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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

Methylation status of the EphA5 gene promoter in prostate cell lines. A, EphA5 methylation status was determined by MSP-PCR analysis. All prostate cancer cell lines exhibited complete methylation of the EphA5 gene. Unmethylated EphA5 alleles were detected in RWPE-1 and PC-3 cell lines. Lanes labeled “M” and “U” denote products amplified with primers recognizing methylated and unmethylated sequences, respectively. B, Schematic depiction of the EphA5 promoter-associated CpG island, which spans the region from -103 to +303 with respect to the TSS (+1). The bisulfite sequencing PCR primers are shown in light blue and bold type. The MSP = PCR primers are highlighted in khaki, italicized, and underlined. There are 38 CpG sites in this region; the CpG sites are numbered in red and bold type. C, Methylation patterns of individual EphA5 promoter clones from prostate cell lines that were sequenced using bisulfite methods. 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). Cell-line names and the percentage of methylation for the corresponding cell line are indicated on the left and right sides, respectively. D, Representative chromatograms of CpG sites 14 to 18 obtained from bisulfite sequencing of the EphA5 fragment. Arrows indicate positions of CpG dinucleotides.
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Fig3: Methylation status of the EphA5 gene promoter in prostate cell lines. A, EphA5 methylation status was determined by MSP-PCR analysis. All prostate cancer cell lines exhibited complete methylation of the EphA5 gene. Unmethylated EphA5 alleles were detected in RWPE-1 and PC-3 cell lines. Lanes labeled “M” and “U” denote products amplified with primers recognizing methylated and unmethylated sequences, respectively. B, Schematic depiction of the EphA5 promoter-associated CpG island, which spans the region from -103 to +303 with respect to the TSS (+1). The bisulfite sequencing PCR primers are shown in light blue and bold type. The MSP = PCR primers are highlighted in khaki, italicized, and underlined. There are 38 CpG sites in this region; the CpG sites are numbered in red and bold type. C, Methylation patterns of individual EphA5 promoter clones from prostate cell lines that were sequenced using bisulfite methods. 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). Cell-line names and the percentage of methylation for the corresponding cell line are indicated on the left and right sides, respectively. D, Representative chromatograms of CpG sites 14 to 18 obtained from bisulfite sequencing of the EphA5 fragment. Arrows indicate positions of CpG dinucleotides.

Mentions: To determine the potential mechanism of EphA5 downregulation in prostate cancer, we analyzed the EphA5 gene 5′ regulatory region. We found a CpG island encompassing the transcription start site (TSS) of EphA5. Then, methylation-specific PCR (MSP-PCR) was performed to examine the methylation status of each of the cell lines. Methylated DNA was detected in all six prostate cancer cell lines, whereas the hypermethylation of EphA5 gene was not detected in nonmalignant RWPE-1 cells (Figure 3A). In addition, both methylated and unmethylated sequences were observed in the PC-3 cell line, indicating partial methylation.Figure 3


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 cell lines. A, EphA5 methylation status was determined by MSP-PCR analysis. All prostate cancer cell lines exhibited complete methylation of the EphA5 gene. Unmethylated EphA5 alleles were detected in RWPE-1 and PC-3 cell lines. Lanes labeled “M” and “U” denote products amplified with primers recognizing methylated and unmethylated sequences, respectively. B, Schematic depiction of the EphA5 promoter-associated CpG island, which spans the region from -103 to +303 with respect to the TSS (+1). The bisulfite sequencing PCR primers are shown in light blue and bold type. The MSP = PCR primers are highlighted in khaki, italicized, and underlined. There are 38 CpG sites in this region; the CpG sites are numbered in red and bold type. C, Methylation patterns of individual EphA5 promoter clones from prostate cell lines that were sequenced using bisulfite methods. 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). Cell-line names and the percentage of methylation for the corresponding cell line are indicated on the left and right sides, respectively. D, Representative chromatograms of CpG sites 14 to 18 obtained from bisulfite sequencing of the EphA5 fragment. Arrows indicate positions of CpG dinucleotides.
© Copyright Policy - open-access
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Fig3: Methylation status of the EphA5 gene promoter in prostate cell lines. A, EphA5 methylation status was determined by MSP-PCR analysis. All prostate cancer cell lines exhibited complete methylation of the EphA5 gene. Unmethylated EphA5 alleles were detected in RWPE-1 and PC-3 cell lines. Lanes labeled “M” and “U” denote products amplified with primers recognizing methylated and unmethylated sequences, respectively. B, Schematic depiction of the EphA5 promoter-associated CpG island, which spans the region from -103 to +303 with respect to the TSS (+1). The bisulfite sequencing PCR primers are shown in light blue and bold type. The MSP = PCR primers are highlighted in khaki, italicized, and underlined. There are 38 CpG sites in this region; the CpG sites are numbered in red and bold type. C, Methylation patterns of individual EphA5 promoter clones from prostate cell lines that were sequenced using bisulfite methods. 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). Cell-line names and the percentage of methylation for the corresponding cell line are indicated on the left and right sides, respectively. D, Representative chromatograms of CpG sites 14 to 18 obtained from bisulfite sequencing of the EphA5 fragment. Arrows indicate positions of CpG dinucleotides.
Mentions: To determine the potential mechanism of EphA5 downregulation in prostate cancer, we analyzed the EphA5 gene 5′ regulatory region. We found a CpG island encompassing the transcription start site (TSS) of EphA5. Then, methylation-specific PCR (MSP-PCR) was performed to examine the methylation status of each of the cell lines. Methylated DNA was detected in all six prostate cancer cell lines, whereas the hypermethylation of EphA5 gene was not detected in nonmalignant RWPE-1 cells (Figure 3A). In addition, both methylated and unmethylated sequences were observed in the PC-3 cell line, indicating partial methylation.Figure 3

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