original article digestive and liver disease...regulação epigenética no envelhecimento e no...

Regulação Epigenética no Envelhecimento e no Câncer Gástrico P á g i n a | 113

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4.2.2 Protein expression and promoter DNA methylation of SIRT1 and 3 in gastric

cancer

Submetido como Original Article à revista “Digestive and Liver Disease” em 08/03/11. Protein expression and promoter DNA methylation of SIRT1 and 3 in gastric cancer

Luara Carolina Frias Lisboa1; Carolina Oliveira Gigek1; Patrícia Natalia Oliveira Silva1;

Mariana Ferreira Leal1; Eleonidas Moura Lima2; Danielle Queiroz Calcagno3; Paulo

Pimentel Assumpção4; Rommel Rodriguez Burbano3; Marília de Arruda Cardoso

Smith1* 1 Disciplina de Genética, Departamento de Morfologia e Genética, Universidade

Federal de São Paulo, SP, Brasil. 2 Departamento de Biologia, Campus Ministro Reis Velloso/Parnaíba, Universidade

Federal do Piauí, PI, Brasil. 3 Laboratório de Citogenética Humana, Instituto de Ciências Biológicas, Universidade

Federal do Pará, PA, Brasil. 4 Serviço de Cirurgia, Hospital Universitário João de Barros Barreto, Universidade

Federal do Pará, PA, Brasil.

ABSTRACT

Background: Sirtuins are an important regulator of chromatin structure and gene

expression. This is the first study evaluating SIRT1 and SIRT3 expression and promoter

methylation in gastric carcinogenesis. Methods: Immunohistochemistry was analyzed

about 50 gastric tumor and in about 20 normal gastric mucosa samples. About 100

gastric cancer and 50 normal mucosa samples were investigated through methylation

specific PCR. Results: SIRT1 expression was associated with gastric cancer (OR=6;

p=0.0003). This is the first study to evaluate SIRT1 promoter methylation. We observed

no association between SIRT1 protein and methylation pattern in gastric tissue.

Concerning SIRT3, we originally report higher protein imunnostaining in 80% of non-

neoplastic, thus differing significantly from neoplastic samples (OR=0.313; p=0.05). The


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frequency of SIRT1 and SIRT3 promoter methylation did not differ between tumor and

normal gastric samples. Conclusion: SIRT1 expression was higher in tumor than in non-

neoplastic gastric tissue, and no association with DNA methylation pattern was

observed. In contrast, SIRT3 may reveal a protective role in gastric carcinogenesis.

Moreover, we originally demonstrated a correlation between SIRT3 methylation pattern

and protein expression in gastric tumor samples.

Keywords: SIRT1, SIRT3, gastric cancer, DNA methylation, epigenetic.

BACKGROUND

Sirtuin is a NAD dependent deacetylase family conserved in many

organisms [1], with seven known components (SIRT1-7) in humans. These proteins

participate in regulation of chromatin structure and gene expression and have a

determinant role on cellular identity [2].

SIRT1 deacetylates histones and non-histone targets, including genes

involved in cell cycle, survival, stress response and apoptosis, as MYC and p53 [3].

Normal SIRT1 expression has a protective effect against DNA damage, enhancing DNA

repair ability and prevention of tumorigenesis [4]. In contrast, in cancer, SIRT1

colocalizes on the promoter region of several aberrantly silenced tumor suppressor

genes whose DNA is hypermethylated, but its ultimate activity has not yet been proved

[5]. Hence, the role of SIRT1 as a tumor promoter or a tumor suppressor remains to be

determined; nevertheless there are strong evidences that SIRT1 may be a critical

regulator of cancer development, being an important therapeutic target [3].

Another sirtuin, SIRT3, has mitochondrial localization and its expression is

associated with reduction of mitochondrial damage [1], growth arrest, senescence and

apoptosis. The role of SIRT3 is still unknown, although high gene expression was

described in breast cancer node positive [6]. SIRT3 is located in 11p15.5, a known

imprinted chromosomal region, which comprises genes related to aging and tumor

suppressor [7].

Gastric cancer is the result of genetic, epigenetic and environmental

onmental factors [8]. Furthermore, gene promoter methylation is the most common


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epigenetic alteration and has been reported in gastric cancer [9-15]. SIRT1 has a CpG

island of about 1Kb and CG content up to 72% in promoter and exon 1 region [16].

