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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 promotes the tumorigenicity of prostate cancer cells. (a) Decreased colony-formation ability is observed in 22Rv-1 cells overexpressing STK4 or antagomiR-18a. (b) PC3 cells expressing STK4 shRNA or pre-miR-18a exhibit increased colony-formation ability. (c) STK4 expression inhibits the oncogenic function of miR-18a. STK4 inhibits anchorage-independent growth of prostate cancer cells. 22Rv1 cells were transfected with STK4 or anti-miR-18a. Soft agar colony-formation assays were performed 24 h after transfection. (d) PWR-1E cells overexpressing pre-miR-18a or STK4 were subjected to a colony-formation assay to validate the effects of STK4 and miR-18a. (e) STK4 inhibits tumor growth of prostate cancer cells in nude mice. 22Rv1 cells were transfected with GFP-STK4, control GFP, anti-miR-18a or control anti-miR, respectively. Twenty-four hours after transfection, 1 × 106 cells were subcutaneously injected into of male nude mice. The dot plot show the tumor weight of each mice, and the inset photographs are representative xenografted tumors 6 weeks after inoculation. *P<0.05; **P<0.01. In vivo studies demonstrated that 22Rv-1 cells transfected with STK4 or antagomiR-18a are associated with decreased tumor volume and growth.
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fig4: miR-18a promotes the tumorigenicity of prostate cancer cells. (a) Decreased colony-formation ability is observed in 22Rv-1 cells overexpressing STK4 or antagomiR-18a. (b) PC3 cells expressing STK4 shRNA or pre-miR-18a exhibit increased colony-formation ability. (c) STK4 expression inhibits the oncogenic function of miR-18a. STK4 inhibits anchorage-independent growth of prostate cancer cells. 22Rv1 cells were transfected with STK4 or anti-miR-18a. Soft agar colony-formation assays were performed 24 h after transfection. (d) PWR-1E cells overexpressing pre-miR-18a or STK4 were subjected to a colony-formation assay to validate the effects of STK4 and miR-18a. (e) STK4 inhibits tumor growth of prostate cancer cells in nude mice. 22Rv1 cells were transfected with GFP-STK4, control GFP, anti-miR-18a or control anti-miR, respectively. Twenty-four hours after transfection, 1 × 106 cells were subcutaneously injected into of male nude mice. The dot plot show the tumor weight of each mice, and the inset photographs are representative xenografted tumors 6 weeks after inoculation. *P<0.05; **P<0.01. In vivo studies demonstrated that 22Rv-1 cells transfected with STK4 or antagomiR-18a are associated with decreased tumor volume and growth.

Mentions: As overexpression of miR-18a implied an oncogenic role in prostate cancer, we decided to examine whether miR-18a has oncogenic functions in prostate cancer cells in vitro and in vivo. We analyzed clonogenic ability of prostate cancer cells by modulation of miR-18a or STK4 in both anchorage-dependent and anchorage-independent conditions. We found that the upregulation of miR-18a in PC3 increased colony formation by 50%, whereas the downregulation of miR-18a in 22Rv-1 and LNCap decreased colony formation by 30% (Figure 4a left panel, P<0.05, Figure 4b right panel, P<0.05 and Supplementary Figure S4A, P<0.001). The contrary effect was also observed in the green fluorescent protein (GFP)-STK4-overexpressing 22Rvl cells, which show that overexpression of STK4 significantly inhibited the proliferation rate of 22Rvl cells (Figure 4a, right panel, P<0.05). The same experiments were performed in another high endogenous miR-18a level prostate cancer cell line (Supplementary Figure S4A, P<0.001 in anti-miR-18a-transfected LNCap cells, P<0.05 in GFP-STK4-transfected LNCap cells). Interestingly, miR-18a suppression affected not only cell growth but also anchorage-independent growth. Transfection with an anti-miR-18a inhibitor and STK4 markedly decreased the plating efficiency of 22Rv-1 cells in soft agar (Figure 4c). Upregulation of miR-18a by the miR-18a precursor of PC-3 and DU145 cell lines and downregulation of miR-18a by the anti-miR-18a of 22Rv1 and LNCap cell lines were confirmed using miRNA-quantitative RT-PCR (Supplementary Figure S4B). Furthermore, we then investigated whether the oncogenic roles of miR-18a were dependent on STK4 expression. We transfected a STK4 construct into immortalized prostatic epithelial PWR-1E