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MicroRNA-34b/c suppresses uveal melanoma cell proliferation and migration through multiple targets.

Dong F, Lou D - Mol. Vis. (2012)

Bottom Line: Recent studies have revealed that miRNAs function as tumor suppressors or oncogenes.In addition, the effect of miR-34b/c on c-Met, cell cycle-related proteins, v-akt murine thymoma viral oncogene homolog (Akt) and extracellular signal-regulated kinase (ERK) pathway was determined by western blotting. miR-34b/c expression, which was dramatically decreased in uveal melanoma cells and clinical samples, can be upregulated by doxorubicin and epigenetic drugs.Furthermore, miR-34b/c was confirmed to downregulate the expression of c-Met, p-Akt, and cell cycle-related proteins by western blotting.

View Article: PubMed Central - PubMed

Affiliation: The First Affiliated Hospital of College of Medicine, Zhejiang University, Hangzhou, China.

ABSTRACT

Purpose: MicroRNAs (miRNAs) are endogenously expressed, small noncoding RNAs that inhibit gene expression by binding to target mRNAs. Recent studies have revealed that miRNAs function as tumor suppressors or oncogenes. In the present study, we investigated the role of miRNA-34b/c in uveal melanoma.

Methods: Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed to detect the expression level of miR-34b/c in uveal melanoma cells and primary samples. Subsequently, uveal melanoma cell proliferation was examined by the MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl] -2H-tetrazolium, inner salt) assay, clone formation assay, and flow cytometry. Cell apoptosis was measured by caspase3/7 assay. Cell migration was evaluated by transwell migration assay. The target of miR-34b/c was predicted by bioinformatics and validated by luciferase assay. In addition, the effect of miR-34b/c on c-Met, cell cycle-related proteins, v-akt murine thymoma viral oncogene homolog (Akt) and extracellular signal-regulated kinase (ERK) pathway was determined by western blotting.

Results: miR-34b/c expression, which was dramatically decreased in uveal melanoma cells and clinical samples, can be upregulated by doxorubicin and epigenetic drugs. The transfection of miR-34b/c into uveal melanoma cells leads to a significant reduction in cell growth and migration. miR-34b/c caused cell cycle G(1) arrest rather than the induction of apoptosis. Met proto-oncogene (c-Met) was identified as a target of miR-34b/c in uveal melanoma cells. Furthermore, miR-34b/c was confirmed to downregulate the expression of c-Met, p-Akt, and cell cycle-related proteins by western blotting.

Conclusions: Our results demonstrate that both miR-34b and miR-34c act as tumor suppressors in uveal melanoma cell proliferation and migration through the downregulation of multiple targets.

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Related in: MedlinePlus

MicroRNA-34b/c was upregulated by doxorubicin and epigenetic drugs. A: Real time reverse transcriptase (RT)-PCR analysis was performed to detect the expression of miR-34b/c in uveal melanoma cell line SP6.5, after treatment with DOX for 24 h and 48 h. B: SP6.5 cells were treated with 5-aza-dC at 1 μM or 5 μM alone, TSA (100 ng/ml) alone, or combinations of both. miR-34b/c expression level was determined by Real-time RT–PCR. The value for miR-34b/c in SP6.5 cells without any treatment was set at 1, and the relative amounts of miR-34b/c in cells treated with drugs were shown as fold induction. U6 snRNA was used as an internal control.
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f2: MicroRNA-34b/c was upregulated by doxorubicin and epigenetic drugs. A: Real time reverse transcriptase (RT)-PCR analysis was performed to detect the expression of miR-34b/c in uveal melanoma cell line SP6.5, after treatment with DOX for 24 h and 48 h. B: SP6.5 cells were treated with 5-aza-dC at 1 μM or 5 μM alone, TSA (100 ng/ml) alone, or combinations of both. miR-34b/c expression level was determined by Real-time RT–PCR. The value for miR-34b/c in SP6.5 cells without any treatment was set at 1, and the relative amounts of miR-34b/c in cells treated with drugs were shown as fold induction. U6 snRNA was used as an internal control.

Mentions: Treatment of tumor cells with DOX (doxorubicin) or inhibitors of DNA methyltransferases and/or histone deacetylase suppresses cell growth by activating multiple tumor suppressor genes [25-27]. We detected the expression levels of miR-34b/c in SP6.5 cells treated with either DOX, 5-aza-dC (a DNA hypomethylating agent), and/or TSA (a histone deacetylase inhibitor). Expression of miR-34b/c increased after treatment with DOX at 24 h, with maximal induction at 48 h (Figure 2A). Similarly, miR-34b/c was also upregulated by 5-aza-dC or TSA (Figure 2B). Furthermore, the effect of drug combination seems to be additive on miR-34b/c expression (Figure 2B). Overall, these results demonstrated that the expression of miR-34b/c in uveal melanoma cells can be affected by DOX and epigenetic drugs.


