Research Paper Volume 13, Issue 10 pp 14258—14276

LncRNA LOC100129620 promotes osteosarcoma progression through regulating CDK6 expression, tumor angiogenesis, and macrophage polarization

LncRNA LOC100129620 regulates the function of miR-335-3p in osteosarcoma cells. (A) MiRDB software predicted the miRNAs that could bind to LOC100129620 and the position of the binding sites. (B) MS2bs RNA pull-down assay to detect the miRNAs that bind to LOC100129620. (C) MS2bs RNA pull-down assay to detect the combination of miRNA with wild-type or mutant LOC100129620. (D) MiR-335-3p pull-down assay to detect the binding of miR-335-3p with LOC100129620. (E) Schematic diagram of the wild-type and mutated binding sites of LOC100129620 with miR-335-3p. (F) The binding of LOC100129620 and miR-335-3p as detected by luciferase reporter gene assay. (G) CCK-8 assay to detect the viability of U2OS cells treated with miR-335-3p mimics or miR-335-3p inhibitors. (H) CCK-8 assay to detect the viability of MG63 cells treated with miR-335-3p mimics or miR-335-3p inhibitors. (I) Colony formation assay to detect the viability of U2OS cells treated with miR-335-3p mimics or miR-335-3p inhibitors. (J) Quantitative analysis of colony formation assay results of U2OS cells treated with miR-335-3p mimics or miR-335-3p inhibitors. (K) Cell scratch assay to detect the migration ability of U2OS cells treated with miR-335-3p mimics or miR-335-3p inhibitors. (L) Transwell assay to detect the invasion ability of U2OS cells treated with miR-335-3p mimics or miR-335-3p inhibitors. (M) Quantitative analysis of the Transwell assay results of U2OS cells treated with miR-335-3p mimics or miR-335-3p inhibitors. (N) Transwell assay to detect the effect of LOC100129620 on the invasion ability of U2OS cells treated with miR-335-3p mimics or miR-335-3p inhibitors. (O) Quantitative analysis to detect the effect of LOC100129620 on the invasion ability of U2OS cells treated with miR-335-3p mimics or miR-335-3p inhibitors. (P) Transwell assay to detect the effects of wild-type LOC100129620 and miR-335-3p binding site mutant LOC100129620 on the invasion ability of U2OS cells. (Q) Quantitative analysis of the Transwell assay results to detect the effects of wild-type LOC100129620 and miR-335-3p binding site mutant LOC100129620 on the invasion ability of U2OS cells. Statistical analysis was conducted using Student’s t-test. Values are means ± SD. *P P

Figure 5. LncRNA LOC100129620 regulates the function of miR-335-3p in osteosarcoma cells. (A) MiRDB software predicted the miRNAs that could bind to LOC100129620 and the position of the binding sites. (B) MS2bs RNA pull-down assay to detect the miRNAs that bind to LOC100129620. (C) MS2bs RNA pull-down assay to detect the combination of miRNA with wild-type or mutant LOC100129620. (D) MiR-335-3p pull-down assay to detect the binding of miR-335-3p with LOC100129620. (E) Schematic diagram of the wild-type and mutated binding sites of LOC100129620 with miR-335-3p. (F) The binding of LOC100129620 and miR-335-3p as detected by luciferase reporter gene assay. (G) CCK-8 assay to detect the viability of U2OS cells treated with miR-335-3p mimics or miR-335-3p inhibitors. (H) CCK-8 assay to detect the viability of MG63 cells treated with miR-335-3p mimics or miR-335-3p inhibitors. (I) Colony formation assay to detect the viability of U2OS cells treated with miR-335-3p mimics or miR-335-3p inhibitors. (J) Quantitative analysis of colony formation assay results of U2OS cells treated with miR-335-3p mimics or miR-335-3p inhibitors. (K) Cell scratch assay to detect the migration ability of U2OS cells treated with miR-335-3p mimics or miR-335-3p inhibitors. (L) Transwell assay to detect the invasion ability of U2OS cells treated with miR-335-3p mimics or miR-335-3p inhibitors. (M) Quantitative analysis of the Transwell assay results of U2OS cells treated with miR-335-3p mimics or miR-335-3p inhibitors. (N) Transwell assay to detect the effect of LOC100129620 on the invasion ability of U2OS cells treated with miR-335-3p mimics or miR-335-3p inhibitors. (O) Quantitative analysis to detect the effect of LOC100129620 on the invasion ability of U2OS cells treated with miR-335-3p mimics or miR-335-3p inhibitors. (P) Transwell assay to detect the effects of wild-type LOC100129620 and miR-335-3p binding site mutant LOC100129620 on the invasion ability of U2OS cells. (Q) Quantitative analysis of the Transwell assay results to detect the effects of wild-type LOC100129620 and miR-335-3p binding site mutant LOC100129620 on the invasion ability of U2OS cells. Statistical analysis was conducted using Student’s t-test. Values are means ± SD. *P < 0.05 and **P < 0.01.