followed by transfection of the miR-18a precursor to perform colony-formation assay. In the colony-formation assay, PWR-1E cells transfected with a control miR and STK4 exhibited lower colony-formation ability than control miR and GFP-transfected cells. Conversely, cells transfected with pre-miR-18a and GFP exhibited an increased colony number compared with those transfected with pre-miR-18a and STK4. However, the effect of pre-miR-18a was not significantly different from that of STK4 (Figure 4d). These results supported the hypothesis that miR-18a executes an oncogenic effect on prostate cancer cells and the expression of miR-18a can inhibit prostate cancer cells' growth in anchorage-dependent as well as -independent conditions. To determine whether miR-18a regulates tumor growth in vivo, we used tumor xenografts by inoculating 22Rv1 cells that had miR-18a suppressed in nude mice. Smaller subcutaneous tumors were observed in nude mice implanted with cells overexpressing antagomiR-18a and STK4 compared with those implanted with control cells (n=7–9/group; GFP compared with STK4 P<0.05, anti-NC compared with anti-miR-18a P<0.01, Figure 4e). At the end of the observation, the average volume (160±40 mm3) of tumors expressing anti-miR-18a was only about one-third of that (410±80 mm3) of the control group (Figure 4e, P<0.01). The similar result was also observed when 22Rv1 cells transfected with GFP-STK4 produced tumors much smaller than that of GFP control group (Figure 4e, right). At the end of the observation, the average tumor volume in the GFP-STK4 group was 110±20 mm3, whereas that in the GFP control group was 400±120 mm3, which represented a 3.0-fold decrease (P<0.05). These results indicate that miR-18a may act as an oncomiR in prostate cancer cells in vivo and in vitro.


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 promotes the tumorigenicity of prostate cancer cells. (a) Decreased colony-formation ability is observed in 22Rv-1 cells overexpressing STK4 or antagomiR-18a. (b) PC3 cells expressing STK4 shRNA or pre-miR-18a exhibit increased colony-formation ability. (c) STK4 expression inhibits the oncogenic function of miR-18a. STK4 inhibits anchorage-independent growth of prostate cancer cells. 22Rv1 cells were transfected with STK4 or anti-miR-18a. Soft agar colony-formation assays were performed 24 h after transfection. (d) PWR-1E cells overexpressing pre-miR-18a or STK4 were subjected to a colony-formation assay to validate the effects of STK4 and miR-18a. (e) STK4 inhibits tumor growth of prostate cancer cells in nude mice. 22Rv1 cells were transfected with GFP-STK4, control GFP, anti-miR-18a or control anti-miR, respectively. Twenty-four hours after transfection, 1 × 106 cells were subcutaneously injected into of male nude mice. The dot plot show the tumor weight of each mice, and the inset photographs are representative xenografted tumors 6 weeks after inoculation. *P<0.05; **P<0.01. In vivo studies demonstrated that 22Rv-1 cells transfected with STK4 or antagomiR-18a are associated with decreased tumor volume and growth.
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fig4: miR-18a promotes the tumorigenicity of prostate cancer cells. (a) Decreased colony-formation ability is observed in 22Rv-1 cells overexpressing STK4 or antagomiR-18a. (b) PC3 cells expressing STK4 shRNA or pre-miR-18a exhibit increased colony-formation ability. (c) STK4 expression inhibits the oncogenic function of miR-18a. STK4 inhibits anchorage-independent growth of prostate cancer cells. 22Rv1 cells were transfected with STK4 or anti-miR-18a. Soft agar colony-formation assays were performed 24 h after transfection. (d) PWR-1E cells overexpressing pre-miR-18a or STK4 were subjected to a colony-formation assay to validate the effects of STK4 and miR-18a. (e) STK4 inhibits tumor growth of prostate cancer cells in nude mice. 22Rv1 cells were transfected with GFP-STK4, control GFP, anti-miR-18a or control anti-miR, respectively. Twenty-four hours after transfection, 1 × 106 cells were subcutaneously injected into of male nude mice. The dot plot show the tumor weight of each mice, and the inset photographs are representative xenografted tumors 6 weeks after inoculation. *P<0.05; **P<0.01. In vivo studies demonstrated that 22Rv-1 cells transfected with STK4 or antagomiR-18a are associated with decreased tumor volume and growth.
