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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

Transfection of microRNA-34 b/c reduced uveal melanoma cell migration. Transwell migration assay of uveal melanoma cell lines was performed. SP6.5 cells were transfected with miR-34b/c or a negative control (NC) for 24 h and plated on cultured inserts in DMEM containing 20 ng/ml of hepatocyte growth factor (HGF) to assess the number of migrating cells. The number of cells that had migrated through the pores was quantified by counting 10 independent visual fields using a 20× microscope objective. *: Differences in cell migration between miR-34b/c and negative control transfected cells were significant, p<0.01.
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f5: Transfection of microRNA-34 b/c reduced uveal melanoma cell migration. Transwell migration assay of uveal melanoma cell lines was performed. SP6.5 cells were transfected with miR-34b/c or a negative control (NC) for 24 h and plated on cultured inserts in DMEM containing 20 ng/ml of hepatocyte growth factor (HGF) to assess the number of migrating cells. The number of cells that had migrated through the pores was quantified by counting 10 independent visual fields using a 20× microscope objective. *: Differences in cell migration between miR-34b/c and negative control transfected cells were significant, p<0.01.

Mentions: We next evaluated the effect of miR-34b/c on uveal melanoma cell migration by transwell migration assay. Following transfection with either miR-34b/c mimic or a negative control, SP6.5 cells were seeded on cultured inserts and the ability of cells to migrate to the underside of the inserts was assessed in the presence of HGF. As shown in Figure 5, the migration of cells transfected with miR-34b/c was significantly inhibited, as compared with negative control (208±15 for NC, 122±10 for miR-34b, 116±9 for miR-34c, n=3 each, p<0.01). Therefore, the introduction of miR-34b/c caused reduced cell migration in response to HGF.


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

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

Transfection of microRNA-34 b/c reduced uveal melanoma cell migration. Transwell migration assay of uveal melanoma cell lines was performed. SP6.5 cells were transfected with miR-34b/c or a negative control (NC) for 24 h and plated on cultured inserts in DMEM containing 20 ng/ml of hepatocyte growth factor (HGF) to assess the number of migrating cells. The number of cells that had migrated through the pores was quantified by counting 10 independent visual fields using a 20× microscope objective. *: Differences in cell migration between miR-34b/c and negative control transfected cells were significant, p<0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Transfection of microRNA-34 b/c reduced uveal melanoma cell migration. Transwell migration assay of uveal melanoma cell lines was performed. SP6.5 cells were transfected with miR-34b/c or a negative control (NC) for 24 h and plated on cultured inserts in DMEM containing 20 ng/ml of hepatocyte growth factor (HGF) to assess the number of migrating cells. The number of cells that had migrated through the pores was quantified by counting 10 independent visual fields using a 20× microscope objective. *: Differences in cell migration between miR-34b/c and negative control transfected cells were significant, p<0.01.
Mentions: We next evaluated the effect of miR-34b/c on uveal melanoma cell migration by transwell migration assay. Following transfection with either miR-34b/c mimic or a negative control, SP6.5 cells were seeded on cultured inserts and the ability of cells to migrate to the underside of the inserts was assessed in the presence of HGF. As shown in Figure 5, the migration of cells transfected with miR-34b/c was significantly inhibited, as compared with negative control (208±15 for NC, 122±10 for miR-34b, 116±9 for miR-34c, n=3 each, p<0.01). Therefore, the introduction of miR-34b/c caused reduced cell migration in response to HGF.

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