SIRT3 promoter region was identified as a bidirectional promoter with a CG content of

67% and comprises two CpG islands [17]. Thus, the investigation of epigenetic

mechanisms involved in regulation of SIRT1 and SIRT3 expression is relevant to

elucidate these genes function in gastric carcinogenesis.

The aim of this study was to evaluate SIRT1 and SIRT3 promoter

methylation pattern in gastric carcinogenesis and correlate with their protein expression

as well as with clinicopathological characteristics. To our knowledge, this is the first

study to investigate the relationship between SIRT1 and SIRT3 promoter methylation

and protein expression in gastric cancer.

METHODS

1. Samples

Gastric samples were obtained surgically from João de Barros Barreto

University Hospital (HUJBB) in Pará State, Brazil. This population is composed of

interethnic crosses among three main origin groups: European (mainly represented by

Portuguese), Africans and Amerindians [18]. Informed consent with approval of the

ethics committee of HUJBB was obtained. All patients had negative histories of

exposure to either chemotherapy or radiotherapy before surgery and there was no other

co-occurrence of diagnosed cancers. All samples were classified according to Laurén

[19] and tumors were staged using standard criteria by TNM [20] staging.

2. Immunohistochemical staining

SIRT1 and SIRT3 protein expression were evaluated in formalin-fixed

paraffin embedded tissues of 70 and 74 samples of gastric mucosa, respectively (Table

1).

Antigen retrieval was performed by microwave treatment 20 min at 900 W

in a citrate buffer, pH 6.0. After cooling, sections were immersed in 0.3% hydrogen

peroxide in phosphate-buffered saline (PBS) for 10 minutes to block endogenous

peroxidase activity. Sections were then incubated in a humid chamber overnight with

SIRT1 primary antibody (C19 DB083, Delta biolabs, USA) or SIRT3 primary antibody


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(C73E3, Cell Signaling Technology, USA). After the PBS rinse, slides were incubated

with secondary antibody and then with streptavidin-biotin-peroxidase complex, both for

30 minutes at room temperature with a PBS wash between each step. Slides were

visualized with diaminobenzidine-hydrogen peroxide and counterstained with Harry’s

hematoxylin.

Positive protein expression was defined as clear nuclear staining in 50%

or more of the cells, whereas negative immunostaining was considered when no

positive cells were observed or in rare cases (less than 25% weakly stained tumor cells)

(Figures 1 and 2). Normal gastric mucosa was used as an internal control. Two

pathologists evaluated the immunostaining results independently.

3. Methylation specific PCR (MSP)

SIRT1 and SIRT3 methylation pattern were evaluated in 162 and 88

gastric tissue samples, respectively. Among these samples, around 70% are from

sporadic gastric adenocarcinomas and 30% of non-neoplastic gastric mucosa of

patients (distant location of primary tumor) (Table 2).

Genomic DNA (200 ng) of gastric tissue samples underwent bisulfite

modification using EpiTect Bisulfite kit (Qiagen, Germany) according to the

manufacturer’s instructions, converting unmethylated cytosines to uracils and leaving

methylated cytosines unchanged. MSP was performed on treated DNA as previously

described [21]. MSP for SIRT3 was performed as previously described [22]. Specific

primers for SIRT1 promoter, were as follows: 5’-GGGGTGAATTTGGTTGTATTATATG-

3’ (sense) and 5’-AAACAAAAACTATTACATCTACCACT-3’ (antisense) for the

unmethylated reactions; 5’-GGCGAATTTGGTTGTATTATACG-3’ (sense) and 5’-

GAACGAAAACTATTACGTCTACCG-3’ (antisense) for the methylated reactions, with

PCR products of 112 bp and 110 bp respectively. Briefly, PCR reaction was carried out

in a 25μL volume with 200μmol/L of MgCl2, 100ng of DNA, 200 pmol/L of primers and

1.25 units of Taq DNA polymerase. After initial denaturation for 5 min at 94ºC, 40 cycles

of 94ºC for 45 s, at 62ºC for 45 s, and 72ºC for 30 s were carried out, followed by a final

extension for 5 min at 72ºC.


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Results were scored when there was a clear and visible band on the

electrophoresis gel with the methylated or unmethylated primers [21]. Hypermethylation

was considered when the presence of a methylated band was observed (Figure 3).

SIRT1 and SIRT3 methylation pattern and respective protein expression

were both in all available gastric cancer samples with immunohistochemistry results

(Table 3).

