Downregulated Long Non-Coding RNA MSC-AS1 Inhibits Osteosarcoma Progression and Increases Sensitivity to Cisplatin by Binding to MicroRNA-142

Abstract

BACKGROUND Osteosarcoma (OS) is the most prevalent malignant primary bone tumor, resulting from severe transformation of primitive mesenchymal cells, which induces osteogenesis. Long non-coding RNA (lncRNA) MSC-AS1 triggers osteogenic differentiation by sponging microRNA (miR)-140-5p. The present study assessed the mechanism of lncRNA MSC-AS1 in OS biological features and sensitivity to cisplatin (DDP) by binding to miR-142. MATERIAL AND METHODS Firstly, lncRNA MSC-AS1 expression in OS tissues and cells was analyzed. OS cells were transfected with silenced MSC-AS1 to determine its role in OS biological behaviors, and we also assessed the effect of MSC-AS1 on OS sensitivity to DDP. Then, website prediction and dual-luciferase reporter gene assay were utilized for verification of the binding site between MSC-AS1 and miR-142. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were performed to determine the effect of MSC-AS1 on expression of miR-142, cyclin-dependent kinase 6 (CDK6), and the PI3K/AKT signaling pathway. Xenograft transplantation was also applied to confirm the in vitro experiments. RESULTS Overexpressed MSC-AS1 was associated with poor prognosis of OS patients. OS cell proliferation, invasion, and migration were reduced after silencing MSC-AS1, while cell apoptosis was enhanced. Moreover, silencing MSC-AS1 made OS cells more sensitive to DDP. Interestingly, MSC-AS1 knockdown induced miR-142 expression and reduced CDK6 levels, thereby decreasing the protein expression of p-PI3K/t-PI3K and p-AKT/t-AKT. Silencing MSC-AS1 repressed OS progression in vivo. CONCLUSIONS Our study demonstrated that silencing MSC-AS1 inhibited OS biological behaviors by enhancing miR-142 to decrease CDK6 and inactivating the PI3K/AKT axis. Our results may provide new insights for OS treatment.

Figure 1

lncRNA MSC-AS1 is upregulated in OS and is associated with poor prognosis. (A) RT-qPCR results showed that lncRNA MSC-AS1 expression was significantly higher in OS tissues than in adjacent normal bone tissues, n=45. (B) Relationship between lncRNA MSC-AS1 expression and prognosis of OS patients was analyzed by Kaplan-Meier assay, n=45. (C) lncRNA MSC-AS1 expression in human normal osteoblast hFOB1.19 cells and OS cell lines were detected by RT-qPCR. The experiments were performed 3 times; compared with normal group/hFOB1.19 cells, ** p<0.05. The independent-samples t test was used for statistical analysis of comparisons in (A), Kaplan-Meier assay was utilized to analyze (B), and one-way ANOVA and Tukey’s multiple comparisons test were applied to determine (C). lncRNA – long non-coding RNA; OS – osteosarcoma; RT-qPCR – reverse transcription-quantitative polymerase chain reaction; ANOVA – analysis of variance.

Figure 2

Silencing of lncRNA MSC-AS1 reduced OS cell proliferation, invasion, migration and EMT and increased OS cell apoptosis. (A) lncRNA MSC-AS1 expression in transfected OS cells MG63 and U2OS was assessed with RT-qPCR. (B) OS cell viability in all groups was detected by MTT assay. (C) Colony formation ability in OS cells from all groups was measured. (D) DNA replication in all groups was detected by EdU assay. (E) OS cell invasion in all groups was assessed with Transwell assay. (F) OS cell migration in all groups was verified by scratch test. (G) EMT-related protein expression in all groups was measured by Western blot analysis. (H) OS cell apoptosis in all groups was assessed with flow cytometry. Compared with control group, * p<0.05, ** p<0.01. The experiments were performed 3 times; one-way ANOVA and Tukey’s multiple comparisons test were applied to determine (A, C–F, H), and two-way ANOVA and Tukey’s multiple comparisons test were applied to determine (B, G). lncRNA – long non-coding RNA; EMT – epithelial-mesenchymal transition; OS – osteosarcoma; RT-qPCR – reverse transcription-quantitative polymerase chain reaction; MTT – 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; EdU – 5-ethynyl-2′-deoxyuridine; ANOVA – analysis of variance.

