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Fibulin-1 is downregulated through promoter hypermethylation in colorectal cancer: a CONSORT study.

Xu Z, Chen H, Liu D, Huo J - Medicine (Baltimore) (2015)

Bottom Line: Furthermore, the methylated level of FBLN1 was analyzed with the clinicopathological characteristics.The methylation rate of FBLN1 promoter was significantly higher in CRC tissues than that in adjacent nontumor tissues (P < 0.001).In addition, the correlation between FBLN1 hypermethylation, protein expression, and overall survival (OS) was statistically significant.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Gastroenterology (ZX, DL, JH), 2nd Xiangya Hospital, Central South University, Changsha, Hunan; and Department of Gastroenterology (ZX, HC), People's Hospital of Taizhou, Taizhou, Jiangsu, China.

ABSTRACT
Fibulin-1 (FBLN1) is involved in the progression of some types of cancer. However, the role of FBLN1 in colorectal cancer (CRC) has not been examined. The purpose of this study was to understand the molecular mechanisms and clinical significance of FBLN1 inactivation in CRC. The expression of FBLN1 in CRC tissues and adjacent normal tissues was analyzed by immunohistochemical analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Methylation-specific polymerase chain reaction (MSP) and bisulfite sequencing PCR (BSP) were performed to examine the methylation status of the FBLN1 gene promoter. Furthermore, the methylated level of FBLN1 was analyzed with the clinicopathological characteristics. Immunohistochemical analysis and qRT-PCR analysis showed that FBLN1 protein and messenger RNA (mRNA) levels in tumor tissues were both significantly decreased compared with that in adjacent nontumor tissues. The methylation rate of FBLN1 promoter was significantly higher in CRC tissues than that in adjacent nontumor tissues (P < 0.001). In addition, the correlation between FBLN1 hypermethylation, protein expression, and overall survival (OS) was statistically significant. Our results indicated that the FBLN1 gene may be a novel candidate of tumor suppressor gene in CRC, and that promoter hypermethylation of FBLN1 is an important reason for its downregulation and is also a good predictor of OS for CRC.

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Determination of FBLN1 methylation status. (A) Schematic diagram of CpG dinucleotides within the FBLN1 promoter. FBLN1 is located on chromosome 22, band q13. The promoter region contains a CpG island. CpG sites are shown as pink bars. The blue line shows the region tested in MSP, and the red line indicates the region tested in BSP. (B) Representative results of the MSP analysis for FBLN1 in selected four pairs of samples, respectively. (U = unmethylated; M = methylated; T = tumor tissue; N = adjacent normal tissue.) (C) Bisulfite sequencing analysis of the upstream regulatory region of FBLN1 in representative tissues (N = adjacent nontumor tissue; T = CRC sample). For each sample, at least five separate clones were sequenced and the results are shown here. Black and white circles represent methylated and unmethylated CpG, respectively. For each row of circles sequence results for an individual clone of the bisulfite-PCR product are given. The methylation level is given as a percentage on the right of each bisulfite result. (D) The methylation status of FBLN1 promoter was compared between CRC tissues and adjacent nontumor tissues. (BSP = bisulfite sequencing polymerase chain reaction; CpG = cytosine-phosphate-guanine, FBLN1 = fibulin-1; MSP = methylation-specific polymerase chain reaction; PCR =  polymerase chain reaction). ∗∗P < 0.001.
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Figure 1: Determination of FBLN1 methylation status. (A) Schematic diagram of CpG dinucleotides within the FBLN1 promoter. FBLN1 is located on chromosome 22, band q13. The promoter region contains a CpG island. CpG sites are shown as pink bars. The blue line shows the region tested in MSP, and the red line indicates the region tested in BSP. (B) Representative results of the MSP analysis for FBLN1 in selected four pairs of samples, respectively. (U = unmethylated; M = methylated; T = tumor tissue; N = adjacent normal tissue.) (C) Bisulfite sequencing analysis of the upstream regulatory region of FBLN1 in representative tissues (N = adjacent nontumor tissue; T = CRC sample). For each sample, at least five separate clones were sequenced and the results are shown here. Black and white circles represent methylated and unmethylated CpG, respectively. For each row of circles sequence results for an individual clone of the bisulfite-PCR product are given. The methylation level is given as a percentage on the right of each bisulfite result. (D) The methylation status of FBLN1 promoter was compared between CRC tissues and adjacent nontumor tissues. (BSP = bisulfite sequencing polymerase chain reaction; CpG = cytosine-phosphate-guanine, FBLN1 = fibulin-1; MSP = methylation-specific polymerase chain reaction; PCR =  polymerase chain reaction). ∗∗P < 0.001.

Mentions: To investigate whether epigenetic alteration could be extrapolated to CRC, we firstly performed MSP analysis to determine the methylation status in the promoter regions of FBLN1 in CRC tissues and adjacent nontumor tissues. The results showed that the FBLN1 promoter was hypermethylated in tumor tissues (Figure 1A and 1B). Furthermore, we designed and validated bisulfate sequencing PCR for FBLN1 methylation within its promoter region which included 36 CpGs (Figure 1A). The results showed that the CpG sites were highly methylated in tumor tissues for FBLN1, and the methylation level varied from 30% to 77.2%, with a mean ratio of 61.54% in the tumor tissue (Figure 1C and 1D). In contrast, the methylation level observed in the adjacent nontumor samples ranged from 10.6% to 42.8%, with a mean of and 25.98% (Figure 1C and 1D). Methylation levels >36.8% were considered hypermethylation. FBLN1 was hypermethylated in 59 (86.8%) of 68 tumors.


