Limits...
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.

Show MeSH

Related in: MedlinePlus

Methylation status of the EphA5 gene promoter in prostate tissue. A, EphA5 methylation status was determined by MSP-PCR. All of the prostate cancer tissues exhibit complete methylation of the EphA5 gene. The unmethylated alleles were detected in adjacent noncancerous (top) and BPH (bottom) tissues. Lanes labeled “M” and “U” denote products amplified by primers recognizing methylated and unmethylated sequences, respectively. B, Methylation patterns of individual EphA5 promoter clones from prostate tissue that were bisulfite sequenced are shown. Six clones from each sample were bisulfite sequenced to obtain a representative sampling of methylation patterns; CpG dinucleotides are represented by squares (■, methylated cytokines; □, unmethylated cytosines). Sample names and the methylation percentage of the corresponding tissue are indicated on the left and right sides, respectively. C, Representative examples of an unmethylated EphA5 CpG island in sample N1 (top) and a highly methylated CpG island in sample T24 are shown, as determined by bisulfite sequencing analysis. Arrows indicate positions of CpG dinucleotides.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Methylation status of the EphA5 gene promoter in prostate tissue. A, EphA5 methylation status was determined by MSP-PCR. All of the prostate cancer tissues exhibit complete methylation of the EphA5 gene. The unmethylated alleles were detected in adjacent noncancerous (top) and BPH (bottom) tissues. Lanes labeled “M” and “U” denote products amplified by primers recognizing methylated and unmethylated sequences, respectively. B, Methylation patterns of individual EphA5 promoter clones from prostate tissue that were bisulfite sequenced are shown. Six clones from each sample were bisulfite sequenced to obtain a representative sampling of methylation patterns; CpG dinucleotides are represented by squares (■, methylated cytokines; □, unmethylated cytosines). Sample names and the methylation percentage of the corresponding tissue are indicated on the left and right sides, respectively. C, Representative examples of an unmethylated EphA5 CpG island in sample N1 (top) and a highly methylated CpG island in sample T24 are shown, as determined by bisulfite sequencing analysis. Arrows indicate positions of CpG dinucleotides.

Mentions: To determine whether EphA5 hypermethylation also occurs in primary prostate tumors, the methylation status of EphA5 was determined by MSP-PCR in 39 BPH tissues, 22 primary prostate tumor tissues and 23 paired noncancerous and tumor tissues. The frequency of EphA5 promoter methylation was significantly higher in prostate cancer samples (32 of 45, 71.1%) than in BPH tissue samples (5 of 39, 12.8%; p < 0.01) and paired noncancerous tissues (2 of 23, 8.7%; p < 0.01). Of these 32 methylated prostate cancer samples, EphA5 expression was markedly downregulated in 25 samples. The correlation between EphA5 expression and hypermethylation of the CpG island was significant (p = 0.001). Among the 23 paired prostate carcinoma specimens, the hypermethylation of EphA5 was detected in 69.6% (16/23) prostate carcinoma tissues. Of these 16 prostate cancer samples, 15(93.8%) exhibited the downregulation of EphA5 expression than that of their respectively matched noncancerous tissues, implying that the hypermethylation of EphA5 was significantly correlated with the downregulation of EphA5 (p < 0.01) (Additional file 1: Table S1). The unmethylated form of EphA5, which was present in all samples, is likely due to the inherent contamination with normal (nonmalignant) cells or partial methylation. Representative results from MSP–PCR analyses in prostate tissue are shown in Figure 4A.Figure 4


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)

Methylation status of the EphA5 gene promoter in prostate tissue. A, EphA5 methylation status was determined by MSP-PCR. All of the prostate cancer tissues exhibit complete methylation of the EphA5 gene. The unmethylated alleles were detected in adjacent noncancerous (top) and BPH (bottom) tissues. Lanes labeled “M” and “U” denote products amplified by primers recognizing methylated and unmethylated sequences, respectively. B, Methylation patterns of individual EphA5 promoter clones from prostate tissue that were bisulfite sequenced are shown. Six clones from each sample were bisulfite sequenced to obtain a representative sampling of methylation patterns; CpG dinucleotides are represented by squares (■, methylated cytokines; □, unmethylated cytosines). Sample names and the methylation percentage of the corresponding tissue are indicated on the left and right sides, respectively. C, Representative examples of an unmethylated EphA5 CpG island in sample N1 (top) and a highly methylated CpG island in sample T24 are shown, as determined by bisulfite sequencing analysis. Arrows indicate positions of CpG dinucleotides.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Methylation status of the EphA5 gene promoter in prostate tissue. A, EphA5 methylation status was determined by MSP-PCR. All of the prostate cancer tissues exhibit complete methylation of the EphA5 gene. The unmethylated alleles were detected in adjacent noncancerous (top) and BPH (bottom) tissues. Lanes labeled “M” and “U” denote products amplified by primers recognizing methylated and unmethylated sequences, respectively. B, Methylation patterns of individual EphA5 promoter clones from prostate tissue that were bisulfite sequenced are shown. Six clones from each sample were bisulfite sequenced to obtain a representative sampling of methylation patterns; CpG dinucleotides are represented by squares (■, methylated cytokines; □, unmethylated cytosines). Sample names and the methylation percentage of the corresponding tissue are indicated on the left and right sides, respectively. C, Representative examples of an unmethylated EphA5 CpG island in sample N1 (top) and a highly methylated CpG island in sample T24 are shown, as determined by bisulfite sequencing analysis. Arrows indicate positions of CpG dinucleotides.
Mentions: To determine whether EphA5 hypermethylation also occurs in primary prostate tumors, the methylation status of EphA5 was determined by MSP-PCR in 39 BPH tissues, 22 primary prostate tumor tissues and 23 paired noncancerous and tumor tissues. The frequency of EphA5 promoter methylation was significantly higher in prostate cancer samples (32 of 45, 71.1%) than in BPH tissue samples (5 of 39, 12.8%; p < 0.01) and paired noncancerous tissues (2 of 23, 8.7%; p < 0.01). Of these 32 methylated prostate cancer samples, EphA5 expression was markedly downregulated in 25 samples. The correlation between EphA5 expression and hypermethylation of the CpG island was significant (p = 0.001). Among the 23 paired prostate carcinoma specimens, the hypermethylation of EphA5 was detected in 69.6% (16/23) prostate carcinoma tissues. Of these 16 prostate cancer samples, 15(93.8%) exhibited the downregulation of EphA5 expression than that of their respectively matched noncancerous tissues, implying that the hypermethylation of EphA5 was significantly correlated with the downregulation of EphA5 (p < 0.01) (Additional file 1: Table S1). The unmethylated form of EphA5, which was present in all samples, is likely due to the inherent contamination with normal (nonmalignant) cells or partial methylation. Representative results from MSP–PCR analyses in prostate tissue are shown in Figure 4A.Figure 4

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