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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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MicroRNA-34b/c expression was downregulated in human uveal melanoma cell line and clinical samples. A: Real time reverse transcriptase (RT)-PCR analysis was performed to detect the expression of miR-34b/c in clinical samples. The value for miR-34b/c in normal uveal tissue was set at 1, and the relative amounts of miR-34b/c in tumor tissues were shown as fold induction. Both miR-34b and miR-34c were dramatically decreased in five specimens as compared with normal tissues. N: normal uveal tissue; T: tumor tissue. B: The expression of miR-34b/c was measured by real time RT–PCR in uveal melanoma cell line SP6.5, as well as the primary uveal melanocytes. miR-34b/c was expressed in uveal melanocytes but dramatically decreased in uveal melanoma cells. U6 snRNA was used as an internal control.
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f1: MicroRNA-34b/c expression was downregulated in human uveal melanoma cell line and clinical samples. A: Real time reverse transcriptase (RT)-PCR analysis was performed to detect the expression of miR-34b/c in clinical samples. The value for miR-34b/c in normal uveal tissue was set at 1, and the relative amounts of miR-34b/c in tumor tissues were shown as fold induction. Both miR-34b and miR-34c were dramatically decreased in five specimens as compared with normal tissues. N: normal uveal tissue; T: tumor tissue. B: The expression of miR-34b/c was measured by real time RT–PCR in uveal melanoma cell line SP6.5, as well as the primary uveal melanocytes. miR-34b/c was expressed in uveal melanocytes but dramatically decreased in uveal melanoma cells. U6 snRNA was used as an internal control.

Mentions: To investigate whether miR-34b/c was involved in the tumorigenesis of uveal melanoma, we first examined miR-34b/c expression levels in primary samples. Real-time reverse transcriptase (RT)- polymerase chain reaction (PCR) analysis was performed to detect miR-34b/c expression in five specimens of uveal melanoma. The adjacent uveal tissue was used as normal control in each case. As a result, miR-34b/c expression was dramatically decreased in specimens 1, 2, and 5, and undetectable in specimens 3 and 4, in contrast to normal tissues (Figure 1A). We also compared the expression of miR-34b/c in uveal melanoma cell line SP6.5 and primary uveal melanocytes by real-time RT–PCR. Consistent with the results from primary samples, miR-34b/c was expressed in uveal melanocytes, but was dramatically decreased in the SP6.5 cell line (Figure 1B). These results indicate that miR-34b/c expression is frequently downregulated in human uveal melanoma.


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

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

MicroRNA-34b/c expression was downregulated in human uveal melanoma cell line and clinical samples. A: Real time reverse transcriptase (RT)-PCR analysis was performed to detect the expression of miR-34b/c in clinical samples. The value for miR-34b/c in normal uveal tissue was set at 1, and the relative amounts of miR-34b/c in tumor tissues were shown as fold induction. Both miR-34b and miR-34c were dramatically decreased in five specimens as compared with normal tissues. N: normal uveal tissue; T: tumor tissue. B: The expression of miR-34b/c was measured by real time RT–PCR in uveal melanoma cell line SP6.5, as well as the primary uveal melanocytes. miR-34b/c was expressed in uveal melanocytes but dramatically decreased in uveal melanoma cells. U6 snRNA was used as an internal control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3298424&req=5

f1: MicroRNA-34b/c expression was downregulated in human uveal melanoma cell line and clinical samples. A: Real time reverse transcriptase (RT)-PCR analysis was performed to detect the expression of miR-34b/c in clinical samples. The value for miR-34b/c in normal uveal tissue was set at 1, and the relative amounts of miR-34b/c in tumor tissues were shown as fold induction. Both miR-34b and miR-34c were dramatically decreased in five specimens as compared with normal tissues. N: normal uveal tissue; T: tumor tissue. B: The expression of miR-34b/c was measured by real time RT–PCR in uveal melanoma cell line SP6.5, as well as the primary uveal melanocytes. miR-34b/c was expressed in uveal melanocytes but dramatically decreased in uveal melanoma cells. U6 snRNA was used as an internal control.
Mentions: To investigate whether miR-34b/c was involved in the tumorigenesis of uveal melanoma, we first examined miR-34b/c expression levels in primary samples. Real-time reverse transcriptase (RT)- polymerase chain reaction (PCR) analysis was performed to detect miR-34b/c expression in five specimens of uveal melanoma. The adjacent uveal tissue was used as normal control in each case. As a result, miR-34b/c expression was dramatically decreased in specimens 1, 2, and 5, and undetectable in specimens 3 and 4, in contrast to normal tissues (Figure 1A). We also compared the expression of miR-34b/c in uveal melanoma cell line SP6.5 and primary uveal melanocytes by real-time RT–PCR. Consistent with the results from primary samples, miR-34b/c was expressed in uveal melanocytes, but was dramatically decreased in the SP6.5 cell line (Figure 1B). These results indicate that miR-34b/c expression is frequently downregulated in human uveal melanoma.

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