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Circulating cell-free cancer-testis MAGE-A RNA, BORIS RNA, let-7b and miR-202 in the blood of patients with breast cancer and benign breast diseases.

Joosse SA, Müller V, Steinbach B, Pantel K, Schwarzenbach H - Br. J. Cancer (2014)

Bottom Line: The serum levels of MAGE-A and BORIS mRNA, as well as let-7b were significantly higher in patients with invasive carcinomas than in patients with benign breast diseases or healthy women (P<0.001), whereas the levels of miR-202 were elevated in both patient cohorts (P<0.001).In uni- and multivariate analyses, high levels of miR-202 significantly correlated with poor overall survival (P=0.0001).Moreover, serum miR-202 is associated with prognosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

ABSTRACT

Background: MAGE-A (melanoma-associated antigen-A) are promising targets for specific immunotherapy and their expression may be induced by the epigenetic factor BORIS.

Methods: To determine their relevance for breast cancer, we quantified the levels of MAGE-A1, -A2, -A3, -A12 and BORIS mRNA, as well as microRNAs let-7b and miR-202 in pre- and postoperative serum of 102 and 34 breast cancer patients, respectively, and in serum of 26 patients with benign breast diseases and 37 healthy women by real-time PCR. The mean follow-up time of the cancer patients was 6.2 years.

Results: The serum levels of MAGE-A and BORIS mRNA, as well as let-7b were significantly higher in patients with invasive carcinomas than in patients with benign breast diseases or healthy women (P<0.001), whereas the levels of miR-202 were elevated in both patient cohorts (P<0.001). In uni- and multivariate analyses, high levels of miR-202 significantly correlated with poor overall survival (P=0.0001). Transfection of breast cancer cells with synthetic microRNAs and their inhibitors showed that let-7b and miR-202 did not affect the protein expression of MAGE-A1.

Conclusions: Based on their cancer-specific increase in breast cancer patients, circulating MAGE-A and BORIS mRNAs may be further explored for early detection of breast cancer and monitoring of MAGE-directed immunotherapies. Moreover, serum miR-202 is associated with prognosis.

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Related in: MedlinePlus

Let-7b and miR-202 do not affect the protein expression of MAGE-A1. Protein levels in MCF-7 cells were analysed by western blot. HSC70 signals served as a loading control. MAGE-A1 protein levels in MCF-7 cells transfected with only transfection agent (basal) or transfected with mimics and inhibitors (Inh.) of let-7b and miR-202 or scrambled RNA (negative control). Representative analysis of three independent experiments is shown, all experiments were performed in duplicate.
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fig4: Let-7b and miR-202 do not affect the protein expression of MAGE-A1. Protein levels in MCF-7 cells were analysed by western blot. HSC70 signals served as a loading control. MAGE-A1 protein levels in MCF-7 cells transfected with only transfection agent (basal) or transfected with mimics and inhibitors (Inh.) of let-7b and miR-202 or scrambled RNA (negative control). Representative analysis of three independent experiments is shown, all experiments were performed in duplicate.

Mentions: The screening of the miRbase databases (Griffiths-Jones et al, 2006) revealed potential binding sites of let-7b and miR-202 in the 3′UTR of MAGE-A1 mRNA. To examine whether expression of MAGE-A1 is regulated by these miRs, we performed transfections of the cell lines MCF-7 and BCM1 using mimics or inhibitors of let-7b and miR-202 and scrambled RNA as a negative control. The mimics are double-stranded RNA molecules that mimic the mature endogenous let-7b and miR-202, whereas the inhibitors are single-stranded, modified RNA molecules, which after transfection, specifically binds to mimics and endogenous let-7b and miR-202 and inhibits their function. The data of quantitative real-time PCR using MAGE-A1-specific primers (data not shown) and of western blotting using antibodies specific for MAGE-A1 (Figure 4) showed no effect of the mimics and inhibitors on the RNA and protein levels of MAGE-A1, respectively. On the western blot the signals derived from MCF-7 cells transfected with mimics and inhibitors of both miRs (let-7b and miR-202) and scrambled control as well as from basal (non-transfected) cells were similar (Figure 4) pointing to the hypothesis that both miRs do not bind to the 3′UTR in the MAGE-A1 mRNA.


