LncRNA MEG3 negatively modified osteosarcoma development through regulation of miR-361-5p and FoxM1.
This study was aimed to figure out whether long noncoding RNA MEG3/miR-361-5p/FoxM1 signaling would contribute to improved proliferation and metastasis of osteosarcoma cells. We altogether collected 204 pairs of osteosarcoma tissues and adjacent normal tissues, and obtained four human osteosarcoma cell lines. Then pcDNA3.1-MEG3, si-MEG3, miR-361-5p mimic, miR-361-5p inhibitor, pcDNA3.1-FoxM1, si-FoxM1, and negative control (NC) were, respectively, transfected into the osteosarcoma cells. Furthermore, real time polymerase chain reaction was utilized to determine the mRNA expressions of maternally expressed gene 3 (MEG3) and miR-361-5p, and western blot analysis was applied for determining the FoxM1 expression. Besides, dual luciferase reporter gene assay was adopted to verify if MEG3 can be directly targeted by miR-361-5p. Finally, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, colony formation assay, flow cytometry, wound healing assay, and transwell assay were conducted to investigate the influence of MEG3, miR-361-5p, and FoxM1 expressions on the viability, proliferation, apoptosis, migration, and invasion of osteosarcoma cells. MEG3 and miR-361-5p were observed to be significantly downregulated within both osteosarcoma tissues and cell lines, whereas FoxM1 was upregulated in osteosarcoma tissues and cell lines (p < 0.05). MEG3 directly bound to miR-361-5p, and significantly upgraded its expression (p < 0.05). The upregulated MEG3 and miR-361-5p or the downregulated FoxM1 appeared to substantially inhibit proliferation, migration, and invasion of osteosarcoma cells (p < 0.05). Finally, the proliferation, migration, invasion, and motility of osteosarcoma cells within the miR-NC + pcDNA3.1-FoxM1 group and pcDNA + pcDNA-FoxM1 group were markedly promoted when compared with the miR-361-5p mimic group and pcDNA3.1-MEG3 group (p < 0.05). The MEG3/miR-361-5p/FoxM1 axis could potentially serve as therapeutic targets or diagnostic biomarkers for osteosarcoma.
Shen B
，Zhou N
，Hu T
，Zhao W
，Wu D
，Wang S
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Up-regulation of microRNA-491-5p suppresses cell proliferation and promotes apoptosis by targeting FOXP4 in human osteosarcoma.
MicroRNAs are small non-coding RNAs involved in pathogenesis and progression of human malignancies. MicroRNA-491-5p (miR-491-5p) is down-regulated in many human cancers where it would serve as a tumour suppressor. However, the role played by miR-491-5p in pathogenesis of human osteosarcoma has remained largely unknown. This study has been conducted to examine effects of miR-491-5p on migration and proliferation of cells of the SAOS-2 and MG63 osteosarcoma lines, and mechanisms of those effects.
Levels of miR-491-5p expression in osteosarcoma tissues and in human osteosarcoma cell lines were studied using qualitative real-time polymerase chain reaction (qRT-PCR) methods. Cell viability was detected using the CCK-8 and EdU assays, while the transwell assay was used to evaluate migration and invasion. Apoptosis was analysed uing flow cytometry and the Hoechst 33342 nuclear staining method. A dual-luciferase reporter system was used to confirm the target gene of miR-491-5p. The electrophoretic mobility shift assay (EMSA) with DIG-labelled double-stranded FOXP4 oligonucleotides was used to confirm whether or not miR-491-5p suppressed FOXP4 activation.
Cells of osteosarcoma tissues and cell lines had low levels of miR-491-5p expression, but high levels of forkhead-box P4 (FOXP4) expression. Transfection of SAOS-2 and MG63 cells with miR-491-5p mimics inhibited expression of FOXP4 protein, which suppressed cell growth and migration, but induced apoptosis. Dual-luciferase reporter assays confirmed FOXP4 as the target gene for miR-491-5p. Overexpression of miR-491-5p suppressed FOXP4 activity in SAOS-2 and MG63 cells. Knockdown of FOXP4 in SAOS-2 and MG63 cells using an RNAi strategy resulted in reduced levels of cell proliferation and migration, but increased levels of apoptosis.
Our in vitro studies showed that up-regulation of miR-491-5p suppressed proliferation of the human osteosarcoma cells and induced apoptosis by targeting FOXP4. These findings suggest that miR-491-5p could be further studied as a potential clinical diagnostic or predictive biomarker for human osteosarcoma.
Yin Z
，Ding H
，He E
，Chen J
，Li M
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MiR-216b inhibits osteosarcoma cell proliferation, migration, and invasion by targeting Forkhead Box M1.
Osteosarcoma (OS) is considered the most common type of primary malignant bone tumor, which has a high rate of mortality in children and adolescents. However, the current treatment methods for OS are ineffective. Therefore, there is an urgent requirement to identify the critical targets. This study aimed to identify the roles and significance of microRNA-216b (miR-216b) in OS. To explore the cellular and molecular functions of miR-216b and Forkhead Box M1 (FoxM1) in OS, the expression of miR-216b and FoxM1 at the transcriptional level was measured using quantitative real-time PCR (qRT-PCR). Wound healing assay, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay (MTT) assay, flow cytometry, and transwell invasion assay were conducted to study the function of miR-216b and FoxM1 in OS cells. Dual luciferase reporter assay was performed to identify the relationships between miR-216b and FoxM1. qRT-PCR results revealed that miR-216b expression was significantly downregulated, and FoxM1 was observed to be significantly upregulated in human OS cell lines (MG-63) and tissues. MTT data showed that upregulation of miR-216b expression led to cell growth inhibition in MG-63 cells. The results of the invasion assay and wound healing assay illustrated that miR-216b upregulation or FoxM1 downregulation could inhibit the invasion and migration in MG-63 cells. In vivo, the tumor volume was significantly decreased by miR-194 mimic treatment compared with the control group. Furthermore, the results of the luciferase assay indicated that FoxM1 is a direct target of miR-216b. These findings may provide novel insights into the molecular mechanism of miR-216b and FoxM1 in the progression of OS, and suggested that miR-216b may serve as a potential tumor inhibitor of OS by targeting FoxM1.
Wang W
，Guo Z
，Yu H
，Fan L
... -
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ResearchGate
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钛学术
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