Molecular Basis of Gene-Gene Interaction: Cyclic Cross-Regulation of Gene Expression and Post-GWAS Gene-Gene Interaction Involved in Atrial Fibrillation
Fig 3
Expression of ZFHX3 is negatively regulated by miR-1.
HCT116 (A-C) and SW620 (D-F) cells were transfected with miR-1 mimics and negative control mimics (NC) and used for isolation of RNA samples for real-time RT-PCR analysis or for isolation of protein extracts for Western blot analysis for the expression levels of ZFHX3 mRNA and protein. A. Real-time RT-PCR analysis revealed that the miR-1 mimics reduced the expression of ZFHX3 by 20% in HCT116 cells (P = 0.004). B, C. Western blot analysis revealed that the miR-1 mimics reduced the expression of the ZFHX3 protein by 54% in HCT116 cells (P = 6.84×10-5). D. Real-time RT-PCR analysis revealed that the miR-1 mimics reduced the expression of ZFHX3 by 27% in SW620 cells (P = 0.01). E, F. Western blot analysis revealed that the miR-1 mimics reduced the expression of the ZFHX3 protein by 45% in SW620 cells (P = 4.887×10-4). G. Identification of two putative miR-1 binding sites at the 3’-UTR of ZFHX3 by bioinformatic analysis and alignment of miR-1 binding sequences across species. H. A schematic diagram shows luciferase reporters containing the potential miR-1 binding site or the related mutated site. I. MiR-1 targets the second miR-1 binding site to regulate expression of ZFHX3. Luciferase assays revealed that compared to negative control mimics, miR-1 mimics significantly reduced luciferase activities from pMIR-ZFHX3–3’-UTR-2, but not from pMIR-ZFHX3-3’-UTR-1. *P<0.05; **P<0.01.