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Up-regulated Dicer expression in patients with cutaneous melanoma.

Ma Z, Swede H, Cassarino D, Fleming E, Fire A, Dadras SS - PLoS ONE (2011)

Bottom Line: The expression of Dicer was significantly higher in melanomas compared to benign melanocytic nevi (P<0.0001).In patients with cutaneous melanomas, Dicer up-regulation was found to be significantly associated with an increased tumor mitotic index (P = 0.04), Breslow's depth of invasion (P = 0.03), nodal metastasis (P = 0.04) and a higher American Joint Committee on Caner (AJCC) clinical stage (P = 0.009).Understanding deregulation of Dicer and its influence on miRNA maturation is needed to predict the susceptibility of melanoma patients to miRNA-based therapy in the future.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Stanford University School of Medicine, Stanford, California, United States of America.

ABSTRACT

Background: MicroRNAs (miRNAs) are small non-coding RNAs (18-24 nucleotides) that have recently been shown to regulate gene expression during cancer progression. Dicer, a central enzyme in the multi-component miRNA biogenesis pathway, is involved in cutting precursor miRNAs to functionally mature forms. Emerging evidence shows that Dicer expression is deregulated in some human malignancies and it correlates with tumor progression, yet this role has not yet been investigated in skin cancers.

Methods and findings: Using an anti-human monoclonal antibody against Dicer and immunohistochemistry, we compared the expression of Dicer protein among 404 clinically annotated controls and skin tumors consisting of melanocytic nevi (n = 71), a variety of melanomas (n = 223), carcinomas (n = 73) and sarcomas (n = 12). Results showed a cell-specific up-regulated Dicer in 81% of cutaneous, 80% of acrolentiginous and 96% of metastatic melanoma specimens compared to carcinoma or sarcoma specimens (P<0.0001). The expression of Dicer was significantly higher in melanomas compared to benign melanocytic nevi (P<0.0001). In patients with cutaneous melanomas, Dicer up-regulation was found to be significantly associated with an increased tumor mitotic index (P = 0.04), Breslow's depth of invasion (P = 0.03), nodal metastasis (P = 0.04) and a higher American Joint Committee on Caner (AJCC) clinical stage (P = 0.009). Using western blot analysis, we confirmed the cell-specific up-regulation of Dicer protein in vitro. A pooled-analysis on mRNA profiling in cutaneous tumors showed up-regulation of Dicer at the RNA level in cutaneous melanoma, also showing deregulation of other enzymes that participate in the biogenesis and maturation of canonical miRNAs.

Conclusions: Increased Dicer expression may be a clinically useful biomarker for patients with cutaneous melanoma. Understanding deregulation of Dicer and its influence on miRNA maturation is needed to predict the susceptibility of melanoma patients to miRNA-based therapy in the future.

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Dicer expression in cell lines recapitulated the observed deregulation in clinical specimens.A) Western blot analysis of Dicer shows a 219-kDa band. Relative band intensity was compared to succinate dehydrogenase (SDHA, 68 kDa) as a loading control. B) Western blot quantification showed >2 to 4-fold change in Dicer levels in melanoma cell lines (WM278, WM1552C and A375P) when compared to melanocyte-L or other melanoma cell lines (WM35 and A375M). C) Dicer mRNA expression did not correlate with mature let-7a expression in vitro. Using qRT-PCR, the relative expression levels of let-7a miRNA and Dicer mRNA were compared to show no significant correlation. All qRT-PCRs were performed in triplicates. Data were normalized to small nuclear RNA RNU6 for let-7a and GAPDH mRNA for Dicer. The samples are: Primary melanocytes were cultured from individuals with light (Melanocyte-L), medium (Melanocyte-M) and dark (Melanocyte-D) skin color, WM983A (primary melanoma), WM278 (primary melanoma), WM35 (primary melanoma), WM1552C (primary melanoma), C32 (amelanotic primary melanoma), A375P (metastatic melanoma), A375SM (metastatic melanoma) and A2058 (metastatic melanoma).
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pone-0020494-g005: Dicer expression in cell lines recapitulated the observed deregulation in clinical specimens.A) Western blot analysis of Dicer shows a 219-kDa band. Relative band intensity was compared to succinate dehydrogenase (SDHA, 68 kDa) as a loading control. B) Western blot quantification showed >2 to 4-fold change in Dicer levels in melanoma cell lines (WM278, WM1552C and A375P) when compared to melanocyte-L or other melanoma cell lines (WM35 and A375M). C) Dicer mRNA expression did not correlate with mature let-7a expression in vitro. Using qRT-PCR, the relative expression levels of let-7a miRNA and Dicer mRNA were compared to show no significant correlation. All qRT-PCRs were performed in triplicates. Data were normalized to small nuclear RNA RNU6 for let-7a and GAPDH mRNA for Dicer. The samples are: Primary melanocytes were cultured from individuals with light (Melanocyte-L), medium (Melanocyte-M) and dark (Melanocyte-D) skin color, WM983A (primary melanoma), WM278 (primary melanoma), WM35 (primary melanoma), WM1552C (primary melanoma), C32 (amelanotic primary melanoma), A375P (metastatic melanoma), A375SM (metastatic melanoma) and A2058 (metastatic melanoma).

