Fig. 4

MiR-27a suppresses PHLPP2 expression by directly targeting the PHLPP2 3′-UTR. a Sequence alignment of miR-27a and its predicted binding sites (green) in PHLPP2 3′-UTR. Predicted miR-27a target sequence (blue) in the 3′-UTR of PHLPP2 (wtPHLPP2-3′-UTR) and position of mutated nucleotides (red) in the 3′-UTR of PHLPP2 (mutPHLPP2-3′-UTR). b Luciferase reporter assay. A vector containing wild type PHLPP2 3′-UTR or mutant PHLPP2 3′-UTR was co-transfected into GC cells together with indicated oligonucleotides. Luciferase activity ratio was presented as firefly luciferase value/renilla luciferase value. c PHLPP2 mRNA levels were detected by qRT-PCR in SGC-7901 and AGS cells transfected with miR-27a antagomir or miR-27a agomir, respectively. d Western blot analysis of PHLPP2 expression. PHLPP2 protein levels were detected by Western blot in SGC-7901 and AGS cells transfected with miR-27a antagomir or miR-27a agomir, respectively. e Relative expression of PHLPP2 in gastric cancer tissues. PHLPP2 expression was tested through qRT-PCR in 50 paired samples. Expression of PHLPP2 mRNA in each tumor tissue was normalized to its matched normal tissue. f Pearson correlation analysis between miR-27a and PHLPP2 mRNA levels in human gastric cancer tissues. (r = -0.293, P = 0.039). g Immunohistochemistry. PHLPP2 protein expression in gastric cancer tissues was analyzed by immunohistochemistry. I, adjacent normal tissue; II, moderately differentiated adenocarcinoma; III, poor differentiated adenocarcinoma; IV, mucous layer infiltration; V, muscular layer infiltration; VI, whole layer infiltration (×200 magnification)