MicroRNA-34b/c suppresses uveal melanoma cell proliferation and migration through multiple targets.

Dong F, Lou D - Mol. Vis. (2012)

MicroRNA-34b/c was upregulated by doxorubicin and epigenetic drugs. A: Real time reverse transcriptase (RT)-PCR analysis was performed to detect the expression of miR-34b/c in uveal melanoma cell line SP6.5, after treatment with DOX for 24 h and 48 h. B: SP6.5 cells were treated with 5-aza-dC at 1 μM or 5 μM alone, TSA (100 ng/ml) alone, or combinations of both. miR-34b/c expression level was determined by Real-time RT–PCR. The value for miR-34b/c in SP6.5 cells without any treatment was set at 1, and the relative amounts of miR-34b/c in cells treated with drugs were shown as fold induction. U6 snRNA was used as an internal control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3298424&req=5

f2: MicroRNA-34b/c was upregulated by doxorubicin and epigenetic drugs. A: Real time reverse transcriptase (RT)-PCR analysis was performed to detect the expression of miR-34b/c in uveal melanoma cell line SP6.5, after treatment with DOX for 24 h and 48 h. B: SP6.5 cells were treated with 5-aza-dC at 1 μM or 5 μM alone, TSA (100 ng/ml) alone, or combinations of both. miR-34b/c expression level was determined by Real-time RT–PCR. The value for miR-34b/c in SP6.5 cells without any treatment was set at 1, and the relative amounts of miR-34b/c in cells treated with drugs were shown as fold induction. U6 snRNA was used as an internal control.
Mentions: Treatment of tumor cells with DOX (doxorubicin) or inhibitors of DNA methyltransferases and/or histone deacetylase suppresses cell growth by activating multiple tumor suppressor genes [25-27]. We detected the expression levels of miR-34b/c in SP6.5 cells treated with either DOX, 5-aza-dC (a DNA hypomethylating agent), and/or TSA (a histone deacetylase inhibitor). Expression of miR-34b/c increased after treatment with DOX at 24 h, with maximal induction at 48 h (Figure 2A). Similarly, miR-34b/c was also upregulated by 5-aza-dC or TSA (Figure 2B). Furthermore, the effect of drug combination seems to be additive on miR-34b/c expression (Figure 2B). Overall, these results demonstrated that the expression of miR-34b/c in uveal melanoma cells can be affected by DOX and epigenetic drugs.

Bottom Line: Recent studies have revealed that miRNAs function as tumor suppressors or oncogenes.In addition, the effect of miR-34b/c on c-Met, cell cycle-related proteins, v-akt murine thymoma viral oncogene homolog (Akt) and extracellular signal-regulated kinase (ERK) pathway was determined by western blotting. miR-34b/c expression, which was dramatically decreased in uveal melanoma cells and clinical samples, can be upregulated by doxorubicin and epigenetic drugs.Furthermore, miR-34b/c was confirmed to downregulate the expression of c-Met, p-Akt, and cell cycle-related proteins by western blotting.

View Article: PubMed Central - PubMed

Affiliation: The First Affiliated Hospital of College of Medicine, Zhejiang University, Hangzhou, China.

ABSTRACT

Purpose: MicroRNAs (miRNAs) are endogenously expressed, small noncoding RNAs that inhibit gene expression by binding to target mRNAs. Recent studies have revealed that miRNAs function as tumor suppressors or oncogenes. In the present study, we investigated the role of miRNA-34b/c in uveal melanoma.

Methods: Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed to detect the expression level of miR-34b/c in uveal melanoma cells and primary samples. Subsequently, uveal melanoma cell proliferation was examined by the MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl] -2H-tetrazolium, inner salt) assay, clone formation assay, and flow cytometry. Cell apoptosis was measured by caspase3/7 assay. Cell migration was evaluated by transwell migration assay. The target of miR-34b/c was predicted by bioinformatics and validated by luciferase assay. In addition, the effect of miR-34b/c on c-Met, cell cycle-related proteins, v-akt murine thymoma viral oncogene homolog (Akt) and extracellular signal-regulated kinase (ERK) pathway was determined by western blotting.

Results: miR-34b/c expression, which was dramatically decreased in uveal melanoma cells and clinical samples, can be upregulated by doxorubicin and epigenetic drugs. The transfection of miR-34b/c into uveal melanoma cells leads to a significant reduction in cell growth and migration. miR-34b/c caused cell cycle G(1) arrest rather than the induction of apoptosis. Met proto-oncogene (c-Met) was identified as a target of miR-34b/c in uveal melanoma cells. Furthermore, miR-34b/c was confirmed to downregulate the expression of c-Met, p-Akt, and cell cycle-related proteins by western blotting.

Conclusions: Our results demonstrate that both miR-34b and miR-34c act as tumor suppressors in uveal melanoma cell proliferation and migration through the downregulation of multiple targets.

Show MeSH
Related in: MedlinePlus