Figure 4
miR-23a/b directly targets Tmem64. (a) Schematic representation of the predicted miR-23a/b target site in the 3′-UTR of mouse Tmem64. The alignment of miR-23a/b with WT and MUT 3′-UTR region is shown by complementary pairing, and three mutated nucleotides are underlined. (b) BMSCs were co-transfected with the luciferase reporter carrying WT-pGL3-Tmem64 or MUT-pGL3-Tmem64 along with agomiR-23a/b or agomiR-NC. The effects of miR-23a/b on the luciferase reporter constructs were determined 48 h after transfection. The firefly luciferase values were normalized to Renilla luciferase; n=5. (c) After BMSCs were transfected with agomiR-23a/b or antagomiR-23a/b, the relative levels of Tmem64 protein expression were determined by western blot; β-actin was used as loading control; n=5. (d) The relative levels of Tmem64 mRNA were determined using qRT-PCR and normalized to β-actin; n=5. (e) Tmem64 protein levels in BMSCs from 3- and 18-month-old mice were measured by western blot and expressed as the densitometry of Tmem64/β-actin. Tmem64 mRNA levels were determined by qRT-PCR and are shown as the fold-induction relative to β-actin; n=3. (f) The increase in ALP activity induced by agomiR-23a/b was blocked by the transfection of MUT Tmem64 3′-UTR into osteogenic-induced-BMSCs. *P<0.05 vs. agomiR-23a/b+WT Tmem64 3′-UTR. n=3. Data are shown as the mean±s.d. *P<0.05 (Student’s t-test or ANOVA). ALP, alkaline phosphatase; ANOVA, analysis of variance; BMSCs, bone marrow mesenchymal stem cells; CDS, coding sequence; qRT-PCR, quantitative reverse transcription PCR; WT, wild type; 3′-UTR, 3′-untranslated region.
