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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

Ectopic microRNA-34 b/c inhibited SP6.5 cell proliferation. A: MTS cell proliferation assay was performed on days 1 to 5 as indicated after transfection into SP6.5 cells with either miR-34 b/c mimic or a negative control (NC). Results represent those obtained in three experiments. B: SP6.5 cells transfected with miR-34b/c or NC were seeded at low density. Colony formation was observed by staining with crystal violet after seven days. Typical results from three independent experiments are depicted. C: SP6.5 cells were transfected with miR-34b/c or NC. After 48 h, cells were collected, stained with propidium iodide, and analyzed by flow cytometry. The most representative results from three independent experiments are shown.
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f3: Ectopic microRNA-34 b/c inhibited SP6.5 cell proliferation. A: MTS cell proliferation assay was performed on days 1 to 5 as indicated after transfection into SP6.5 cells with either miR-34 b/c mimic or a negative control (NC). Results represent those obtained in three experiments. B: SP6.5 cells transfected with miR-34b/c or NC were seeded at low density. Colony formation was observed by staining with crystal violet after seven days. Typical results from three independent experiments are depicted. C: SP6.5 cells were transfected with miR-34b/c or NC. After 48 h, cells were collected, stained with propidium iodide, and analyzed by flow cytometry. The most representative results from three independent experiments are shown.

Mentions: After determining the miR-34b/c expression pattern, we sought to investigate the effects on uveal melanoma cells by restoration of miR-34b/c. SP6.5 cells were transfected with either the miR-34b/c mimic or a negative control. After transfection, the MTS assay was performed to assess cell numbers at days 1 to 5. MiR-34b/c caused a dramatic inhibition of cell proliferation in SP6.5 uveal melanoma cells over a five-day interval (Figure 3A) as compared with the negative control. We detected a decrease of cell numbers in the SP6.5 cell line by day 2 following transfection, and the growth of cells transfected with the miR-34b/c mimic was statistically significantly retarded, as compared with the negative control. A significant reduction in cell number persisted through day 5 (48.85±5.39% decrease for miR-34b and 61.72±3.6% for miR-34c, p<0.01, Figure 3A). With clone formation assay, we were able to visually depict the growth retardation effect of miR-34b/c on SP6.5 cells by crystal violet staining after 7 days of culture (Figure 3B). The mechanism by which miR-34b/c inhibited cell growth was attributed to induction of G1 cell cycle arrest. Forty-eight hours after transfection, cell were stained with propidium iodide and analyzed by flow cytometry. In cells transfected with miR-34b, 73.22% of cells accumulated in G1 compared with 59.71% of cells for the negative control. In cells transfected with miR-34c, 80.76% of cells accumulated in G1 compared with 59.71% of cells for the negative control (Figure 3C). These results indicate that miR-34b/c inhibits cell proliferation and plays an important role in regulating uveal melanoma cell cycle G1 phase.


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

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

Ectopic microRNA-34 b/c inhibited SP6.5 cell proliferation. A: MTS cell proliferation assay was performed on days 1 to 5 as indicated after transfection into SP6.5 cells with either miR-34 b/c mimic or a negative control (NC). Results represent those obtained in three experiments. B: SP6.5 cells transfected with miR-34b/c or NC were seeded at low density. Colony formation was observed by staining with crystal violet after seven days. Typical results from three independent experiments are depicted. C: SP6.5 cells were transfected with miR-34b/c or NC. After 48 h, cells were collected, stained with propidium iodide, and analyzed by flow cytometry. The most representative results from three independent experiments are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Ectopic microRNA-34 b/c inhibited SP6.5 cell proliferation. A: MTS cell proliferation assay was performed on days 1 to 5 as indicated after transfection into SP6.5 cells with either miR-34 b/c mimic or a negative control (NC). Results represent those obtained in three experiments. B: SP6.5 cells transfected with miR-34b/c or NC were seeded at low density. Colony formation was observed by staining with crystal violet after seven days. Typical results from three independent experiments are depicted. C: SP6.5 cells were transfected with miR-34b/c or NC. After 48 h, cells were collected, stained with propidium iodide, and analyzed by flow cytometry. The most representative results from three independent experiments are shown.
Mentions: After determining the miR-34b/c expression pattern, we sought to investigate the effects on uveal melanoma cells by restoration of miR-34b/c. SP6.5 cells were transfected with either the miR-34b/c mimic or a negative control. After transfection, the MTS assay was performed to assess cell numbers at days 1 to 5. MiR-34b/c caused a dramatic inhibition of cell proliferation in SP6.5 uveal melanoma cells over a five-day interval (Figure 3A) as compared with the negative control. We detected a decrease of cell numbers in the SP6.5 cell line by day 2 following transfection, and the growth of cells transfected with the miR-34b/c mimic was statistically significantly retarded, as compared with the negative control. A significant reduction in cell number persisted through day 5 (48.85±5.39% decrease for miR-34b and 61.72±3.6% for miR-34c, p<0.01, Figure 3A). With clone formation assay, we were able to visually depict the growth retardation effect of miR-34b/c on SP6.5 cells by crystal violet staining after 7 days of culture (Figure 3B). The mechanism by which miR-34b/c inhibited cell growth was attributed to induction of G1 cell cycle arrest. Forty-eight hours after transfection, cell were stained with propidium iodide and analyzed by flow cytometry. In cells transfected with miR-34b, 73.22% of cells accumulated in G1 compared with 59.71% of cells for the negative control. In cells transfected with miR-34c, 80.76% of cells accumulated in G1 compared with 59.71% of cells for the negative control (Figure 3C). These results indicate that miR-34b/c inhibits cell proliferation and plays an important role in regulating uveal melanoma cell cycle G1 phase.

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