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MicroRNA-18a is elevated in prostate cancer and promotes tumorigenesis through suppressing STK4 in vitro and in vivo.

Hsu TI, Hsu CH, Lee KH, Lin JT, Chen CS, Chang KC, Su CY, Hsiao M, Lu PJ - Oncogenesis (2014)

Bottom Line: Our bioinformatics analysis showed that the serine/threonine-protein kinase 4 (STK4) 3' untranslated region contains a highly conserved binding site for the miR-18a seed region.Luciferase reporter assays were performed to indicate that STK4 is a direct target of miR-18a.Interestingly, miR-18a knockdown decreased cell growth in prostate cancer cells and significantly decreased prostate tumor growth in in vivo nude mice experiments through STK4-mediated dephosphorylation of AKT and thereby inducing apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Basic Medical Sciences, Medical College, National Cheng Kung University, Tainan, Taiwan.

ABSTRACT
MicroRNAs (miRNAs) comprise a class of short, non-coding RNAs that regulate protein synthesis through posttranscriptional modifications. In this study, we found significant upregulation of miR-18a in prostate cancer specimens and prostate cancer cell lines compared with the normal controls. MiRNAs can be separated into two groups based on whether they regulate tumor suppressors or oncogenes. In our previous study, we found that miR-18a, which belongs to the miR17-92 cluster, is upregulated in prostate cancer; the objective of this study was to investigate the associated regulatory mechanisms. We found that miR-18a is upregulated in clinical tumor specimens and cancer cell lines. Our bioinformatics analysis showed that the serine/threonine-protein kinase 4 (STK4) 3' untranslated region contains a highly conserved binding site for the miR-18a seed region. Luciferase reporter assays were performed to indicate that STK4 is a direct target of miR-18a. Interestingly, miR-18a knockdown decreased cell growth in prostate cancer cells and significantly decreased prostate tumor growth in in vivo nude mice experiments through STK4-mediated dephosphorylation of AKT and thereby inducing apoptosis. Our results suggest that miR-18a acts as an oncomiR targeting STK4 in prostate cancer, and inhibition of miR-18a expression may offer therapeutically beneficial option for prostate cancer treatment.

No MeSH data available.


Related in: MedlinePlus

miR-18a directly binds the STK4 3′UTR to suppress the protein level. (a) Bioinformatics analysis demonstrates that the miR-18a seed region is highly conserved in different species. The computer-predicted binding energy is −21.1 Kcal/mol. (b) Luciferase reporter assay in HEK293T (upper panel) and DU145 (bottom panel) cells, with the cotransfection of WT-reporter and control-miR or pre-miR-18a as well as cotransfection of WT-reporter and control anti-miR or anti-miR-18a as indicated. Each bar represents the mean and s.d. of three independent experiments. *P<0.05, **P<0.01, ***P<0.001. (c) HEK-293T cells were transfected with the mutant STK4 3′UTR to examine the binding affinities of these sequences. The mutant UTRs of STK4 had no effect on reporter activity. (d) STK4 protein level after miR-18a knockdown in 22Rv1 and DU145 cell lines or overexpression of miR-18a in RWPE-1 and PWR-1E cell lines, respectively. At 48 h after transfection, total protein and RNA were extracted and used for western blotting (upper panel) and miRNA-quantitative RT-PCR (bottom panel) analysis.
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fig3: miR-18a directly binds the STK4 3′UTR to suppress the protein level. (a) Bioinformatics analysis demonstrates that the miR-18a seed region is highly conserved in different species. The computer-predicted binding energy is −21.1 Kcal/mol. (b) Luciferase reporter assay in HEK293T (upper panel) and DU145 (bottom panel) cells, with the cotransfection of WT-reporter and control-miR or pre-miR-18a as well as cotransfection of WT-reporter and control anti-miR or anti-miR-18a as indicated. Each bar represents the mean and s.d. of three independent experiments. *P<0.05, **P<0.01, ***P<0.001. (c) HEK-293T cells were transfected with the mutant STK4 3′UTR to examine the binding affinities of these sequences. The mutant UTRs of STK4 had no effect on reporter activity. (d) STK4 protein level after miR-18a knockdown in 22Rv1 and DU145 cell lines or overexpression of miR-18a in RWPE-1 and PWR-1E cell lines, respectively. At 48 h after transfection, total protein and RNA were extracted and used for western blotting (upper panel) and miRNA-quantitative RT-PCR (bottom panel) analysis.

