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Inactivation of bone morphogenetic protein 2 may predict clinical outcome and poor overall survival for renal cell carcinoma through epigenetic pathways.

Mitsui Y, Hirata H, Arichi N, Hiraki M, Yasumoto H, Chang I, Fukuhara S, Yamamura S, Shahryari V, Deng G, Saini S, Majid S, Dahiya R, Tanaka Y, Shiina H - Oncotarget (2015)

Bottom Line: The prevalence of BMP-2 promoter methylation was significantly greater and BMP-2 mRNA expression was significantly lower in RCC samples as compared to normal kidney samples.Furthermore, a significant correlation was found between BMP-2 promoter methylation and mRNA transcription in tumors.Aberrant BMP-2 methylation and the resultant loss of BMP-2 expression may be a useful molecular marker for designing improved diagnostic and therapeutic strategies for RCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Shimane University Faculty of Medicine, Enya-cho, Izumo, Japan.

ABSTRACT
We investigated whether impaired regulation of bone morphogenetic protein-2 (BMP-2) via epigenetic pathways is associated with renal cell carcinoma (RCC) pathogenesis. Expression and CpG methylation of the BMP-2 gene were analyzed using RCC cell lines, and 96 matched RCC and normal renal tissues. We also performed functional analysis using BMP-2 restored RCC cells. A significant association of BMP-2 mRNA expression was also found with advanced tumor stage and lymph node involvement, while lower BMP-2 mRNA expression was significantly associated with poor overall survival after radical nephrectomy. In RCC cells, BMP-2 restoration significantly inhibited cell proliferation, migration, invasion, and colony formation. In addition, BMP-2 overexpression induced p21(WAF1/CIP1) and p27(KIP1) expression, and cellular apoptosis in RCC cells. BMP-2 mRNA expression was significantly enhanced in RCC cells by 5-aza-2'-deoxycitidine treatment. The prevalence of BMP-2 promoter methylation was significantly greater and BMP-2 mRNA expression was significantly lower in RCC samples as compared to normal kidney samples. Furthermore, a significant correlation was found between BMP-2 promoter methylation and mRNA transcription in tumors. Aberrant BMP-2 methylation and the resultant loss of BMP-2 expression may be a useful molecular marker for designing improved diagnostic and therapeutic strategies for RCC.

No MeSH data available.


Related in: MedlinePlus

Analysis of BMP-2 methylation in RCC cell lines and clinical samples(A) Schema of BMP-2 promoter and locations of the primers. The universal primers (F, forward; R, reverse) used did not contain any CpG sites within the primer sequence. The F and R methylation-specific PCR (MSP) primers contained 3 and 4 CpG sites, respectively, with un-methylation-specific PCR (USP) primers designed in the same manner. (B) Primer sequences. Boldface indicates difference with modified sequence, and underline indicates changes between methylated and un-methylated sequences after bisulfite modification. (C) Alteration of BMP-2 expression before and after de-methylation. In both Caki-1 and Caki-2 cell lines, the expression level of the mRNA transcript of BMP-2 was significantly increased after 5-aza-dC treatment as compared with that before de-methylation. *P<0.0001. (D) MSP and USP bands in Caki-1 and Caki-2 cells, which were partially methylated. (E) Representative results of MSP and USP of the BMP-2 promoter in clinical samples. Top and bottom show MSP and USP bands, respectively, from the same samples. (F) BMP-2 methylation status in clinical samples. The prevalence of BMP-2 methylation was significantly higher in RCC samples than normal renal tissues (P<0.0001). (G) Typical bisulfite DNA sequencing in normal kidney and RCC samples. In the normal kidney samples, the majority of cytosines within the CpG sites were completely converted to thymines after bisulfite modification, whereas in the RCC samples, the majority of cytosines remained virtually unchanged after bisulfite modification. Horizontal bar, CpG sites; UM, un-methylation; M, methylation.
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Figure 2: Analysis of BMP-2 methylation in RCC cell lines and clinical samples(A) Schema of BMP-2 promoter and locations of the primers. The universal primers (F, forward; R, reverse) used did not contain any CpG sites within the primer sequence. The F and R methylation-specific PCR (MSP) primers contained 3 and 4 CpG sites, respectively, with un-methylation-specific PCR (USP) primers designed in the same manner. (B) Primer sequences. Boldface indicates difference with modified sequence, and underline indicates changes between methylated and un-methylated sequences after bisulfite modification. (C) Alteration of BMP-2 expression before and after de-methylation. In both Caki-1 and Caki-2 cell lines, the expression level of the mRNA transcript of BMP-2 was significantly increased after 5-aza-dC treatment as compared with that before de-methylation. *P<0.0001. (D) MSP and USP bands in Caki-1 and Caki-2 cells, which were partially methylated. (E) Representative results of MSP and USP of the BMP-2 promoter in clinical samples. Top and bottom show MSP and USP bands, respectively, from the same samples. (F) BMP-2 methylation status in clinical samples. The prevalence of BMP-2 methylation was significantly higher in RCC samples than normal renal tissues (P<0.0001). (G) Typical bisulfite DNA sequencing in normal kidney and RCC samples. In the normal kidney samples, the majority of cytosines within the CpG sites were completely converted to thymines after bisulfite modification, whereas in the RCC samples, the majority of cytosines remained virtually unchanged after bisulfite modification. Horizontal bar, CpG sites; UM, un-methylation; M, methylation.

