A functional Variant (Rs35592567) in TP63 at 3q28 is Associated with Gastric Cancer Risk via Modifying its Regulation by MicroRNA-140

Abstract

Background/Aims: TP63 was believed to play an important role in the development of many malignancies, while the polymorphisms located at the miRNA binding sites within the 3’UTR of TP63 mRNA may interfere with its expression. In this study, we aimed to study the role of TP63 regulation in the tumorigenesis of gastric cancer (GC). Methods: Computational and luciferase analysis were used to search and confirm the target of miR-140. Real-time PCR, western-blot, MTT assay, and flow cytometry cell cycle analysis were utilized to explore the molecular pathway of miR-140 involved in the progression of GC. Results: TP63 was identified as a direct target gene of miR-140. In HT-29 cells over-expressing miR-140, the luciferase activity was decreased when the cells were transfected with wild-type TP63 3’UTR, but remained unchanged when the cells were transfected with mutant 1 and mutant 2 TP63 3’UTR. In addition, the level of TP63 in HT-29 cells transfected with miR-140 mimic was evidently down-regulated, whereas the level of TP63 in HT-29 cells transfected with miR-140 inhibitor was significantly up-regulated. Furthermore, based on the results from MTT assay and flow cytometry cell cycle analysis, HT-29 cells transfected with miR-140 mimics were associated with significantly higher viability compared to the cells transfected with the control plasmid, suggesting that an increased expression of miR-140 protected HT-29 cells against apoptosis. Finally, when miR-140 expression was high, the number of cells at the G1 phase was notably increased, accompanied by a remarkably diminished number of cells at the S phase. Conclusions: The rs35592567 polymorphism in TP63 affected the expression of TP63 by interfering with its interaction with miR-140, and could serve as an explanation for the increased risk of GC.

Keywords

• Rs35592567
• TP63
• Gastric cancer
• MiR-140
• 3’-UTR