Fig. 6

CREB3 is a direct target of miR-203a-3p. a Clustered heatmap of significantly differentially expressed mRNAs in HOS transfected with sh-control and with sh-circTADA2A. Each sample was mixed with three replicates. b Significantly overexpressed genes are represented by red dots, and significant downregulated genes are represented by green dots in the volcano plot. c Schematic flowchart shows the overlapping of the downregulated mRNAs collection and target gene analyses of miR-203a-3p from TargetScan database. d & e The expression levels of 6 selected genes in HOS and 143B cells transfected with N.C. or sh-circTADA2A were determined by qRT-PCR. The histograms show relative mRNA levels. f Both HOS and 143B cells were transfected with siRNA of 6 selected genes. Apoptotic rates after 48 h were evaluated by Annexin V-FITC/PI staining. g H&E staining and immunohistochemistry (IHC) results of chondroma and OS tissues. CREB3, c-Jun, mmp9 and Bcl-2 expression was higher in human OS tissue than in chondroma tissue, while cleaved-caspase 3 expression is relatively lower in OS. Representative images are shown; scale bars, 100 μm. h qRT-PCR demonstrated the differential expression of CREB3 at the mRNA level between osteosarcoma and chondroma tissues (n = 10), (*P < 0.01, Student’s t-test). i Schematic illustration of the complementary sequence between miR-203a-3p and CREB3. Mutated nucleotides of CREB3 3’UTR are shown in lowercase letters. j HEK-293 cells were cotransfected with miR-203a-3p mimics (or N.C.) and a luciferase reporter construct containing wild-type (or mutated) CREB3 3′-UTRs. The luciferase activities of the cells were detected. k The protein levels of CREB3, c-Jun, Bcl-2, mmp9, mmp2, E-cadherin, N-cadherin and Vimentin in OS cells transfected with miR-203a-3p sponge (or N.C. sponge) or pre-mir-203a-3p (or N.C.) were respectively evaluated by Western blotting. l HOS and 143B cells were transfected with sh-circTADA2A or cotransfected with both sh-circTADA2A and the miR-203a-sponge. Western blotting was used to detect the ability of miR-203a-3p to rescue the expression of CREB3, c-Jun, Bcl-2, mmp9, mmp2, E-cadherin, N-cadherin and Vimentin at the protein level. Data are from three independent experiments (mean ± SEM) (d-f and j) or are representative of three independent experiments with similar results (g, k and l) (*P < 0.01 vs control or as indicated by Student’s t-test)