Figure 3

HOXA10 as a target of miR-135a. (A) Online prediction of miR-135a potential binding sites on HOXA10 3'-UTR. The nucleotide sequence illustrates the predicted base-pairing between miR-135a and the HOXA10 3'-UTR. (B) The endogenous mRNA expression of HOXA10 was detected by RT-PCR in breast cancer cell lines with β-actin used as an expression control. (C) In vitro luciferase reporter assay in HEK293 cells. pS-135a or pS-N was co-transfected with the pGL3-3'-UTR reporter plasmids containing the HOXB7 3'-UTR, APC 3'-UTR, wide-type (wt) HOXA10 3'-UTR, and mutant(mu) HOXA10 3'-UTR. The 3'-UTR HOXB7 reporter plasmid was the negative control, and the APC reporter plasmid was the positive control. (D) In vitro luciferase reporter assays in BT549 cells. Negative (pGL3-promoter), wild-type (wt) and mutated (mu) 3'-UTR reporter plasmids of HOXA10 were cotransfected with an anti-sense inhibitor of miR-135a (135a inhibitor) or no inhibitor into BT549 cells, which express endogenous miR-135a. n = 3, *, p < 0.05, significantly decreased/increased activity.