Figures & data
Figure 1. Wnt7b expression is upregulated in osteosarcoma and promotes OS cell proliferation
(a) Wnt7b expression in a total of 6 paired normal and OS tissues, as well as in normal HOB and OS U2OS and MG63 cell lines determined by real-time PCR. (b) Wnt7b protein levels in a total of 6 paired normal and OS tissues, as well as in normal HOB and OS U2OS and MG63 cell lines determined by Immunoblotting. (c–d) U2OS and MG63 cells were transfected with si-NC or si-Wnt7b. The interfering efficiency of si-Wnt7b in U2OS and MG63 cells was determined by RT-PCR (c) and Immunoblotting (d). (e–f) The cell proliferation was determined by MTT assays. **P < 0.01.
Figure 2. miR-342-5p binds to the 3′-UTR of Wnt7b to inhibit its expression
(a) A schematic diagram showing the process of selecting miRNAs that may target Wnt7b. (b) The correlation of miR-342-5p and Wnt7b expression based on GSE70415. (c) U2OS and MG63 cells were transfected with miR-342-5p or anti-miR-342-5p to conduct miR-342-5p overexpression or miR-342-5p inhibition, as confirmed by real-time PCR. (d) Wnt7b expression in miR-342-5p-overexpressed or miR-342-5p-inhibited U2OS and MG63 cells were determined by real-time PCR. (e) Wild and mutant types of Wnt7b 3′-UTR luciferase reporter vectors containing wild or mutated miR-342-5p binding site were constructed and co-transfected in 293T cells with miR-342-5p or anti-miR-342-5p. The luciferase activity was determined. **P < 0.01, compared to miR-NC group; ##P < 0.01, compared to anti-NC group.
Figure 3. Effects of miR-342-5p on OS cells and Wnt7b
OS cell lines, U2OS and MG63, were transfected with miR-342-5p and examined for (a) DNA synthesis ability by EdU assay; (b) cell invasion by invasion assay; (c) cell migration by Wound healing assay; (D) cell apoptosis by Flow cytometry; (e) Doxorubicin inhibitory effects on cell viability MTT assay; (f) the protein levels of Wnt7b, β-catenin, c-myc, cyclin D1, and E-cadherin by Immunoblotting. *P < 0.05, **P < 0.01.
Figure 4. Dynamic effects of miR-342-5p and Wnt7b on OS cells
OS cell lines, U2OS and MG63, were co-transfected with anti-miR-342-5p and si-Wnt7b and examined for (a) Wnt7b expression by real-time PCR; (b) the protein levels of Wnt7b, β-catenin, c-myc, cyclin D1, and E-cadherin by Immunoblotting; (c) DNA synthesis ability by EdU assay; (d) cell invasion by Invasion assay. *P < 0.05, **P < 0.01, compared to control group; #P < 0.05, ##P < 0.01, compared to anti-NC + si-Wnt7b group.
Figure 5. Dynamic effects of miR-342-5p and Wnt7b on OS cell migration, apoptosis, and sensitivity to Doxorubicin
OS cells were co-transfected with anti-miR-342-5p and si-Wnt7b and examined for (a) cell migration by Wound healing assay; (b) cell apoptosis by Flow cytometry; (c) Doxorubicin inhibitory effects on cell viability MTT assay. *P < 0.05, **P < 0.01, compared to control group; ##P < 0.01, compared to anti-NC + si-Wnt7b group.
Supplemental material
