Figure 5.

The EP4 receptor is a direct target of miR-101. (A) Predicted miR-101 binding sequence in the human PTGER4 3′-UTR, encoding EP4 receptor. The PTGER4 wildtype and mutant constructs were cloned into the pGL3 plasmid and downstream of the Luciferase gene at the Xba1 restriction site. The location of the wild-type constructs in the plasmid is denoted with yellow and mutant with red. Ninety percent of the seed sequence is mutated in the mutant construct. (B) The LS174T colon cells were co-transfected with pGL3carrying the constructs (wild-type or mutant EP4-3′-UTR and either a miR-101 expressing plasmid or empty vector, and Renilla. Co-transfection of the wildtype construct and miR-101 suppressed EP4 Luciferase expression. The relative luciferase activities were measured and normalized against their control. (C) Western analysis of endogenous EP4 receptor protein expression levels after ectopic miR-101 expression in LS174T cells. The levels of endogenous EP4 receptor protein decreases in a time-dependent manner