Mentions: We next investigated whether the 3′UTR of STK4 was a functional target of miR-18a. We performed bioinformatics searches to find whether miR-18a may target nt 85–115 of 3′UTR of STK4. The schematic diagram of miR-18a binding sites in the 3′UTR of STK4 is shown in Figure 3a that was highly conserved across seven species. Because miR-18a has a predicted target site in the 3′UTR of STK4, we generated pMIR-reporter luciferase vectors containing the STK4 3′UTR sequence. In these vectors, the firefly luciferase coding sequence is followed by a 300 nucleotide PCR fragment encompassing the predicted miRNA binding site within the STK4 3′UTR, from nucleotides 17 to 317, as amplified from the wild type or seed region mutant constructs (Figure 3a, bottom panel, marked as Mut. 3′UTR). The recombinant STK4 3′UTR constructs were transfected into HEK-293T and DU-145 cells together with miR-18a or antagomiR-18a. The luciferase activity in HEK-293T and DU-145 cells transfected with STK4 wild type constructs plus miR-18a mimics was significantly lower than that in cells transfected with the control miR (Figure 3b, upper panel, lanes 2 and 3, P<0.05). The opposite result was observed in cells transfected with antagomiR-18a. In contrast, luciferase activities of mutant 3′UTR remained unchanged in miR-18a precursor overexpressing HEK293T cells (Figure 3c, lanes 4 and 5). The miR-18a expression level was examined by real-time PCR in all of the reporter assays (Supplementary Figures S3A and B). These results suggest that the STK4 mRNA is a direct target of miR-18a. To determine whether miR-18a inhibits endogenous STK4 expression, we used established human prostate cancer cell lines. As anticipated, the endogenous miR-18a levels were high in all the cells tested, making loss-of-function experiments challenging. The transfection efficiency of miR-18a precursor and anti-miR-18a in the cells has been examined by miRNA-quantitative RT-PCR (Supplementary Figures S3 A and B). We also examined the STK4 mRNA expression levels of reporter assay. In the real time PCR data showed STK4 mRNA levels did not change occur with miR-18a expression level (Supplementary Figures S3A and S3B). However, antagomiR-18a consistently and strongly increased STK4 protein and mRNA expression (Figure 3d and Supplementary Figure S3C). The selectivity was validated using a miRNA mimic approach. When the miR-18a mimic was transfected into RWPE-1 and PWR-1E cells, decreases in the STK4 protein and mRNA levels were observed (Figure 3d and Supplementary Figure S3C). The STK4 mRNA level alternation could be side effect. But the reporter assay showed that luciferase activities of mutant 3′UTR remained unchanged in miR-18a precursor overexpressing HEK293T cells (Figure 3c, lanes 4 and 5). These results indicated that miR-18a directly targets the STK4 3′UTR to suppress STK4 protein translation.


MicroRNA-18a is elevated in prostate cancer and promotes tumorigenesis through suppressing STK4 in vitro and in vivo.

Hsu TI, Hsu CH, Lee KH, Lin JT, Chen CS, Chang KC, Su CY, Hsiao M, Lu PJ - Oncogenesis (2014)

miR-18a directly binds the STK4 3′UTR to suppress the protein level. (a) Bioinformatics analysis demonstrates that the miR-18a seed region is highly conserved in different species. The computer-predicted binding energy is −21.1 Kcal/mol. (b) Luciferase reporter assay in HEK293T (upper panel) and DU145 (bottom panel) cells, with the cotransfection of WT-reporter and control-miR or pre-miR-18a as well as cotransfection of WT-reporter and control anti-miR or anti-miR-18a as indicated. Each bar represents the mean and s.d. of three independent experiments. *P<0.05, **P<0.01, ***P<0.001. (c) HEK-293T cells were transfected with the mutant STK4 3′UTR to examine the binding affinities of these sequences. The mutant UTRs of STK4 had no effect on reporter activity. (d) STK4 protein level after miR-18a knockdown in 22Rv1 and DU145 cell lines or overexpression of miR-18a in RWPE-1 and PWR-1E cell lines, respectively. At 48 h after transfection, total protein and RNA were extracted and used for western blotting (upper panel) and miRNA-quantitative RT-PCR (bottom panel) analysis.
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Related In: Results  -  Collection

