Fig. 4

MiR-509-3p inhibits the expression of PHLPP2. A A list of top 10 predicted miR-509-3p target genes were probed in HCT116 or SW480 CRC cell lines stably transfected with miR-509-3p (pre-mir) or empty vector control using qPCR. Only one gene, PHLPP2 was found to be downregulated in both cell lines transfected with miR-509-3p as compared to negative control. B Upon administration of an inhibitor against miR-509-3p, the expression levels of PHLPP2 were restored. C Real-time PCR-based expression analysis of PHLPP2 in CRC tumor and NAT tissue in 25 patients. D Expression of PHLPP2 negatively correlated with the expression of miR-509-3p within the 25 CRC patients. For (A-D), expression of miR-509-3p was normalized to the expression of RNU6B, while expression of PHLPP2 and other putative target genes were normalized to the expression of GAPDH. Expression results are shown as -∆CT. E Two predicted binding sites for miR-509-3p within the 3′-UTR of PHLPP2 were identified by TargetScan. Within the original sequence, the uppercase characters indicate the original putative binding site of miR-509-3p within the 3′-UTR of PHLPP2. Within the mutant sequences, the underlined characters indicate the mutated bases within the potential binding site. Lowercase characters are the flanking 3′-UTR bases. CFTR was used as the positive control within the Luciferase Reporter assay. F HCT116 cells were co-transfected with PHLPP2 (1) or (2) 3′-UTR original or mutant sequence reporter constructs with miR-509-3p mimic or empty vector control and Renilla Luciferase control vector. For each transfectant, Firefly Lucferase signal was normalized with the Renilla Luciferase signal and expressed as Relative Luciferase Units. *p value < 0.05; **p value < 0.01; ***p value < 0.001