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MicroRNA‑20a promotes the proliferation and cell cycle of human osteosarcoma cells by suppressing early growth response 2 expression.

Zhuo W, Ge W, Meng G, Jia S, Zhou X, Liu J - Mol Med Rep (2015)

Bottom Line: It was determined that miR‑20a expression is markedly upregulated in OS tissues and cells compared with the matched adjacent normal tissues and h‑FOB human osteoblast cell lines.Data from luciferase reporter assays showed that miR‑20a directly binds to the 3'‑untranslated region (3'‑UTR) of EGR2 mRNA and represses expression at the transcriptional and translational levels.In conclusion, the data provide compelling evidence that miR‑20a functions as an onco‑miRNA, which is important in promoting cell proliferation in OS, and its oncogenic effect is mediated primarily through direct suppression of EGR2 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedics and Traumatology, Institute of Orthopedics and Traumatology, Xijing Hospital, The Fourth Military Medical University, Xi'an, Shanxi 710000, P.R. China.

ABSTRACT
MicroRNAs (miRNAs) are crucial in cancer development. However, the underlying mechanisms of miRNAs in osteosarcoma (OS) remain largely uncharacterized. The present study investigated the role of miR‑20a in OS cell proliferation. It was determined that miR‑20a expression is markedly upregulated in OS tissues and cells compared with the matched adjacent normal tissues and h‑FOB human osteoblast cell lines. Ectopic expression of miR‑20a promoted the proliferation and anchorage‑independent growth of OS cells, whereas inhibition of miR‑20a reduced this effect. Bioinformatics analysis further revealed early growth response 2 (EGR2), as a potential target of miR‑20a. Data from luciferase reporter assays showed that miR‑20a directly binds to the 3'‑untranslated region (3'‑UTR) of EGR2 mRNA and represses expression at the transcriptional and translational levels. In functional assays, miR‑20a promoted OS cell proliferation and the cell cycle, which could be suppressed by an inhibitor of miR‑20a. In conclusion, the data provide compelling evidence that miR‑20a functions as an onco‑miRNA, which is important in promoting cell proliferation in OS, and its oncogenic effect is mediated primarily through direct suppression of EGR2 expression.

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miR-20a downregulation inhibited OS cell proliferation. (A) Validation of miR-20a expression levels after transfection by PCR analysis. (B) MTT assays revealed that downregulation of miR-20a inhibited the growth of Saos-2 OS cells. (C) Representative quantification of crystal violet-stained cell colonies. (D) Cell cycle distribution in cells with miR-20a-in transfection. Each bar represents the mean of three independent experiments. *P<0.05, compared with NC. miR, microRNA; OS, osteosarcoma; PCR, polymerase chain reaction.
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f3-mmr-12-04-4989: miR-20a downregulation inhibited OS cell proliferation. (A) Validation of miR-20a expression levels after transfection by PCR analysis. (B) MTT assays revealed that downregulation of miR-20a inhibited the growth of Saos-2 OS cells. (C) Representative quantification of crystal violet-stained cell colonies. (D) Cell cycle distribution in cells with miR-20a-in transfection. Each bar represents the mean of three independent experiments. *P<0.05, compared with NC. miR, microRNA; OS, osteosarcoma; PCR, polymerase chain reaction.

Mentions: As miR-20a was significantly upregulated in OS tissues and OS cell lines, it was investigated whether miR-20a promotes cell proliferation and the cell cycle of OS cells (Figs. 2 and 3). Saos-2 cells were transfected with miR-20a mimics, miR-20a inhibitor or the respective controls, both of them showed great transfection efficiency (Figs. 2A and 3A). The MTT assay showed that the cell growth rate was significantly higher in miR-20a-transduced Saos-2 cells than in miR-NC-transfected cells (Fig. 2B). Colony formation assays consistently showed that enforced expression of miR-20a significantly enhanced the number of Saos-2 cell colonies after 14 days of culture compared with the controls (Fig. 2C). By contrast, the cell growth rates and colony numbers of Saos-2 cells transfected with miR-20a-in were significantly lower than those transfected with NC (Fig. 3B and C). To determine whether modulating cell viability was a result of the cell cycle, flow cytometry was used. After 48 h transfection, miR-20a mimics decreased the proportion of Saos-2 cells in the G0/G1-phase and increased the proportion of Saos-2 cells in the S-phase compared with those transfected with NC (Fig. 2D). However, miR-20a-in increased the proportion of Saos-2 cells in the G0/G1-phase and decreased the proportion of Saos-2 cells in S-phase compared with those transfected with NC (Fig. 3D). These results showed that miR-20a promoted OS cell tumorigenicity in vitro.


