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Downregulation of EphA5 by promoter methylation in human prostate cancer.

Li S, Zhu Y, Ma C, Qiu Z, Zhang X, Kang Z, Wu Z, Wang H, Xu X, Zhang H, Ren G, Tang J, Li X, Guan M - BMC Cancer (2015)

Bottom Line: EphA5 is a member of the Eph/ephrin family and plays a critical role in the regulation of carcinogenesis.Among 23 paired prostate carcinoma tissues, 16 tumor samples exhibited the hypermethylation of EphA5, and 15 of these 16 specimens (93.8%) shown the downregulation of EphA5 expression than that of their respectively matched noncancerous samples.Immunostaining analysis demonstrated that the EphA5 protein was absent or down-regulated in 10 of 13 (76.9%) available carcinoma samples, and 8 of these 10 samples (80.0%) exhibited hypermethylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Huashan Hospital, Shanghai Medical School, Fudan University, 12 Central Urumqi Road, Shanghai, 200040, China. sdjnshlb@126.com.

ABSTRACT

Background: EphA5 is a member of the Eph/ephrin family and plays a critical role in the regulation of carcinogenesis. A significant reduction of EphA5 transcripts in high-grade prostate cancer tissue was shown using a transcriptomic analysis, compared to the low-grade prostate cancer tissue. As less is known about the mechanism of EphA5 downregulation and the function of EphA5, here we investigated the expression and an epigenetic change of EphA5 in prostate cancer and determined if these findings were correlated with clinicopathologic characteristics of prostate cancer.

Methods: Seven prostate cell lines (RWPE-1, LNCap, LNCap-LN3, CWR22rv-1, PC-3, PC-3M-LN4, and DU145), thirty-nine BPH, twenty-two primary prostate carcinomas, twenty-three paired noncancerous and cancerous prostate tissues were examined via qRT-PCR, methylation-specific PCR, bisulfite sequencing, immunohistochemistry and western blotting. The role of EphA5 in prostate cancer cell migration and invasion was examined by wound healing and transwell assay.

Results: Downregulation or loss of EphA5 mRNA or protein expression was detected in 28 of 45 (62.2%) prostate carcinomas, 2 of 39 (5.1%) hyperplasias, and all 6 prostate cancer cell lines. Methylation of the EphA5 promoter region was present in 32 of 45 (71.1%) carcinoma samples, 3 of 39 (7.7%) hyperplasias, and the 6 prostate cancer cell lines. Among 23 paired prostate carcinoma tissues, 16 tumor samples exhibited the hypermethylation of EphA5, and 15 of these 16 specimens (93.8%) shown the downregulation of EphA5 expression than that of their respectively matched noncancerous samples. Immunostaining analysis demonstrated that the EphA5 protein was absent or down-regulated in 10 of 13 (76.9%) available carcinoma samples, and 8 of these 10 samples (80.0%) exhibited hypermethylation. The frequency of EphA5 methylation was higher in cancer patients with an elevated Gleason score or T3-T4 staging. Following the treatment of 6 prostate cancer cell lines with 5-aza-2'-deoxycytidine, the levels of EphA5 mRNA were significantly increased. Prostate cancer cells invasion and migration were significantly suppressed by ectopic expression of EphA5 in vitro.

Conclusion: Our study provides evidence that EphA5 is a potential target for epigenetic silencing in primary prostate cancer and is a potentially valuable prognosis predictor and thereapeutic marker for prostate cancer.

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Related in: MedlinePlus

Western blotting analysis of EphA5 protein expression in clinical prostate specimens. A, EphA5 protein (114 kDa band) levels in representative resected fresh prostate cancer tissues and matched non-tumor tissues were analyzed by western blotting and normalized to β-actin expression (43 kDa band). Expression was further normalized to the expression level observed in the first noncancerous specimen. B, EphA5 protein levels (114 kDa band) in representative fresh prostate cancer tissues (collected via biopsy) and benign prostate hyperplasias were analyzed by western blotting and normalized to β-actin (43 kDa band). Protein levels were further normalized to the expression level observed in the first benign prostate hyperplasia specimen. Representative results from triplicate experiments are shown as mean ± SD (n = 3). *P < 0.05, vs. the respective noncancerous tissue and BPH. N = non-tumor tissue; T = tumor tissue; BPH = benign prostate hyperplasia.
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Fig2: Western blotting analysis of EphA5 protein expression in clinical prostate specimens. A, EphA5 protein (114 kDa band) levels in representative resected fresh prostate cancer tissues and matched non-tumor tissues were analyzed by western blotting and normalized to β-actin expression (43 kDa band). Expression was further normalized to the expression level observed in the first noncancerous specimen. B, EphA5 protein levels (114 kDa band) in representative fresh prostate cancer tissues (collected via biopsy) and benign prostate hyperplasias were analyzed by western blotting and normalized to β-actin (43 kDa band). Protein levels were further normalized to the expression level observed in the first benign prostate hyperplasia specimen. Representative results from triplicate experiments are shown as mean ± SD (n = 3). *P < 0.05, vs. the respective noncancerous tissue and BPH. N = non-tumor tissue; T = tumor tissue; BPH = benign prostate hyperplasia.

Mentions: To further validate the expression of EphA5 in human PCa tissue, we also analysed 4 BPH tissues, 5 primary prostate tumors tissues and 4 paired normal tissues in our study by Western blotting assay. Similar to the results of qRT–PCR in the corresponding tissues, EphA5 protein level in prostate tumour samples was significantly lower than that of matched adjacent normal tissues or BPH tissues (Figure 2).Figure 2


Downregulation of EphA5 by promoter methylation in human prostate cancer.

