Figure 1
Long non-coding RNA TUG1 promoted hFOB1.19 cell proliferation. A-C, TUG1 was overexpressed or knocked down in hFOB1.19 cells after introduction of TUG1 or si-TUG1. A, The expression of TUG1 was determined by qRT-PCR. B, Cell viability was detected by CCK-8 assay. C, The expression of PCNA was measured by western blot assay. Data are reported as means±SD of three independent experiments. *P<0.05 (Student's t-test).
Figure 2
TUG1 and CNR2 were upregulated, while miR-545-3p was down-regulated in osteogenic differentiated hFOB1.19 cells. The hFOB1.19 cells were incubated in osteogenic medium (OM) for 0, 1, 4, 7, 14, or 21 d. A and B, TUG1 and miR-545-3p expression was detected by qRT-PCR. C, CNR2 protein level was examined by western blot assay. Data are reported as means±SD of three independent experiments. *P<0.05 (ANOVA).
Figure 3
ALP activity and marker expression were increased during osteogenesis. A, The activity of ALP was determined in hFOB1.19 cells cultured with growth medium (GM) or osteogenic medium (OM) (14d). B, The expression of osteogenic differentiation markers was determined by western blot assay. Data are reported as means±SD of three independent experiments. *P<0.05 (t-test).
Figure 4
TUG1 facilitated hFOB1.19 cell differentiation. A, The activity of ALP in osteogenic differentiated hFOB1.19 cells was detected after TUG1 was upregulated or down-regulated. B, The protein levels of ALP, Runx2, osteocalcin (OCN), and osteopontin (OPN) in osteogenic differentiated hFOB1.19 cells were measured after transfection with TUG1 or si-TUG1. Data are reported as means±SD of three independent experiments. *P<0.05 (t-test).
Figure 5
TUG1 directly targeted miR-545-3p. A, The predicted binding sites between TUG1 and miR-545-3p are shown. B, Luciferase activity was detected in hFOB1.19 cells co-transfected with TUG1-wt (wild type) or TUG1-mut (mutated) and miR-545-3p or negative control (NC). C, RIP assay was used to measure the enrichment of TUG1 and miR-545-3p in the immunoprecipitation complex. D, Expression of miR-545-3p was examined in hFOB1.19 cells transfected with vector, TUG1, si-NC, or si-TUG1. Data are reported as means±SD of three independent experiments. *P<0.05 (t-test).
Figure 6
TUG1 restored the effect of miR-545-3p on osteogenic differentiation. A, Expression of miR-545-3p was measured in hFOB1.19 cells transfected with negative control (NC), miR-545-3p, vector+miR-545-3p, or TUG1+miR-545-3p. Also, miR-545-3p expression was tested in hFOB1.19 cells transfected with anti-NC, anti-miR-545-3p, si-NC+anti-miR-545-3p, or si-TUG1+anti-miR-545-3p. B, The activity of ALP was detected in osteogenic differentiated hFOB1.19 cells following miR-545-3p upregulation and/or TUG1 upregulation. C, ALP activity was examined in osteogenic differentiated hFOB1.19 cells after miR-545-3p down-regulation and/or TUG1 down-regulation. D, Protein levels of ALP, Runx2, osteocalcin (OCN), and osteopontin (OPN) were analyzed in osteogenic differentiated hFOB1.19 cells transfected with miR-545-3p and/or TUG1. E, The levels of ALP, Runx2, OCN, and OPN were tested in osteogenic differentiated hFOB1.19 cells introduced with anti-miR-545-3p and/or si-TUG1 by western blot assay. Data are reported as means±SD of three independent experiments. *P<0.05 (ANOVA).
Figure 7
CNR2 was a target of miR-545-3p. A, The putative binding sites for miR-545-3p and CNR2 3'UTR are shown. B, Dual-luciferase reporter assay was conducted to confirm the relationship between miR-545-3p and CNR2. C, RIP assay was utilized to validate the correlation between miR-545-3p and CNR2. D, The protein expression of CNR2 was examined in hFOB1.19 cells transfected with negative control (NC), miR-545-3p, anti-NC, or anti-miR-545-3p. E, After transfection with vector, TUG1, si-NC, or si-TUG1, CNR2 expression was tested by western blot assay. Data are reported as means±SD of three independent experiments. *P<0.05 (t-test).
Figure 8
MiR-545-3p inversed the effect of CNR2 on osteogenic differentiation. A, The protein level of CNR2 was measured in hFOB1.19 cells transfected with si-NC (negative control), si-CNR2, anti-NC+si-CNR2, or anti-miR-545-3p+si-CNR2. Additionally, CNR2 expression was detected in hFOB1.19 cells introduced with vector, CNR2, NC+CNR2, or miR-545-3p+CNR2. B, ALP activity was determined in osteogenic differentiated hFOB1.19 cells transfected with si-CNR2 and/or anti-miR-545-3p. C, After introduction with CNR2 and/or miR-545-3p, ALP activity was evaluated in osteogenic differentiated hFOB1.19 cells. D, Protein levels of ALP, Runx2, osteocalcin (OCN), and osteopontin (OPN) were measured in osteogenic differentiated hFOB1.19 cells following CNR2 down-regulation and/or miR-545-3p down-regulation. E, Expression of ALP, Runx2, OCN, and OPN was detected in osteogenic differentiated hFOB1.19 cells following CNR2 overexpression and/or miR-545-3p overexpression. Data are reported as means±SD of three independent experiments. *P<0.05 (ANOVA).