Fibulin-1 is downregulated through promoter hypermethylation in colorectal cancer: a CONSORT study.

Xu Z, Chen H, Liu D, Huo J - Medicine (Baltimore) (2015)

Determination of FBLN1 methylation status. (A) Schematic diagram of CpG dinucleotides within the FBLN1 promoter. FBLN1 is located on chromosome 22, band q13. The promoter region contains a CpG island. CpG sites are shown as pink bars. The blue line shows the region tested in MSP, and the red line indicates the region tested in BSP. (B) Representative results of the MSP analysis for FBLN1 in selected four pairs of samples, respectively. (U = unmethylated; M = methylated; T = tumor tissue; N = adjacent normal tissue.) (C) Bisulfite sequencing analysis of the upstream regulatory region of FBLN1 in representative tissues (N = adjacent nontumor tissue; T = CRC sample). For each sample, at least five separate clones were sequenced and the results are shown here. Black and white circles represent methylated and unmethylated CpG, respectively. For each row of circles sequence results for an individual clone of the bisulfite-PCR product are given. The methylation level is given as a percentage on the right of each bisulfite result. (D) The methylation status of FBLN1 promoter was compared between CRC tissues and adjacent nontumor tissues. (BSP = bisulfite sequencing polymerase chain reaction; CpG = cytosine-phosphate-guanine, FBLN1 = fibulin-1; MSP = methylation-specific polymerase chain reaction; PCR =  polymerase chain reaction). ∗∗P < 0.001.
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Related In: Results  -  Collection

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Figure 1: Determination of FBLN1 methylation status. (A) Schematic diagram of CpG dinucleotides within the FBLN1 promoter. FBLN1 is located on chromosome 22, band q13. The promoter region contains a CpG island. CpG sites are shown as pink bars. The blue line shows the region tested in MSP, and the red line indicates the region tested in BSP. (B) Representative results of the MSP analysis for FBLN1 in selected four pairs of samples, respectively. (U = unmethylated; M = methylated; T = tumor tissue; N = adjacent normal tissue.) (C) Bisulfite sequencing analysis of the upstream regulatory region of FBLN1 in representative tissues (N = adjacent nontumor tissue; T = CRC sample). For each sample, at least five separate clones were sequenced and the results are shown here. Black and white circles represent methylated and unmethylated CpG, respectively. For each row of circles sequence results for an individual clone of the bisulfite-PCR product are given. The methylation level is given as a percentage on the right of each bisulfite result. (D) The methylation status of FBLN1 promoter was compared between CRC tissues and adjacent nontumor tissues. (BSP = bisulfite sequencing polymerase chain reaction; CpG = cytosine-phosphate-guanine, FBLN1 = fibulin-1; MSP = methylation-specific polymerase chain reaction; PCR =  polymerase chain reaction). ∗∗P < 0.001.
Mentions: To investigate whether epigenetic alteration could be extrapolated to CRC, we firstly performed MSP analysis to determine the methylation status in the promoter regions of FBLN1 in CRC tissues and adjacent nontumor tissues. The results showed that the FBLN1 promoter was hypermethylated in tumor tissues (Figure 1A and 1B). Furthermore, we designed and validated bisulfate sequencing PCR for FBLN1 methylation within its promoter region which included 36 CpGs (Figure 1A). The results showed that the CpG sites were highly methylated in tumor tissues for FBLN1, and the methylation level varied from 30% to 77.2%, with a mean ratio of 61.54% in the tumor tissue (Figure 1C and 1D). In contrast, the methylation level observed in the adjacent nontumor samples ranged from 10.6% to 42.8%, with a mean of and 25.98% (Figure 1C and 1D). Methylation levels >36.8% were considered hypermethylation. FBLN1 was hypermethylated in 59 (86.8%) of 68 tumors.

Bottom Line: Furthermore, the methylated level of FBLN1 was analyzed with the clinicopathological characteristics.The methylation rate of FBLN1 promoter was significantly higher in CRC tissues than that in adjacent nontumor tissues (P < 0.001).In addition, the correlation between FBLN1 hypermethylation, protein expression, and overall survival (OS) was statistically significant.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Gastroenterology (ZX, DL, JH), 2nd Xiangya Hospital, Central South University, Changsha, Hunan; and Department of Gastroenterology (ZX, HC), People's Hospital of Taizhou, Taizhou, Jiangsu, China.

ABSTRACT
Fibulin-1 (FBLN1) is involved in the progression of some types of cancer. However, the role of FBLN1 in colorectal cancer (CRC) has not been examined. The purpose of this study was to understand the molecular mechanisms and clinical significance of FBLN1 inactivation in CRC. The expression of FBLN1 in CRC tissues and adjacent normal tissues was analyzed by immunohistochemical analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Methylation-specific polymerase chain reaction (MSP) and bisulfite sequencing PCR (BSP) were performed to examine the methylation status of the FBLN1 gene promoter. Furthermore, the methylated level of FBLN1 was analyzed with the clinicopathological characteristics. Immunohistochemical analysis and qRT-PCR analysis showed that FBLN1 protein and messenger RNA (mRNA) levels in tumor tissues were both significantly decreased compared with that in adjacent nontumor tissues. The methylation rate of FBLN1 promoter was significantly higher in CRC tissues than that in adjacent nontumor tissues (P < 0.001). In addition, the correlation between FBLN1 hypermethylation, protein expression, and overall survival (OS) was statistically significant. Our results indicated that the FBLN1 gene may be a novel candidate of tumor suppressor gene in CRC, and that promoter hypermethylation of FBLN1 is an important reason for its downregulation and is also a good predictor of OS for CRC.

Show MeSH
Related in: MedlinePlus