Figure 3.

PTEN is a direct target of miR-136. (A) Binding sequences for miR-136 in the 3′UTR of PTEN, and the mutations in the 3′UTR of PTEN are presented. (B) A luciferase reporter assay was performed in 293T cells at 48 h after co-transfection with miR-136 inhibitor or NC inhibitor, and pGL3-PTEN-3′UTR Wt or pGL3-PTEN-3′UTR Mut. The treatment with a miR-136 inhibitor was able to increase luciferase activity in cells that were transfected with pGL3-PTEN-3′UTR Wt. By contrast, miR-136 inhibitor did not affect the luciferase activity in in cells that were transfected with pGL3-PTEN-3′UTR Mut. *P<0.05 vs. NC inhibitor. (C) RT-qPCR analysis of PTEN mRNA in MGC-803 and SGC-7901 cells following transfection with miR-136 inhibitor or NC inhibitor. *P<0.05 vs. NC inhibitor. (D) Western blotting was used to detect PTEN protein expression in MGC-803 and SGC-7901 cells following transfection with miR-136 inhibitor or NC inhibitor. (E) The relative PTEN mRNA expression levels were determined using RT-qPCR in gastric cancer tissues and adjacent non-tumorous gastric mucosae tissues. The data are presented as box plots. The top of the box indicates the upper quartile, and the bottom indicates the lower quartile. The central line in the box indicates median, and the whiskers indicate the range. *P<0.05 vs. non-tumorous gastric mucosae tissues. (F) Analysis of correlation between miR-136 and PTEN mRNA expression in gastric cancer tissues. r=−0.6035, P=0.0011. The data are presented as the mean ± standard deviation. PTEN, phosphatase and tensin homolog deleted on chromosome ten; miR-136, microRNA-136; NC inhibitor, negative control inhibitor; 3′UTR, 3′-untranslated regions; Wt, wild-type; Mut, mutant; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.