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Methylation-associated gene silencing of RARB in areca carcinogens induced mouse oral squamous cell carcinoma.

Lai ZL, Tsou YA, Fan SR, Tsai MH, Chen HL, Chang NW, Cheng JC, Chen CM - Biomed Res Int (2014)

Bottom Line: These results showed that retinoic acid receptor β (RARB) was indicated in hypermethylation at the promoter region and the loss of expression during cancer development.According to the results of real-time PCR, it was shown that de novo DNA methyltransferases were involved in gene epigenetic alternations of OSCC.Collectively, our results showed that RARB hypermethylation was involved in the areca-associated oral carcinogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Agricultural Biotechnology Center, National Chung Hsing University, No. 250 Kao-Kuang Road, Taichung 402, Taiwan.

ABSTRACT
Regarding oral squamous cell carcinoma (OSCC) development, chewing areca is known to be a strong risk factor in many Asian cultures. Therefore, we established an OSCC induced mouse model by 4-nitroquinoline-1-oxide (4-NQO), or arecoline, or both treatments, respectively. These are the main two components of the areca nut that could increase the occurrence of OSCC. We examined the effects with the noncommercial MCGI (mouse CpG islands) microarray for genome-wide screening the DNA methylation aberrant in induced OSCC mice. The microarray results showed 34 hypermethylated genes in 4-NQO plus arecoline induced OSCC mice tongue tissues. The examinations also used methylation-specific polymerase chain reaction (MS-PCR) and bisulfite sequencing to realize the methylation pattern in collected mouse tongue tissues and human OSCC cell lines of different grades, respectively. These results showed that retinoic acid receptor β (RARB) was indicated in hypermethylation at the promoter region and the loss of expression during cancer development. According to the results of real-time PCR, it was shown that de novo DNA methyltransferases were involved in gene epigenetic alternations of OSCC. Collectively, our results showed that RARB hypermethylation was involved in the areca-associated oral carcinogenesis.

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Methylation status in human oral cancer cell lines of RARB. (a) The schematic for CpG island presentation of RARB. There was a CpG island located in promoter region. We designed the MS-PCR and bisulfite sequencing primer sets in this region. (b) The RARB methylation status in different human oral cancer cell lines. The cell lines were also treated with 5′-aza-dC. The methylation status was recovered because of 5′-aza-dC treatment. (c) The RARB mRNA expression in different human oral cancer cell lines. The RARB was not expressed in Ca922, OC2, and HSC3 obviously. (d) The RARB bisulfite sequencing in different human oral cancer cell lines. There were more methylated RARB in TW2.6, Ca922, OC2, and HSC3 than in NHOK and DOK. The hollow circle is the unmethylated CpG site and the full circle is the methylated CpG site.
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fig4: Methylation status in human oral cancer cell lines of RARB. (a) The schematic for CpG island presentation of RARB. There was a CpG island located in promoter region. We designed the MS-PCR and bisulfite sequencing primer sets in this region. (b) The RARB methylation status in different human oral cancer cell lines. The cell lines were also treated with 5′-aza-dC. The methylation status was recovered because of 5′-aza-dC treatment. (c) The RARB mRNA expression in different human oral cancer cell lines. The RARB was not expressed in Ca922, OC2, and HSC3 obviously. (d) The RARB bisulfite sequencing in different human oral cancer cell lines. There were more methylated RARB in TW2.6, Ca922, OC2, and HSC3 than in NHOK and DOK. The hollow circle is the unmethylated CpG site and the full circle is the methylated CpG site.

Mentions: According to the tumor progression percentages and H&E staining results, we selected the tongues tissues to be targets from OSCC mouse model with N and NA at 26 and 28 weeks for MCGI microarray screening. Figure 2(a) depicts representative data from the OSCC study. The expanded hybridization views showed the usefulness of the MCGI microarrays cohybridized with fluorescently labeled T (tumor part) and NT (nontumor part) with N at week 28 and NA at weeks 26 and 28, respectively. Spots hybridized predominantly with tumor amplicon, but not with nontumor amplicon, would appear red and are indicative of hypermethylated CpG island loci, present in the tumor genome. The hybridization results showed that treatment with NA at week 28 represented much more spots that were obviously more hypermethylated than other treatments. Yellow spots (Cy5 : Cy3 = 1) represent equal amounts of bound DNA from each amplicon, an indication of no methylation differences between tumor and nontumor genomes. Selection of genes was based on the criteria described in the Materials and Methods. Figures 6(b) and 6(c) showed specific genes of selection results in hypermethylation and hypomethylation, respectively. We conducted a confirmation study to determine whether the cutoff ratio (≧2) could accurately identify hypermethylation. The hierarchical clustering presented the 109 gene loci of hypermethylation in the classifier in N and NA (Figure 2(b)). This methylation profile analysis has led to the identification of CpG island clusters that could evaluate many new genes correlating with OSCC progression in mouse model. These newly collected genes are shown in detail in Table 2. Upon further examination, we selected the RARB gene to examine in greater detail by MS-PCR bisulfite sequencing and semiquantitative RT-PCR. The locations of CpG islands in mouse and human RARB genes were predicted using MethPrimer (http://www.urogene.org/methprimer/index1.html), respectively (Figures 3(a) and 4(a)).