Mentions: As overexpression of miR-18a implied an oncogenic role in prostate cancer, we decided to examine whether miR-18a has oncogenic functions in prostate cancer cells in vitro and in vivo. We analyzed clonogenic ability of prostate cancer cells by modulation of miR-18a or STK4 in both anchorage-dependent and anchorage-independent conditions. We found that the upregulation of miR-18a in PC3 increased colony formation by 50%, whereas the downregulation of miR-18a in 22Rv-1 and LNCap decreased colony formation by 30% (Figure 4a left panel, P<0.05, Figure 4b right panel, P<0.05 and Supplementary Figure S4A, P<0.001). The contrary effect was also observed in the green fluorescent protein (GFP)-STK4-overexpressing 22Rvl cells, which show that overexpression of STK4 significantly inhibited the proliferation rate of 22Rvl cells (Figure 4a, right panel, P<0.05). The same experiments were performed in another high endogenous miR-18a level prostate cancer cell line (Supplementary Figure S4A, P<0.001 in anti-miR-18a-transfected LNCap cells, P<0.05 in GFP-STK4-transfected LNCap cells). Interestingly, miR-18a suppression affected not only cell growth but also anchorage-independent growth. Transfection with an anti-miR-18a inhibitor and STK4 markedly decreased the plating efficiency of 22Rv-1 cells in soft agar (Figure 4c). Upregulation of miR-18a by the miR-18a precursor of PC-3 and DU145 cell lines and downregulation of miR-18a by the anti-miR-18a of 22Rv1 and LNCap cell lines were confirmed using miRNA-quantitative RT-PCR (Supplementary Figure S4B). Furthermore, we then investigated whether the oncogenic roles of miR-18a were dependent on STK4 expression. We transfected a STK4 construct into immortalized prostatic epithelial PWR-1E followed by transfection of the miR-18a precursor to perform colony-formation assay. In the colony-formation assay, PWR-1E cells transfected with a control miR and STK4 exhibited lower colony-formation ability than control miR and GFP-transfected cells. Conversely, cells transfected with pre-miR-18a and GFP exhibited an increased colony number compared with those transfected with pre-miR-18a and STK4. However, the effect of pre-miR-18a was not significantly different from that of STK4 (Figure 4d). These results supported the hypothesis that miR-18a executes an oncogenic effect on prostate cancer cells and the expression of miR-18a can inhibit prostate cancer cells' growth in anchorage-dependent as well as -independent conditions. To determine whether miR-18a regulates tumor growth in vivo, we used tumor xenografts by inoculating 22Rv1 cells that had miR-18a suppressed in nude mice. Smaller subcutaneous tumors were observed in nude mice implanted with cells overexpressing antagomiR-18a and STK4 compared with those implanted with control cells (n=7–9/group; GFP compared with STK4 P<0.05, anti-NC compared with anti-miR-18a P<0.01, Figure 4e). At the end of the observation, the average volume (160±40 mm3) of tumors expressing anti-miR-18a was only about one-third of that (410±80 mm3) of the control group (Figure 4e, P<0.01). The similar result was also observed when 22Rv1 cells transfected with GFP-STK4 produced tumors much smaller than that of GFP control group (Figure 4e, right). At the end of the observation, the average tumor volume in the GFP-STK4 group was 110±20 mm3, whereas that in the GFP control group was 400±120 mm3, which represented a 3.0-fold decrease (P<0.05). These results indicate that miR-18a may act as an oncomiR in prostate cancer cells in vivo and in vitro.

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