Mentions: We used 5-aza-dC to screen for the epigenetic status of BMP-2 in RCC cell lines. In Caki-1 and Caki-2 cells, the expression level of the BMP-2 mRNA transcript was significantly increased after 5-aza-dC treatment (Fig. 2C), suggesting that promoter CpG methylation may be associated with BMP-2 expression in these cells. To confirm the relationship between CpG methylation and expression of the BMP-2 mRNA transcript, we performed MSP analysis. As shown in Fig. 2A and B, MSP and USP primers were designed based on a previous report [14]. Caki-1 and Caki-2 cells, which slightly express the BMP-2 gene, were partially methylated (Fig. 2D).


Inactivation of bone morphogenetic protein 2 may predict clinical outcome and poor overall survival for renal cell carcinoma through epigenetic pathways.

Mitsui Y, Hirata H, Arichi N, Hiraki M, Yasumoto H, Chang I, Fukuhara S, Yamamura S, Shahryari V, Deng G, Saini S, Majid S, Dahiya R, Tanaka Y, Shiina H - Oncotarget (2015)

Analysis of BMP-2 methylation in RCC cell lines and clinical samples(A) Schema of BMP-2 promoter and locations of the primers. The universal primers (F, forward; R, reverse) used did not contain any CpG sites within the primer sequence. The F and R methylation-specific PCR (MSP) primers contained 3 and 4 CpG sites, respectively, with un-methylation-specific PCR (USP) primers designed in the same manner. (B) Primer sequences. Boldface indicates difference with modified sequence, and underline indicates changes between methylated and un-methylated sequences after bisulfite modification. (C) Alteration of BMP-2 expression before and after de-methylation. In both Caki-1 and Caki-2 cell lines, the expression level of the mRNA transcript of BMP-2 was significantly increased after 5-aza-dC treatment as compared with that before de-methylation. *P<0.0001. (D) MSP and USP bands in Caki-1 and Caki-2 cells, which were partially methylated. (E) Representative results of MSP and USP of the BMP-2 promoter in clinical samples. Top and bottom show MSP and USP bands, respectively, from the same samples. (F) BMP-2 methylation status in clinical samples. The prevalence of BMP-2 methylation was significantly higher in RCC samples than normal renal tissues (P<0.0001). (G) Typical bisulfite DNA sequencing in normal kidney and RCC samples. In the normal kidney samples, the majority of cytosines within the CpG sites were completely converted to thymines after bisulfite modification, whereas in the RCC samples, the majority of cytosines remained virtually unchanged after bisulfite modification. Horizontal bar, CpG sites; UM, un-methylation; M, methylation.
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Figure 2: Analysis of BMP-2 methylation in RCC cell lines and clinical samples(A) Schema of BMP-2 promoter and locations of the primers. The universal primers (F, forward; R, reverse) used did not contain any CpG sites within the primer sequence. The F and R methylation-specific PCR (MSP) primers contained 3 and 4 CpG sites, respectively, with un-methylation-specific PCR (USP) primers designed in the same manner. (B) Primer sequences. Boldface indicates difference with modified sequence, and underline indicates changes between methylated and un-methylated sequences after bisulfite modification. (C) Alteration of BMP-2 expression before and after de-methylation. In both Caki-1 and Caki-2 cell lines, the expression level of the mRNA transcript of BMP-2 was significantly increased after 5-aza-dC treatment as compared with that before