Mentions: Our findings in clinical melanoma specimens raised the question of whether Dicer up-regulation might be intrinsic to the tumor cells. We compared Dicer protein levels between primary melanocytes, primary (n = 3) and metastatic (n = 3) melanoma cell lines. Western blot analysis combined with measured relative band intensity, normalized against succinate dehydrogenase (SDHA), showed >2 to 4-fold higher Dicer levels in melanoma cell lines (WM278, WM1552C and A375P) when compared to melanocyte-L or other melanoma cell lines (WM35 and A375M) (Fig. 5A-B). Furthermore, we tested additional cell lines; combined western blot analysis and measured relative band intensity showed >2-fold higher Dicer levels in melanoma cell lines (WM1552C and A2058) compared to basal cell carcinoma (BCC), primary melanocytes (n = 3) or other melanoma cell lines (WM35 and C32) (results not shown). The melanocytes were derived from three different individuals with light, medium and dark skin color. Dicer levels were comparable among the three melanocytes, despite the skin color. Overall, Dicer expression in cell lines recapitulated the observed deregulation in clinical specimens, confirming higher Dicer immunostaining in melanomas when compared to melanocytic nevi or carcinomas of the skin (Figs. 1, 2 and 3).


Up-regulated Dicer expression in patients with cutaneous melanoma.

Ma Z, Swede H, Cassarino D, Fleming E, Fire A, Dadras SS - PLoS ONE (2011)

Dicer expression in cell lines recapitulated the observed deregulation in clinical specimens.A) Western blot analysis of Dicer shows a 219-kDa band. Relative band intensity was compared to succinate dehydrogenase (SDHA, 68 kDa) as a loading control. B) Western blot quantification showed >2 to 4-fold change in Dicer levels in melanoma cell lines (WM278, WM1552C and A375P) when compared to melanocyte-L or other melanoma cell lines (WM35 and A375M). C) Dicer mRNA expression did not correlate with mature let-7a expression in vitro. Using qRT-PCR, the relative expression levels of let-7a miRNA and Dicer mRNA were compared to show no significant correlation. All qRT-PCRs were performed in triplicates. Data were normalized to small nuclear RNA RNU6 for let-7a and GAPDH mRNA for Dicer. The samples are: Primary melanocytes were cultured from individuals with light (Melanocyte-L), medium (Melanocyte-M) and dark (Melanocyte-D) skin color, WM983A (primary melanoma), WM278 (primary melanoma), WM35 (primary melanoma), WM1552C (primary melanoma), C32 (amelanotic primary melanoma), A375P (metastatic melanoma), A375SM (metastatic melanoma) and A2058 (metastatic melanoma).
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Related In: Results  -  Collection