Methylation-associated gene silencing of RARB in areca carcinogens induced mouse oral squamous cell carcinoma.

Lai ZL, Tsou YA, Fan SR, Tsai MH, Chen HL, Chang NW, Cheng JC, Chen CM - Biomed Res Int (2014)

Methylation status in human oral cancer cell lines of RARB. (a) The schematic for CpG island presentation of RARB. There was a CpG island located in promoter region. We designed the MS-PCR and bisulfite sequencing primer sets in this region. (b) The RARB methylation status in different human oral cancer cell lines. The cell lines were also treated with 5′-aza-dC. The methylation status was recovered because of 5′-aza-dC treatment. (c) The RARB mRNA expression in different human oral cancer cell lines. The RARB was not expressed in Ca922, OC2, and HSC3 obviously. (d) The RARB bisulfite sequencing in different human oral cancer cell lines. There were more methylated RARB in TW2.6, Ca922, OC2, and HSC3 than in NHOK and DOK. The hollow circle is the unmethylated CpG site and the full circle is the methylated CpG site.
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Related In: Results  -  Collection

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fig4: Methylation status in human oral cancer cell lines of RARB. (a) The schematic for CpG island presentation of RARB. There was a CpG island located in promoter region. We designed the MS-PCR and bisulfite sequencing primer sets in this region. (b) The RARB methylation status in different human oral cancer cell lines. The cell lines were also treated with 5′-aza-dC. The methylation status was recovered because of 5′-aza-dC treatment. (c) The RARB mRNA expression in different human oral cancer cell lines. The RARB was not expressed in Ca922, OC2, and HSC3 obviously. (d) The RARB bisulfite sequencing in different human oral cancer cell lines. There were more methylated RARB in TW2.6, Ca922, OC2, and HSC3 than in NHOK and DOK. The hollow circle is the unmethylated CpG site and the full circle is the methylated CpG site.
Mentions: According to the tumor progression percentages and H&E staining results, we selected the tongues tissues to be targets from OSCC mouse model with N and NA at 26 and 28 weeks for MCGI microarray screening. Figure 2(a) depicts representative data from the OSCC study. The expanded hybridization views showed the usefulness of the MCGI microarrays cohybridized with fluorescently labeled T (tumor part) and NT (nontumor part) with N at week 28 and NA at weeks 26 and 28, respectively. Spots hybridized predominantly with tumor amplicon, but not with nontumor amplicon, would appear red and are indicative of hypermethylated CpG island loci, present in the tumor genome. The hybridization results showed that treatment with NA at week 28 represented much more spots that were obviously more hypermethylated than other treatments. Yellow spots (Cy5 : Cy3 = 1) represent equal amounts of bound DNA from each amplicon, an indication of no methylation differences between tumor and nontumor genomes. Selection of genes was based on the criteria described in the Materials and Methods. Figures 6(b) and 6(c) showed specific genes of selection results in hypermethylation and hypomethylation, respectively. We conducted a confirmation study to determine whether the cutoff ratio (≧2) could accurately identify hypermethylation. The hierarchical clustering presented the 109 gene loci of hypermethylation in the classifier in N and NA (Figure 2(b)). This methylation profile analysis has led to the identification of CpG island clusters that could evaluate many new genes correlating with OSCC progression in mouse model. These newly collected genes are shown in detail in Table 2. Upon further examination, we selected the RARB gene to examine in greater detail by MS-PCR bisulfite sequencing and semiquantitative RT-PCR. The locations of CpG islands in mouse and human RARB genes were predicted using MethPrimer (http://www.urogene.org/methprimer/index1.html), respectively (Figures 3(a) and 4(a)).

Bottom Line: These results showed that retinoic acid receptor β (RARB) was indicated in hypermethylation at the promoter region and the loss of expression during cancer development.According to the results of real-time PCR, it was shown that de novo DNA methyltransferases were involved in gene epigenetic alternations of OSCC.Collectively, our results showed that RARB hypermethylation was involved in the areca-associated oral carcinogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Agricultural Biotechnology Center, National Chung Hsing University, No. 250 Kao-Kuang Road, Taichung 402, Taiwan.

ABSTRACT
Regarding oral squamous cell carcinoma (OSCC) development, chewing areca is known to be a strong risk factor in many Asian cultures. Therefore, we established an OSCC induced mouse model by 4-nitroquinoline-1-oxide (4-NQO), or arecoline, or both treatments, respectively. These are the main two components of the areca nut that could increase the occurrence of OSCC. We examined the effects with the noncommercial MCGI (mouse CpG islands) microarray for genome-wide screening the DNA methylation aberrant in induced OSCC mice. The microarray results showed 34 hypermethylated genes in 4-NQO plus arecoline induced OSCC mice tongue tissues. The examinations also used methylation-specific polymerase chain reaction (MS-PCR) and bisulfite sequencing to realize the methylation pattern in collected mouse tongue tissues and human OSCC cell lines of different grades, respectively. These results showed that retinoic acid receptor β (RARB) was indicated in hypermethylation at the promoter region and the loss of expression during cancer development. According to the results of real-time PCR, it was shown that de novo DNA methyltransferases were involved in gene epigenetic alternations of OSCC. Collectively, our results showed that RARB hypermethylation was involved in the areca-associated oral carcinogenesis.

Show MeSH
Related in: MedlinePlus