de-methylation. *P<0.0001. (D) MSP and USP bands in Caki-1 and Caki-2 cells, which were partially methylated. (E) Representative results of MSP and USP of the BMP-2 promoter in clinical samples. Top and bottom show MSP and USP bands, respectively, from the same samples. (F) BMP-2 methylation status in clinical samples. The prevalence of BMP-2 methylation was significantly higher in RCC samples than normal renal tissues (P<0.0001). (G) Typical bisulfite DNA sequencing in normal kidney and RCC samples. In the normal kidney samples, the majority of cytosines within the CpG sites were completely converted to thymines after bisulfite modification, whereas in the RCC samples, the majority of cytosines remained virtually unchanged after bisulfite modification. Horizontal bar, CpG sites; UM, un-methylation; M, methylation.
Mentions: We used 5-aza-dC to screen for the epigenetic status of BMP-2 in RCC cell lines. In Caki-1 and Caki-2 cells, the expression level of the BMP-2 mRNA transcript was significantly increased after 5-aza-dC treatment (Fig. 2C), suggesting that promoter CpG methylation may be associated with BMP-2 expression in these cells. To confirm the relationship between CpG methylation and expression of the BMP-2 mRNA transcript, we performed MSP analysis. As shown in Fig. 2A and B, MSP and USP primers were designed based on a previous report [14]. Caki-1 and Caki-2 cells, which slightly express the BMP-2 gene, were partially methylated (Fig. 2D).

Bottom Line: The prevalence of BMP-2 promoter methylation was significantly greater and BMP-2 mRNA expression was significantly lower in RCC samples as compared to normal kidney samples.Furthermore, a significant correlation was found between BMP-2 promoter methylation and mRNA transcription in tumors.Aberrant BMP-2 methylation and the resultant loss of BMP-2 expression may be a useful molecular marker for designing improved diagnostic and therapeutic strategies for RCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Shimane University Faculty of Medicine, Enya-cho, Izumo, Japan.

ABSTRACT
We investigated whether impaired regulation of bone morphogenetic protein-2 (BMP-2) via epigenetic pathways is associated with renal cell carcinoma (RCC) pathogenesis. Expression and CpG methylation of the BMP-2 gene were analyzed using RCC cell lines, and 96 matched RCC and normal renal tissues. We also performed functional analysis using BMP-2 restored RCC cells. A significant association of BMP-2 mRNA expression was also found with advanced tumor stage and lymph node involvement, while lower BMP-2 mRNA expression was significantly associated with poor overall survival after radical nephrectomy. In RCC cells, BMP-2 restoration significantly inhibited cell proliferation, migration, invasion, and colony formation. In addition, BMP-2 overexpression induced p21(WAF1/CIP1) and p27(KIP1) expression, and cellular apoptosis in RCC cells. BMP-2 mRNA expression was significantly enhanced in RCC cells by 5-aza-2'-deoxycitidine treatment. The prevalence of BMP-2 promoter methylation was significantly greater and BMP-2 mRNA expression was significantly lower in RCC samples as compared to normal kidney samples. Furthermore, a significant correlation was found between BMP-2 promoter methylation and mRNA transcription in tumors. Aberrant BMP-2 methylation and the resultant loss of BMP-2 expression may be a useful molecular marker for designing improved diagnostic and therapeutic strategies for RCC.

No MeSH data available.


Related in: MedlinePlus