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pone-0020494-g005: Dicer expression in cell lines recapitulated the observed deregulation in clinical specimens.A) Western blot analysis of Dicer shows a 219-kDa band. Relative band intensity was compared to succinate dehydrogenase (SDHA, 68 kDa) as a loading control. B) Western blot quantification showed >2 to 4-fold change in Dicer levels in melanoma cell lines (WM278, WM1552C and A375P) when compared to melanocyte-L or other melanoma cell lines (WM35 and A375M). C) Dicer mRNA expression did not correlate with mature let-7a expression in vitro. Using qRT-PCR, the relative expression levels of let-7a miRNA and Dicer mRNA were compared to show no significant correlation. All qRT-PCRs were performed in triplicates. Data were normalized to small nuclear RNA RNU6 for let-7a and GAPDH mRNA for Dicer. The samples are: Primary melanocytes were cultured from individuals with light (Melanocyte-L), medium (Melanocyte-M) and dark (Melanocyte-D) skin color, WM983A (primary melanoma), WM278 (primary melanoma), WM35 (primary melanoma), WM1552C (primary melanoma), C32 (amelanotic primary melanoma), A375P (metastatic melanoma), A375SM (metastatic melanoma) and A2058 (metastatic melanoma).
Mentions: Our findings in clinical melanoma specimens raised the question of whether Dicer up-regulation might be intrinsic to the tumor cells. We compared Dicer protein levels between primary melanocytes, primary (n = 3) and metastatic (n = 3) melanoma cell lines. Western blot analysis combined with measured relative band intensity, normalized against succinate dehydrogenase (SDHA), showed >2 to 4-fold higher Dicer levels in melanoma cell lines (WM278, WM1552C and A375P) when compared to melanocyte-L or other melanoma cell lines (WM35 and A375M) (Fig. 5A-B). Furthermore, we tested additional cell lines; combined western blot analysis and measured relative band intensity showed >2-fold higher Dicer levels in melanoma cell lines (WM1552C and A2058) compared to basal cell carcinoma (BCC), primary melanocytes (n = 3) or other melanoma cell lines (WM35 and C32) (results not shown). The melanocytes were derived from three different individuals with light, medium and dark skin color. Dicer levels were comparable among the three melanocytes, despite the skin color. Overall, Dicer expression in cell lines recapitulated the observed deregulation in clinical specimens, confirming higher Dicer immunostaining in melanomas when compared to melanocytic nevi or carcinomas of the skin (Figs. 1, 2 and 3).

Bottom Line: The expression of Dicer was significantly higher in melanomas compared to benign melanocytic nevi (P<0.0001).In patients with cutaneous melanomas, Dicer up-regulation was found to be significantly associated with an increased tumor mitotic index (P = 0.04), Breslow's depth of invasion (P = 0.03), nodal metastasis (P = 0.04) and a higher American Joint Committee on Caner (AJCC) clinical stage (P = 0.009).Understanding deregulation of Dicer and its influence on miRNA maturation is needed to predict the susceptibility of melanoma patients to miRNA-based therapy in the future.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Stanford University School of Medicine, Stanford, California, United States of America.

ABSTRACT

Background: MicroRNAs (miRNAs) are small non-coding RNAs (18-24 nucleotides) that have recently been shown to regulate gene expression during cancer progression. Dicer, a central enzyme in the multi-component miRNA biogenesis pathway, is involved in cutting precursor miRNAs to functionally mature forms. Emerging evidence shows that Dicer expression is deregulated in some human malignancies and it correlates with tumor progression, yet this role has not yet been investigated in skin cancers.

Methods and findings: Using an anti-human monoclonal antibody against Dicer and immunohistochemistry, we compared the expression of Dicer protein among 404 clinically annotated controls and skin tumors consisting of melanocytic nevi (n = 71), a variety of melanomas (n = 223), carcinomas (n = 73) and sarcomas (n = 12). Results showed a cell-specific up-regulated Dicer in 81% of cutaneous, 80% of acrolentiginous and 96% of metastatic melanoma specimens compared to carcinoma or sarcoma specimens (P<0.0001). The expression of Dicer was significantly higher in melanomas compared to benign melanocytic nevi (P<0.0001). In patients with cutaneous melanomas, Dicer up-regulation was found to be significantly associated with an increased tumor mitotic index (P = 0.04), Breslow's depth of invasion (P = 0.03), nodal metastasis (P = 0.04) and a higher American Joint Committee on Caner (AJCC) clinical stage (P = 0.009). Using western blot analysis, we confirmed the cell-specific up-regulation of Dicer protein in vitro. A pooled-analysis on mRNA profiling in cutaneous tumors showed up-regulation of Dicer at the RNA level in cutaneous melanoma, also showing deregulation of other enzymes that participate in the biogenesis and maturation of canonical miRNAs.

Conclusions: Increased Dicer expression may be a clinically useful biomarker for patients with cutaneous melanoma. Understanding deregulation of Dicer and its influence on miRNA maturation is needed to predict the susceptibility of melanoma patients to miRNA-based therapy in the future.

Show MeSH
Related in: MedlinePlus