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fig3: miR-18a directly binds the STK4 3′UTR to suppress the protein level. (a) Bioinformatics analysis demonstrates that the miR-18a seed region is highly conserved in different species. The computer-predicted binding energy is −21.1 Kcal/mol. (b) Luciferase reporter assay in HEK293T (upper panel) and DU145 (bottom panel) cells, with the cotransfection of WT-reporter and control-miR or pre-miR-18a as well as cotransfection of WT-reporter and control anti-miR or anti-miR-18a as indicated. Each bar represents the mean and s.d. of three independent experiments. *P<0.05, **P<0.01, ***P<0.001. (c) HEK-293T cells were transfected with the mutant STK4 3′UTR to examine the binding affinities of these sequences. The mutant UTRs of STK4 had no effect on reporter activity. (d) STK4 protein level after miR-18a knockdown in 22Rv1 and DU145 cell lines or overexpression of miR-18a in RWPE-1 and PWR-1E cell lines, respectively. At 48 h after transfection, total protein and RNA were extracted and used for western blotting (upper panel) and miRNA-quantitative RT-PCR (bottom panel) analysis.
Mentions: We next investigated whether the 3′UTR of STK4 was a functional target of miR-18a. We performed bioinformatics searches to find whether miR-18a may target nt 85–115 of 3′UTR of STK4. The schematic diagram of miR-18a binding sites in the 3′UTR of STK4 is shown in Figure 3a that was highly conserved across seven species. Because miR-18a has a predicted target site in the 3′UTR of STK4, we generated pMIR-reporter luciferase vectors containing the STK4 3′UTR sequence. In these vectors, the firefly luciferase coding sequence is followed by a 300 nucleotide PCR fragment encompassing the predicted miRNA binding site within the STK4 3′UTR, from nucleotides 17 to 317, as amplified from the wild type or seed region mutant constructs (Figure 3a, bottom panel, marked as Mut. 3′UTR). The recombinant STK4 3′UTR constructs were transfected into HEK-293T and DU-145 cells together with miR-18a or antagomiR-18a. The luciferase activity in HEK-293T and DU-145 cells transfected with STK4 wild type constructs plus miR-18a mimics was significantly lower than that in cells transfected with the control miR (Figure 3b, upper panel, lanes 2 and 3, P<0.05). The opposite result was observed in cells transfected with antagomiR-18a. In contrast, luciferase activities of mutant 3′UTR remained unchanged in miR-18a precursor overexpressing HEK293T cells (Figure 3c, lanes 4 and 5). The miR-18a expression level was examined by real-time PCR in all of the reporter assays (Supplementary Figures S3A and B). These results suggest that the STK4 mRNA is a direct target of miR-18a. To determine whether miR-18a inhibits endogenous STK4 expression, we used established human prostate cancer cell lines. As anticipated, the endogenous miR-18a levels were high in all the cells tested, making loss-of-function experiments challenging. The transfection efficiency of miR-18a precursor and anti-miR-18a in the cells has been examined by miRNA-quantitative RT-PCR (Supplementary Figures S3 A and B). We also examined the STK4 mRNA expression levels of reporter assay. In the real time PCR data showed STK4 mRNA levels did not change occur with miR-18a expression level (Supplementary Figures S3A and S3B). However, antagomiR-18a consistently and strongly increased STK4 protein and mRNA expression (Figure 3d and Supplementary Figure S3C). The selectivity was validated using a miRNA mimic approach. When the miR-18a mimic was transfected into RWPE-1 and PWR-1E cells, decreases in the STK4 protein and mRNA levels were observed (Figure 3d and Supplementary Figure S3C). The STK4 mRNA level alternation could be side effect. But the reporter assay showed that luciferase activities of mutant 3′UTR remained unchanged in miR-18a precursor overexpressing HEK293T cells (Figure 3c, lanes 4 and 5). These results indicated that miR-18a directly targets the STK4 3′UTR to suppress STK4 protein translation.

Bottom Line: Our bioinformatics analysis showed that the serine/threonine-protein kinase 4 (STK4) 3' untranslated region contains a highly conserved binding site for the miR-18a seed region.Luciferase reporter assays were performed to indicate that STK4 is a direct target of miR-18a.Interestingly, miR-18a knockdown decreased cell growth in prostate cancer cells and significantly decreased prostate tumor growth in in vivo nude mice experiments through STK4-mediated dephosphorylation of AKT and thereby inducing apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Basic Medical Sciences, Medical College, National Cheng Kung University, Tainan, Taiwan.

ABSTRACT
MicroRNAs (miRNAs) comprise a class of short, non-coding RNAs that regulate protein synthesis through posttranscriptional modifications. In this study, we found significant upregulation of miR-18a in prostate cancer specimens and prostate cancer cell lines compared with the normal controls. MiRNAs can be separated into two groups based on whether they regulate tumor suppressors or oncogenes. In our previous study, we found that miR-18a, which belongs to the miR17-92 cluster, is upregulated in prostate cancer; the objective of this study was to investigate the associated regulatory mechanisms. We found that miR-18a is upregulated in clinical tumor specimens and cancer cell lines. Our bioinformatics analysis showed that the serine/threonine-protein kinase 4 (STK4) 3' untranslated region contains a highly conserved binding site for the miR-18a seed region. Luciferase reporter assays were performed to indicate that STK4 is a direct target of miR-18a. Interestingly, miR-18a knockdown decreased cell growth in prostate cancer cells and significantly decreased prostate tumor growth in in vivo nude mice experiments through STK4-mediated dephosphorylation of AKT and thereby inducing apoptosis. Our results suggest that miR-18a acts as an oncomiR targeting STK4 in prostate cancer, and inhibition of miR-18a expression may offer therapeutically beneficial option for prostate cancer treatment.

No MeSH data available.


Related in: MedlinePlus