MicroRNA‑20a promotes the proliferation and cell cycle of human osteosarcoma cells by suppressing early growth response 2 expression.

Zhuo W, Ge W, Meng G, Jia S, Zhou X, Liu J - Mol Med Rep (2015)

miR-20a downregulation inhibited OS cell proliferation. (A) Validation of miR-20a expression levels after transfection by PCR analysis. (B) MTT assays revealed that downregulation of miR-20a inhibited the growth of Saos-2 OS cells. (C) Representative quantification of crystal violet-stained cell colonies. (D) Cell cycle distribution in cells with miR-20a-in transfection. Each bar represents the mean of three independent experiments. *P<0.05, compared with NC. miR, microRNA; OS, osteosarcoma; PCR, polymerase chain reaction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581803&req=5

f3-mmr-12-04-4989: miR-20a downregulation inhibited OS cell proliferation. (A) Validation of miR-20a expression levels after transfection by PCR analysis. (B) MTT assays revealed that downregulation of miR-20a inhibited the growth of Saos-2 OS cells. (C) Representative quantification of crystal violet-stained cell colonies. (D) Cell cycle distribution in cells with miR-20a-in transfection. Each bar represents the mean of three independent experiments. *P<0.05, compared with NC. miR, microRNA; OS, osteosarcoma; PCR, polymerase chain reaction.
Mentions: As miR-20a was significantly upregulated in OS tissues and OS cell lines, it was investigated whether miR-20a promotes cell proliferation and the cell cycle of OS cells (Figs. 2 and 3). Saos-2 cells were transfected with miR-20a mimics, miR-20a inhibitor or the respective controls, both of them showed great transfection efficiency (Figs. 2A and 3A). The MTT assay showed that the cell growth rate was significantly higher in miR-20a-transduced Saos-2 cells than in miR-NC-transfected cells (Fig. 2B). Colony formation assays consistently showed that enforced expression of miR-20a significantly enhanced the number of Saos-2 cell colonies after 14 days of culture compared with the controls (Fig. 2C). By contrast, the cell growth rates and colony numbers of Saos-2 cells transfected with miR-20a-in were significantly lower than those transfected with NC (Fig. 3B and C). To determine whether modulating cell viability was a result of the cell cycle, flow cytometry was used. After 48 h transfection, miR-20a mimics decreased the proportion of Saos-2 cells in the G0/G1-phase and increased the proportion of Saos-2 cells in the S-phase compared with those transfected with NC (Fig. 2D). However, miR-20a-in increased the proportion of Saos-2 cells in the G0/G1-phase and decreased the proportion of Saos-2 cells in S-phase compared with those transfected with NC (Fig. 3D). These results showed that miR-20a promoted OS cell tumorigenicity in vitro.

Bottom Line: It was determined that miR‑20a expression is markedly upregulated in OS tissues and cells compared with the matched adjacent normal tissues and h‑FOB human osteoblast cell lines.Data from luciferase reporter assays showed that miR‑20a directly binds to the 3'‑untranslated region (3'‑UTR) of EGR2 mRNA and represses expression at the transcriptional and translational levels.In conclusion, the data provide compelling evidence that miR‑20a functions as an onco‑miRNA, which is important in promoting cell proliferation in OS, and its oncogenic effect is mediated primarily through direct suppression of EGR2 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedics and Traumatology, Institute of Orthopedics and Traumatology, Xijing Hospital, The Fourth Military Medical University, Xi'an, Shanxi 710000, P.R. China.

ABSTRACT
MicroRNAs (miRNAs) are crucial in cancer development. However, the underlying mechanisms of miRNAs in osteosarcoma (OS) remain largely uncharacterized. The present study investigated the role of miR‑20a in OS cell proliferation. It was determined that miR‑20a expression is markedly upregulated in OS tissues and cells compared with the matched adjacent normal tissues and h‑FOB human osteoblast cell lines. Ectopic expression of miR‑20a promoted the proliferation and anchorage‑independent growth of OS cells, whereas inhibition of miR‑20a reduced this effect. Bioinformatics analysis further revealed early growth response 2 (EGR2), as a potential target of miR‑20a. Data from luciferase reporter assays showed that miR‑20a directly binds to the 3'‑untranslated region (3'‑UTR) of EGR2 mRNA and represses expression at the transcriptional and translational levels. In functional assays, miR‑20a promoted OS cell proliferation and the cell cycle, which could be suppressed by an inhibitor of miR‑20a. In conclusion, the data provide compelling evidence that miR‑20a functions as an onco‑miRNA, which is important in promoting cell proliferation in OS, and its oncogenic effect is mediated primarily through direct suppression of EGR2 expression.

Show MeSH
Related in: MedlinePlus