Figure 3.

miR-29a suppresses GSK3β expression by direct binding to its 3′UTR. (A) Sequence of hsa-miR-29a-3p and its putative binding sites at 3′UTR of human GSK3B and mouse Gsk3b mRNAs. CDS, coding sequence. Open rectangles represent the region spanning putative miR-29a-3p binding sites. Base-paired nucleotides between 3′UTR and miR-29a-3p are presented in the upper case. (B) A schematic representation of the plasmid for cytomegalovirus (CMV)-driven luciferase reporter assay. 3′-UTR of Gsk3b or Gsk3b mutant was chemically synthesized into the pMIR-REPORT^TM luciferase plasmid. Base-paired nucleotides are presented in upper case, while those in red denotes mismatched nucleotides. Plasmid was introduced into human hepatocyte cell line HepG2, followed by transfection of miR-29a mimic, miR negative control or no-treatment. (C) Suppression of reporter activities by miR-29a-3p overexpression in HepG2 cells. Cells were no treated (NT) or treated with miR negative control (CONT) or miR-29a mimic for 24h. Five independent experiments for each group were conducted. *p < 0.05 compared with CONT. #p < 0.05 between indicated groups. (D) Gsk3b mRNA level was determined by qRT-PCR. GSK3β protein abundance was measured by Western blot. Data collected from six to ten independent experiments per group are expressed as mean ± SE. * p < 0.05 between indicated groups. MCD, methionine/choline-deficient; MCS, methionine/choline-sufficient.