its function in BNLCL.2 fetal liver cells and C3H10 cells.
Methods:
Four pairs of double-stranded DNA fragments for silencing mouse BMP9 were annealed in vitro and cloned into pSOS-BMP9 vector with BMP9 gene to construct pSOS-simBMP9 plasmid. The 4 pSOS-simBMP9 plasmids were separately transfected in HEK293 cells via Lipofectamine, and the gene silencing efficiency was assessed by GFP detection. BNLCL.2 fetal liver cells were infected with the constructed adenovirus simBMP9s, and their BMP9 expression was detected with RT-PCR and Western blotting. C3H10 cells were co-infected with Ad-simBMP9 and Ad-BMP9, and the inhibitory effect on BMP9-induced osteoblasts was evaluated by alkaline phosphatase (ALP) activity.
Results:
GFP expression in the two simBMP9 groups was significantly decreased in HEK293 cells, and the endogenous expression of BMP9 was reduced by 50%-70% by adenovirus-mediated simBMP9 in the fetal liver cells. ALP activity in C3H10 cells was significantly higher in BMP9 group than in the control group (P<0.01), while the activity of the two Ad-simBMP9-infected groups was significantly lower than that in Ad-BMP9-infected group (P<0.01).
Conclusion:
Two specific siRNA targeting mouse BMP9 gene have been obtained, which can effectively inhibit both endogenous and exogenous expressions of BMP9 to facilitate the study of the mechanisms of BMP9 in liver cell differentiation.