Li S, Zhu Y, Ma C, Qiu Z, Zhang X, Kang Z, Wu Z, Wang H, Xu X, Zhang H, Ren G, Tang J, Li X, Guan M - BMC Cancer (2015)

Western blotting analysis of EphA5 protein expression in clinical prostate specimens. A, EphA5 protein (114 kDa band) levels in representative resected fresh prostate cancer tissues and matched non-tumor tissues were analyzed by western blotting and normalized to β-actin expression (43 kDa band). Expression was further normalized to the expression level observed in the first noncancerous specimen. B, EphA5 protein levels (114 kDa band) in representative fresh prostate cancer tissues (collected via biopsy) and benign prostate hyperplasias were analyzed by western blotting and normalized to β-actin (43 kDa band). Protein levels were further normalized to the expression level observed in the first benign prostate hyperplasia specimen. Representative results from triplicate experiments are shown as mean ± SD (n = 3). *P < 0.05, vs. the respective noncancerous tissue and BPH. N = non-tumor tissue; T = tumor tissue; BPH = benign prostate hyperplasia.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4307617&req=5

Fig2: Western blotting analysis of EphA5 protein expression in clinical prostate specimens. A, EphA5 protein (114 kDa band) levels in representative resected fresh prostate cancer tissues and matched non-tumor tissues were analyzed by western blotting and normalized to β-actin expression (43 kDa band). Expression was further normalized to the expression level observed in the first noncancerous specimen. B, EphA5 protein levels (114 kDa band) in representative fresh prostate cancer tissues (collected via biopsy) and benign prostate hyperplasias were analyzed by western blotting and normalized to β-actin (43 kDa band). Protein levels were further normalized to the expression level observed in the first benign prostate hyperplasia specimen. Representative results from triplicate experiments are shown as mean ± SD (n = 3). *P < 0.05, vs. the respective noncancerous tissue and BPH. N = non-tumor tissue; T = tumor tissue; BPH = benign prostate hyperplasia.
Mentions: To further validate the expression of EphA5 in human PCa tissue, we also analysed 4 BPH tissues, 5 primary prostate tumors tissues and 4 paired normal tissues in our study by Western blotting assay. Similar to the results of qRT–PCR in the corresponding tissues, EphA5 protein level in prostate tumour samples was significantly lower than that of matched adjacent normal tissues or BPH tissues (Figure 2).Figure 2

Bottom Line: EphA5 is a member of the Eph/ephrin family and plays a critical role in the regulation of carcinogenesis.Among 23 paired prostate carcinoma tissues, 16 tumor samples exhibited the hypermethylation of EphA5, and 15 of these 16 specimens (93.8%) shown the downregulation of EphA5 expression than that of their respectively matched noncancerous samples.Immunostaining analysis demonstrated that the EphA5 protein was absent or down-regulated in 10 of 13 (76.9%) available carcinoma samples, and 8 of these 10 samples (80.0%) exhibited hypermethylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Huashan Hospital, Shanghai Medical School, Fudan University, 12 Central Urumqi Road, Shanghai, 200040, China. sdjnshlb@126.com.

ABSTRACT

Background: EphA5 is a member of the Eph/ephrin family and plays a critical role in the regulation of carcinogenesis. A significant reduction of EphA5 transcripts in high-grade prostate cancer tissue was shown using a transcriptomic analysis, compared to the low-grade prostate cancer tissue. As less is known about the mechanism of EphA5 downregulation and the function of EphA5, here we investigated the expression and an epigenetic change of EphA5 in prostate cancer and determined if these findings were correlated with clinicopathologic characteristics of prostate cancer.

Methods: Seven prostate cell lines (RWPE-1, LNCap, LNCap-LN3, CWR22rv-1, PC-3, PC-3M-LN4, and DU145), thirty-nine BPH, twenty-two primary prostate carcinomas, twenty-three paired noncancerous and cancerous prostate tissues were examined via qRT-PCR, methylation-specific PCR, bisulfite sequencing, immunohistochemistry and western blotting. The role of EphA5 in prostate cancer cell migration and invasion was examined by wound healing and transwell assay.

Results: Downregulation or loss of EphA5 mRNA or protein expression was detected in 28 of 45 (62.2%) prostate carcinomas, 2 of 39 (5.1%) hyperplasias, and all 6 prostate cancer cell lines. Methylation of the EphA5 promoter region was present in 32 of 45 (71.1%) carcinoma samples, 3 of 39 (7.7%) hyperplasias, and the 6 prostate cancer cell lines. Among 23 paired prostate carcinoma tissues, 16 tumor samples exhibited the hypermethylation of EphA5, and 15 of these 16 specimens (93.8%) shown the downregulation of EphA5 expression than that of their respectively matched noncancerous samples. Immunostaining analysis demonstrated that the EphA5 protein was absent or down-regulated in 10 of 13 (76.9%) available carcinoma samples, and 8 of these 10 samples (80.0%) exhibited hypermethylation. The frequency of EphA5 methylation was higher in cancer patients with an elevated Gleason score or T3-T4 staging. Following the treatment of 6 prostate cancer cell lines with 5-aza-2'-deoxycytidine, the levels of EphA5 mRNA were significantly increased. Prostate cancer cells invasion and migration were significantly suppressed by ectopic expression of EphA5 in vitro.

Conclusion: Our study provides evidence that EphA5 is a potential target for epigenetic silencing in primary prostate cancer and is a potentially valuable prognosis predictor and thereapeutic marker for prostate cancer.

Show MeSH
Related in: MedlinePlus