Circulating cell-free cancer-testis MAGE-A RNA, BORIS RNA, let-7b and miR-202 in the blood of patients with breast cancer and benign breast diseases.

Joosse SA, Müller V, Steinbach B, Pantel K, Schwarzenbach H - Br. J. Cancer (2014)

Let-7b and miR-202 do not affect the protein expression of MAGE-A1. Protein levels in MCF-7 cells were analysed by western blot. HSC70 signals served as a loading control. MAGE-A1 protein levels in MCF-7 cells transfected with only transfection agent (basal) or transfected with mimics and inhibitors (Inh.) of let-7b and miR-202 or scrambled RNA (negative control). Representative analysis of three independent experiments is shown, all experiments were performed in duplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150270&req=5

fig4: Let-7b and miR-202 do not affect the protein expression of MAGE-A1. Protein levels in MCF-7 cells were analysed by western blot. HSC70 signals served as a loading control. MAGE-A1 protein levels in MCF-7 cells transfected with only transfection agent (basal) or transfected with mimics and inhibitors (Inh.) of let-7b and miR-202 or scrambled RNA (negative control). Representative analysis of three independent experiments is shown, all experiments were performed in duplicate.
Mentions: The screening of the miRbase databases (Griffiths-Jones et al, 2006) revealed potential binding sites of let-7b and miR-202 in the 3′UTR of MAGE-A1 mRNA. To examine whether expression of MAGE-A1 is regulated by these miRs, we performed transfections of the cell lines MCF-7 and BCM1 using mimics or inhibitors of let-7b and miR-202 and scrambled RNA as a negative control. The mimics are double-stranded RNA molecules that mimic the mature endogenous let-7b and miR-202, whereas the inhibitors are single-stranded, modified RNA molecules, which after transfection, specifically binds to mimics and endogenous let-7b and miR-202 and inhibits their function. The data of quantitative real-time PCR using MAGE-A1-specific primers (data not shown) and of western blotting using antibodies specific for MAGE-A1 (Figure 4) showed no effect of the mimics and inhibitors on the RNA and protein levels of MAGE-A1, respectively. On the western blot the signals derived from MCF-7 cells transfected with mimics and inhibitors of both miRs (let-7b and miR-202) and scrambled control as well as from basal (non-transfected) cells were similar (Figure 4) pointing to the hypothesis that both miRs do not bind to the 3′UTR in the MAGE-A1 mRNA.

Bottom Line: The serum levels of MAGE-A and BORIS mRNA, as well as let-7b were significantly higher in patients with invasive carcinomas than in patients with benign breast diseases or healthy women (P<0.001), whereas the levels of miR-202 were elevated in both patient cohorts (P<0.001).In uni- and multivariate analyses, high levels of miR-202 significantly correlated with poor overall survival (P=0.0001).Moreover, serum miR-202 is associated with prognosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

ABSTRACT

Background: MAGE-A (melanoma-associated antigen-A) are promising targets for specific immunotherapy and their expression may be induced by the epigenetic factor BORIS.

Methods: To determine their relevance for breast cancer, we quantified the levels of MAGE-A1, -A2, -A3, -A12 and BORIS mRNA, as well as microRNAs let-7b and miR-202 in pre- and postoperative serum of 102 and 34 breast cancer patients, respectively, and in serum of 26 patients with benign breast diseases and 37 healthy women by real-time PCR. The mean follow-up time of the cancer patients was 6.2 years.

Results: The serum levels of MAGE-A and BORIS mRNA, as well as let-7b were significantly higher in patients with invasive carcinomas than in patients with benign breast diseases or healthy women (P<0.001), whereas the levels of miR-202 were elevated in both patient cohorts (P<0.001). In uni- and multivariate analyses, high levels of miR-202 significantly correlated with poor overall survival (P=0.0001). Transfection of breast cancer cells with synthetic microRNAs and their inhibitors showed that let-7b and miR-202 did not affect the protein expression of MAGE-A1.

Conclusions: Based on their cancer-specific increase in breast cancer patients, circulating MAGE-A and BORIS mRNAs may be further explored for early detection of breast cancer and monitoring of MAGE-directed immunotherapies. Moreover, serum miR-202 is associated with prognosis.

Show MeSH
Related in: MedlinePlus