Figure 3

Silencing of lncRNA MSC-AS1 makes OS cells more sensitive to cisplatin. (A) lncRNA MSC-AS1 expression in transfected U2OS cells and U2OS/DDP cells was detected by RT-qPCR. (B) U2OS and U2OS/DDP cell viabilities in different DDP concentrations (0, 2, 4, 8, 16, 32, 64 ug/mL) were measured by MTT assay. (C) U2OS cell colony formation ability treated by 4 μg/mL DDP and U2OS/DDP cell colony formation ability treated by 8 μg/mL DDP were assessed by colony formation assay. (D) U2OS cell apoptosis treated with 4 μg/mL DDP and U2OS/DDP cell apoptosis treated with 8 μg/mL DDP was detected by flow cytometry. Compared with control group, * p<0.05, ** p<0.01, the experiments were performed 3 times, one-way ANOVA and the t test were applied to determine (A, C, D), and two-way ANOVA and Tukey’s multiple comparisons test were applied to determine (B). lncRNA – long non-coding RNA; OS – osteosarcoma; RT-qPCR – reverse transcription-quantitative polymerase chain reaction; MTT – 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; ANOVA – analysis of variance.

Figure 4

lncRNA MSC-AS1 binds to miR-142, thereby reducing miR-142-targeted inhibition of CDK6 and modulating PI3K/AKT signaling pathway activation. (A) A bioinformatics website was used to determine that lncRNA MSC-AS1 was mainly localized in the cytoplasm. (B) Nucleoplasmic separation assay indicated that lncRNA MSC-AS1 was mainly found in cytoplasm. (C) The bioinformatics website and dual-luciferase reporter gene assay helped to discover that both lncRNA MSC-AS1 and CDK6 were combined to miR-142 at some sites. (D) RNA pull-down assay showed the adsorptive effect of lncRNA MSC-AS1 on miR-142. (E) miR-142 expression in U2OS cells and U2OS/DDP cells, as well as mRNA expression of CDK6, were measured by RT-qPCR. (F) CDK6, p-PI3K, t-PI3K, p-AKT, and t-AKT expression in U2OS cells and U2OS/DDP cells were verified with Western blot analysis. Compared with the control group, ** p<0.01, the experiments were performed 3 times, one-way ANOVA and Tukey’s multiple comparisons test were applied to determine (D, E); two-way ANOVA and Tukey’s multiple comparisons test were applied to determine (B, F), two-way ANOVA and Sidak’s multiple comparisons test were applied to determine (C). lncRNA – long non-coding RNA; miR – microRNA; CDK6 – cyclin-dependent kinase 6; PI3K – phosphatidylinositol 3-kinase; RT-qPCR – reverse transcription-quantitative polymerase chain reaction; ANOVA – analysis of variance.

Figure 5

Silenced lncRNA MSC-AS1 inhibits OS cell in vivo. (A) Tumor volumes in all groups were calculated every 3 days using the formula V=L×W²×0.5. (B) On the 21^st day, the tumors were taken out and weighed. (C) Ki67-positive expression of tumors in each group was detected by immunohistochemistry. N=6 in each group, compared with the control group, * p<0.05, ** p<0.01. Two-way ANOVA and Tukey’s multiple comparisons test were applied to determine (A), and one-way ANOVA and Tukey’s multiple comparisons test were applied to determine (B, C). lncRNA – long non-coding RNA; OS – osteosarcoma; ANOVA – analysis of variance.