Research Paper Volume 13, Issue 4 pp 5297—5311

LncRNA MIR205HG regulates melanomagenesis via the miR-299-3p/VEGFA axis

VEGFA is the direct target of miR-299-3p. (A) The predicted binding site for miR-299-3p in the 3'UTR of the VEGFA transcript between nucleotides 359-366. (B) Dual luciferase reporter assay results show the luciferase activity in A375 cells co-transfected with the plasmid containing wild-type (WT) or mutant (MT) VEGFA 3′-UTR and miR-299-3p. (C) QRT-PCR analysis shows the expression levels of VEGFA mRNA in control, 5 nM miR-299-3p mimics-transfected, 10 nM miR-299-3p mimics-transfected and 20 nM miR-299-3p mimics-transfected A375 cells. (D) QRT-PCR analysis shows the expression levels of VEGFA mRNA in control, MIR205HG shRNA2-transfected, MIR205HG shRNA2 plus miR-299-3p inhibitor transfected A375 cells. (E, F) Representative western blots show the levels of VEGFA, cleaved caspase3, E-cadherin, α-SMA, and Vimentin proteins in control, MIR205HG shRNA2-transfected, MIR205HG shRNA2 plus miR-299-3p inhibitor transfected A375 cells. The relative expression of these proteins was estimated using β-actin as the loading control. Note: All experiments were performed thrice. **P##P

Figure 5. VEGFA is the direct target of miR-299-3p. (A) The predicted binding site for miR-299-3p in the 3'UTR of the VEGFA transcript between nucleotides 359-366. (B) Dual luciferase reporter assay results show the luciferase activity in A375 cells co-transfected with the plasmid containing wild-type (WT) or mutant (MT) VEGFA 3′-UTR and miR-299-3p. (C) QRT-PCR analysis shows the expression levels of VEGFA mRNA in control, 5 nM miR-299-3p mimics-transfected, 10 nM miR-299-3p mimics-transfected and 20 nM miR-299-3p mimics-transfected A375 cells. (D) QRT-PCR analysis shows the expression levels of VEGFA mRNA in control, MIR205HG shRNA2-transfected, MIR205HG shRNA2 plus miR-299-3p inhibitor transfected A375 cells. (E, F) Representative western blots show the levels of VEGFA, cleaved caspase3, E-cadherin, α-SMA, and Vimentin proteins in control, MIR205HG shRNA2-transfected, MIR205HG shRNA2 plus miR-299-3p inhibitor transfected A375 cells. The relative expression of these proteins was estimated using β-actin as the loading control. Note: All experiments were performed thrice. **P<0.01 vs. control; ##P<0.01 vs. MIR205HG knockdown A375 cells.