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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

c-Met was a target of microRNA-34 b/c. A: Two specific binding sites of miR-34b/c in the c-Met 3′ untranslated region (UTR) was marked with black color. Alignment between the predicted miR-34b/c target sites and miR-34b/c, the common 8 bp seed sequence for miR-34b/c:mRNA (mRNA) pairing is shown. B: Design of the pMIR luciferase reporter constructs, containing c-Met 3′ UTR, which was used to verify the putative miR-34b/c binding sites. C: SP6.5 cells were cotransfected with miR-34b/c, pLuc-MET 3′ UTR, and a pRL-SV40 reporter plasmid. The luciferase activity was measured after 24 h. Values are presented as relative luciferase activity after normalization to Renilla luciferase activity. *: Differences in luciferase activity between miR-34b/c and negative control transfected cells were significant, p<0.01.
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f6: c-Met was a target of microRNA-34 b/c. A: Two specific binding sites of miR-34b/c in the c-Met 3′ untranslated region (UTR) was marked with black color. Alignment between the predicted miR-34b/c target sites and miR-34b/c, the common 8 bp seed sequence for miR-34b/c:mRNA (mRNA) pairing is shown. B: Design of the pMIR luciferase reporter constructs, containing c-Met 3′ UTR, which was used to verify the putative miR-34b/c binding sites. C: SP6.5 cells were cotransfected with miR-34b/c, pLuc-MET 3′ UTR, and a pRL-SV40 reporter plasmid. The luciferase activity was measured after 24 h. Values are presented as relative luciferase activity after normalization to Renilla luciferase activity. *: Differences in luciferase activity between miR-34b/c and negative control transfected cells were significant, p<0.01.

Mentions: To explore the molecular mechanisms underlying miR-34b/c mediated cell proliferation and migration, we used TargetScan for miR-34b/c target prediction. As shown in the Figure 6A, two potential binding sites of miR-34b/c, as well as the miR-34b/c: mRNA pairing model, were predicted in the 3′ UTR of the c-Met mRNA (Figure 6A). To examine the specific regulation of c-Met through the two predicted binding sites, we cloned the c-Met 3′ UTR into the pMIR-REPORT vector (Figure 6B). As expected, the luciferase activity of the wild-type pLuc-MET 3′ UTR construct was significantly suppressed following the transfection of miR-34b/c into SP6.5 cells, in contrast to the negative control (Figure 4C). Mutations of the two 8 bp binding sites in c-Met 3′ UTR completely abolished miR-34b/c-mediated inhibition of luciferase activity (Figure 6C). These results demonstrated that c-Met was a direct target of miR-34b/c.


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

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

c-Met was a target of microRNA-34 b/c. A: Two specific binding sites of miR-34b/c in the c-Met 3′ untranslated region (UTR) was marked with black color. Alignment between the predicted miR-34b/c target sites and miR-34b/c, the common 8 bp seed sequence for miR-34b/c:mRNA (mRNA) pairing is shown. B: Design of the pMIR luciferase reporter constructs, containing c-Met 3′ UTR, which was used to verify the putative miR-34b/c binding sites. C: SP6.5 cells were cotransfected with miR-34b/c, pLuc-MET 3′ UTR, and a pRL-SV40 reporter plasmid. The luciferase activity was measured after 24 h. Values are presented as relative luciferase activity after normalization to Renilla luciferase activity. *: Differences in luciferase activity 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

f6: c-Met was a target of microRNA-34 b/c. A: Two specific binding sites of miR-34b/c in the c-Met 3′ untranslated region (UTR) was marked with black color. Alignment between the predicted miR-34b/c target sites and miR-34b/c, the common 8 bp seed sequence for miR-34b/c:mRNA (mRNA) pairing is shown. B: Design of the pMIR luciferase reporter constructs, containing c-Met 3′ UTR, which was used to verify the putative miR-34b/c binding sites. C: SP6.5 cells were cotransfected with miR-34b/c, pLuc-MET 3′ UTR, and a pRL-SV40 reporter plasmid. The luciferase activity was measured after 24 h. Values are presented as relative luciferase activity after normalization to Renilla luciferase activity. *: Differences in luciferase activity between miR-34b/c and negative control transfected cells were significant, p<0.01.
Mentions: To explore the molecular mechanisms underlying miR-34b/c mediated cell proliferation and migration, we used TargetScan for miR-34b/c target prediction. As shown in the Figure 6A, two potential binding sites of miR-34b/c, as well as the miR-34b/c: mRNA pairing model, were predicted in the 3′ UTR of the c-Met mRNA (Figure 6A). To examine the specific regulation of c-Met through the two predicted binding sites, we cloned the c-Met 3′ UTR into the pMIR-REPORT vector (Figure 6B). As expected, the luciferase activity of the wild-type pLuc-MET 3′ UTR construct was significantly suppressed following the transfection of miR-34b/c into SP6.5 cells, in contrast to the negative control (Figure 4C). Mutations of the two 8 bp binding sites in c-Met 3′ UTR completely abolished miR-34b/c-mediated inhibition of luciferase activity (Figure 6C). These results demonstrated that c-Met was a direct target of miR-34b/c.

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