4. Statistical analyses

Logistic regression was performed to evaluate imunnohistochemistry

results and clinicopathological characteristics χ2 test or Fisher’s exact was used to

assess associations between methylation status and clinicopathological characteristics.

p values less than 0.05 were considered significant.

RESULTS

SIRT1 expression was more frequently observed in neoplastic than in non-

neoplastic samples (0.885 vs 0.556). The odds ratio for SIRT1 was 6 (95% CI 2.7-

21.17). (p=0.003; χ2 = 8.79, df=1). Compared to normal mucosa, frequency of SIRT1

staining was significantly higher in diffuse type gastric cancer (p=0.0079). However,

SIRT1 expression did not differ between diffuse and intestinal type gastric cancer

(p=0.4109) (Table 1).

Eighty per cent of normal gastric mucosa and 55.5% of gastric cancer

samples showed SIRT3 expression. Thus, SIRT3 we observed a difference between

neoplastic and non-neoplastic samples, with OR = 0.313 (0.092-1.059) (p=0.05; χ2 =

3.708, df=1). There was no difference of SIRT3 protein staining between diffuse and

intestinal type gastric cancer (p=1). Gastric cancer samples of the noncardia region

presented more frequent SIRT3 expression than the cardia region (p=0.0459). There

was no clear association between the frequency of SIRT1 and SIRT3 expression and

age, gender, H. pylori infection, tumor extension and presence of distant metastasis

(Table 1).

The frequency of SIRT1 and SIRT3 promoter methylation did not differ

between tumor and normal samples. SIRT3 promoter methylation was more frequently

observed in cardia than in noncardia tumors (p=0.0363), occurring mostly in cardia


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intestinal type gastric cancer (p=0.0152). Promoter methylation of SIRT1 and SIRT3 did

not show association with age, gender, H. pylori infection, tumor extension and

presence of distant metastasis (Table 2).

To clarify the relationship between promoter methylation and gene

expression, we compared the presence of methylation of SIRT1 and SIRT3 promoter

regions with their protein expression in gastric cancer and normal mucosa specimens.

There was no correlation between SIRT1 expression and its promoter methylation

pattern in our sample (p=0.8409, r=0.0308). In gastric specimens, SIRT3 methylation

pattern was associated with lack of protein expression (p=0.0033, r=0.4461) (Table 3).

DISCUSSION

In the present study, we observed higher immunostaining of SIRT1 protein

in 88.5% of neoplastic samples, thus indicating that presence of this protein increases in

6 times the risk of gastric cancer. A recent study reported that 73% of gastric cancer

samples presented positive SIRT1 immunoreactivity. Moreover, SIRT1 expression was

associated with poor prognosis, histological type and p53 expression [23].

Overexpression of SIRT1 allows rapid proliferation and loss of checkpoints, promoting

the progression of prostate [24] and colon cancer [25]. Taken together, these data

suggest that this protein participate in tumor promotion in carcinogenesis processes,

including gastric cancer.

Tumor growth depends on the loss of control of differentiation and

inhibition of apoptosis. Tumor suppressor p53 is a known substrate of SIRT1, and plays

a role in cell cycle regulation and apoptosis, regulating proliferation and stress response

[4]. We observed higher SIRT1 protein in diffuse type gastric cancer. This observation

might be correlated with our previous data in the same sample population: an increased

frequency of immunostaining of p53 in the intestinal type gastric cancer [26]. Cha et al

[23] demonstrated that 100% of p53-positive gastric cancer cases showed lower SIRT1

expression. Thereby, SIRT1 appears to promote gastric tumorigenesis via p53.

MYC is another protein involved in gastric tumorigenesis and binds to

SIRT1 promoter region, inducing SIRT1 expression [27]. We have previously described

MYC immunoreactivity in all gastric adenocarcinoma samples in individuals from Pará


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State [28-30], suggesting that MYC overexpression might contribute to higher SIRT1

levels found in our sample.

Furthermore, MYC induces SIRT1 expression in HeLa cells and SIRT1, in

turn, deacetylates and downregulates MYC, resulting in decreased MYC target gene

expression and cellular transformation. Consequently, this MYC-SIRT1 negative

feedback loop can restrain MYC’s transformational activities. However, MYC

overexpression and poor prognosis of advanced gastric neoplasia suggest that this

possible MYC-SIRT1 feedback is not sufficient to restrain the gastric malignization.

Therefore, SIRT1 might have both tumor suppression and promoting role depending on

the stage of cell transformation [27].

