The expression level of miR-483-5p was verified by a quantitative reverse transcription-polymerase chain reaction. Growth, apoptosis, and colony formation ability were also examined in SHEEC cells after transfection with inhibitors targeting miR-483-5p. Target genes of miR-483-5p were predicted using bioinformatics approaches and the expression profile of SHEEC cells transfected with the miRNA inhibitors. Protein levels of the target gene in SHEEC cells with a control or miRNA inhibitors were measured using Western blotting. The expression of miR-483-5p was elevated in SHEEC cells as compared to the SHEE cells. Silencing of miR-483-5p expression in SHEEC cells inhibited both the proliferation and formation of colonies and increased apoptosis. They also identified hepatocyte nuclear factor 4α (HNF4A) as a target of miR-483-5p in SHEEC cells. Knockdown of HNF4A recapitulated the effects of miR-483-5p.
This data showed that the miR-483-5p/HNF4A axis affected the malignant transformation of immortalized human esophageal epithelial cells and is a potential therapeutic target for ESCC.
HNF4A is a direct target of miR-483-5p. miR-483-5p
binding sites in the HNF4A 3’UTR region
A Western blot analysis demonstrated that the transfection of miR-483-5p reduced HNF4A protein expression (A). HNF4A mutation indicates the HNF4A 3’UTR with a mutation in miR-483-5p binding sites (B). Relative luciferase assay comparing the pGL3-HNF4A and pGL3-HNF4A mutation vectors in SHEEC cells. Firefy luciferase activity was normalized to Renilla luciferase activity (C). Values are expressed as the mean ± standard deviation. *P<0.05 vs. control.