Figure 2

miR-372 and miR-373-mediated regulation of large tumor suppressor homolog 2 (LATS2) translation and AGS cell proliferation. (A) Schematic representation of the pGL3 vector (control) and the pGL3-LATS2 sensor containing the 3' untranslated region (UTR) of the LATS2 human gene, which harbors two predicted miR-372 and miR-373 target sequences [36]. Also shown is the predicted base pairing formed between miR-372 or miR-373 with each of the LATS2 3'UTR binding sites as predicted by http://pictar.mdc-berlin.de. Seed regions of miR-372 and miR-373 are underlined. (B) LATS2 levels and mature miR-372 and miR-373 expression in AGS, MKN-74 and HeLa cells were determined by immunoblot and northern blot, respectively. α-Tubulin and U6 small nuclear RNA (snRNA) were used as loading controls. (C) The pGL3 and pGL3-LATS2 vectors were transfected into either parental AGS (mock), or cells treated with 100 nM of as372-373 or the sc372-373 control oligonucleotides. Transfection efficiency was assessed with the pRL-SV40 vector. Luciferase activities were measured 48 h post transfection. Bars indicate the relative firefly luciferase activity normalized to the Renilla activity and compared to pGL3. Data are mean ± SD from three independent experiments. *P < 0.05; **P < 0.01. (D) The DNA synthesis rate was measured by 5-bromo-2'-deoxyuridine (BrdU) incorporation (10 nM for 1 h) in untreated (mock) AGS cells or cells treated for 48 h with 100 nM as372-373 or sc372-373 or transfected with the pCMVmyc-LATS2 vector. Bars represent the relative BrdU incorporation compared to that of untreated cells. Data are mean ± SD of three independent experiments. **P < 0.01.