Fig. 3
miR-500a-5p suppresses cell proliferation and invasion by targeting HDAC2 in vitro. a The putative miR-500a-5p-binding sequence within the 3′-UTR of HDAC2 mRNA. Mutations in the complementary site for the seed region of miR-500a-5p in the 3′-UTR of the HDAC2 gene are underlined. b Reporter plasmids containing either the wild-type 3′-UTR or a mutated 3′-UTR of the HDAC2 gene were co-transfected into CRC cells with an miR-500a-5p-encoding plasmid, and luciferase activity was measured. WT, wild type; MUT, mutant; site 1, the miR-500a-5p-motif spanning nt 4703–4709; site 2, the motif spanning nt 5244–5250 in the HDAC2 3ʹ-UTR; the data are presented as the mean ± SD of three experiments. Student’s t test; *P > 0.05; *** P < 0.01; ****P < 0.001. c Western blot analysis of HDAC2 protein expression in colon epithelial cells transfected with miR-500a-5p mimics or inhibitor. Cells transfected with a control mimic (m-NC) or inhibitor (i-NC) plasmids were used as a control. d Representative results (up) and quantification (down) of crystal violet-stained cell colonies formed by the indicated CRC cells on the 12th day after seeding. Student’s t test; ****P < 0.001. e The proliferation of LoVo cells by the WST-1 assay. Student’s t test; ***P < 0.01; ****P < 0.001. f The invasive activity of cells transfected with an HDAC2 expression plasmid and/or miR-500a-5p mimics was evaluated by the transwell assay. HPF, high-power field. Student’s t test; **P < 0.05 and ***P < 0.01. All experiments were repeated at least three times with identical findings
