Bufalin Inhibits Proliferation and Induces Apoptosis in Osteosarcoma Cells by Downregulating MicroRNA-221

Figure 3

Functional screening of miR-221 target sites using luciferase reporter assay. (a) The selection criteria of the miRNA targets were based on their common detection in the target prediction online databases (TargetScan7.0 http://www.targetscan.org/) as well as the full complementarity between the seed region of miR-221 and 3′-UTR of BBC3. (b) HEK 293 cells cotransfected with miR-221-MIMIC, psi-Check2, psi-Check2-BBC3, or MUT-psi-Check2-BBC3. The luciferase activity levels were measured 24 h after transfection; results from at least three separate experiments are presented as means ± SE. The results show the regulation of BBC3 by miR-221 ( compared with the control). (c) After transfection for 24 h, the BBC3 mRNA expression levels in Saos-2 cells transfected with miR-221-MIMIC and miR-221-AMO were determined by qRT-PCR ( compared with the control). (d) The BBC3 protein expression levels in Saos-2 cells transfected with miR-221-MIMIC and miR-221-AMO were analyzed by Western blot at the same time to qRT-PCR.

(a)

(b)

(c)

(d)