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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

EphA5 overexpression inhibited the migration and invasiveness of Du145 cells. A. Western blot analysis of EphA5 protein (114 kDa band) expression in DU145 cells after transfection with pCMV6-GFP-EphA5. B. Cell migration activity determined with the wound healing assay. C. Cell invasion activity determined with the Matrigel invasion assay (×100). All experiments were performed in triplicate. Data are shown as the mean ± SD. *P < 0.05 vs. pCMV6-AC-GFP group.
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Fig7: EphA5 overexpression inhibited the migration and invasiveness of Du145 cells. A. Western blot analysis of EphA5 protein (114 kDa band) expression in DU145 cells after transfection with pCMV6-GFP-EphA5. B. Cell migration activity determined with the wound healing assay. C. Cell invasion activity determined with the Matrigel invasion assay (×100). All experiments were performed in triplicate. Data are shown as the mean ± SD. *P < 0.05 vs. pCMV6-AC-GFP group.

Mentions: To assess the effect of EphA5 expression on invasion and migration, we transiently infected Du145 cells lacking expression of EphA5 with plasmid expressing pCMV6-AC-GFP or pCMV6-AC-GFP-EphA5 and generated stably transfected cells using G418 selection. Western blotting analysis demonstrated that EphA5 was over-expressed in DU145 cells transfected with pCMV6-AC-GFP-EphA5 (Figure 7A). The wound healing assay demonstrated that the wound-closure rate of pCMV6-AC-GFP-EphA5 cells decreased by 33.7% (p <0.05) when compared with control pCMV6-AC-GFP cell (Figure 7C). The transwell assay showed that the number of invasive cells in pCMV6-AC-GFP-EphA5 cells decreased by 38.1% (p <0.05) when compared with control pCMV6-AC-GFP cell (Figure 7B). These data implied that EphA5 expression inhibited cell migration and invasion in vitro.Figure 7


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)

EphA5 overexpression inhibited the migration and invasiveness of Du145 cells. A. Western blot analysis of EphA5 protein (114 kDa band) expression in DU145 cells after transfection with pCMV6-GFP-EphA5. B. Cell migration activity determined with the wound healing assay. C. Cell invasion activity determined with the Matrigel invasion assay (×100). All experiments were performed in triplicate. Data are shown as the mean ± SD. *P < 0.05 vs. pCMV6-AC-GFP group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: EphA5 overexpression inhibited the migration and invasiveness of Du145 cells. A. Western blot analysis of EphA5 protein (114 kDa band) expression in DU145 cells after transfection with pCMV6-GFP-EphA5. B. Cell migration activity determined with the wound healing assay. C. Cell invasion activity determined with the Matrigel invasion assay (×100). All experiments were performed in triplicate. Data are shown as the mean ± SD. *P < 0.05 vs. pCMV6-AC-GFP group.
Mentions: To assess the effect of EphA5 expression on invasion and migration, we transiently infected Du145 cells lacking expression of EphA5 with plasmid expressing pCMV6-AC-GFP or pCMV6-AC-GFP-EphA5 and generated stably transfected cells using G418 selection. Western blotting analysis demonstrated that EphA5 was over-expressed in DU145 cells transfected with pCMV6-AC-GFP-EphA5 (Figure 7A). The wound healing assay demonstrated that the wound-closure rate of pCMV6-AC-GFP-EphA5 cells decreased by 33.7% (p <0.05) when compared with control pCMV6-AC-GFP cell (Figure 7C). The transwell assay showed that the number of invasive cells in pCMV6-AC-GFP-EphA5 cells decreased by 38.1% (p <0.05) when compared with control pCMV6-AC-GFP cell (Figure 7B). These data implied that EphA5 expression inhibited cell migration and invasion in vitro.Figure 7

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