To our knowledge, this is the first study evaluating SIRT1 promoter

methylation, as well as its relation with protein expression. Here we observed that 11%

of all gastric tissue (non-neoplastic and neoplastic) presented SIRT1 promoter

methylation. The fact that both non-neoplastic and neoplastic samples presented SIRT1

promoter methylation might be due to the regular methylation levels previously

observed in health stomach tissue [31]. Moreover, SIRT1 overexpression was

previously associated with high-CpG island methylator phenotype in colorectal cancers

[32]. Hypermethylation of multiple tumor-related genes is frequently detected in gastric

carcinoma and adjacent normal tissues and is likely to be involved in the early gastric

carcinogenesis process [9-14, 33].

To clarify the relationship between promoter methylation and protein

expression, we compared the presence of methylation of SIRT1 promoter region with

protein immunostaining in gastric cancer and normal mucosa specimens. Therefore, no

association between protein expression and promoter methylation pattern was found in

our sample, indicating that this type of epigenetic control does not have any influence in

protein expression.

Concerning SIRT3, we found that 80% of non-neoplastic gastric mucosa

presented this protein immunostaining, diffenring significantly from neoplastic samples,

therefore having a protective role in this type of cancer. The mitochondrial localization of

SIRT3 is consistent with its role in active process to minimize mitochondrial damage

[34]. In colorectal carcinoma, SIRT3 mediates apoptosis by silencing of BCL2, a


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mitochondrial anti-apoptotic protein [1]. Likewise, high levels of BCL2 has been reported

in the early stage of gastric carcinogenesis process [35], which may explain the frequent

lack of SIRT3 protein in our neoplastic samples.

In addition, SIRT3 overexpression increases FOXO3A DNA-binding

activity, and thus, FOXO3A dependent gene expression [34]. In gastric cancer,

FOXO3A induces apoptosis cooperating with RUNX3 [36], a transcription factor

frequently inactivated in 40% of early stage and almost 90% of advanced gastric cancer

by hemizygous deletion and hypermethylation of its promoter [37]. In our population, we

also described RUNX3 methylation in about 50% of gastric cancer samples [10].

Our data corroborates with previous report that SIRT3 acts as tumor

suppressor, and its loss amplifies the phenotypic effects of oncogene expression [38].

The frequency of SIRT3 promoter methylation did not differ between tumor

and normal samples. In the present study, we originally reported that SIRT3 methylation

pattern association with lack of protein expression in our gastric specimens. We

observed SIRT3 methylation in more than 60% all of gastric samples and an inverse

relationship between promoter methylation and protein expression was also found. No

methylation in lymphocytes of healthy elderly individuals was observed in a previous

research and only 6.8% of young individuals presented no methylation in lymphocytes

[22]. In the present study, we identified an association between SIRT3 expression and

unmethylated status in gastric tumor samples. Taken together, our data suggest a

tissue-specific regulation of SIRT3.

SIRT1 expression was higher in tumor cells than in normal gastric tissue,

increasing the risk of this type of cancer. No association between protein and DNA

methylation pattern was observed. Moreover, we originally demonstrated the SIRT3

methylation pattern and protein expression was correlated with gastric tumor samples.

Further studies may reveal a possible therapy via sirtuin in gastric cancer.

REFERENCES

1. Allison, S.J. and J. Milner, SIRT3 is pro-apoptotic and participates in distinct

basal apoptotic pathways. Cell Cycle, 2007. 6(21): p. 2669-77.


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2. Metoyer, C.F. and K. Pruitt, The role of sirtuin proteins in obesity.

Pathophysiology, 2008. 15(2): p. 103-8.

3. Kim, E.J. and S.J. Um, SIRT1: roles in aging and cancer. BMB Rep, 2008.

41(11): p. 751-6.

4. Zeng, L., et al., Silent information regulator, Sirtuin 1, and age-related diseases.

Geriatr Gerontol Int, 2009. 9(1): p. 7-15.

5. Jones, P.A. and S.B. Baylin, The fundamental role of epigenetic events in cancer.

Nat Rev Genet, 2002. 3(6): p. 415-28.

6. Ashraf, N., et al., Altered sirtuin expression is associated with node-positive

breast cancer. Br J Cancer, 2006. 95(8): p. 1056-61.

7. Schwer, B., et al., The human silent information regulator (Sir)2 homologue

hSIRT3 is a mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase. J

Cell Biol, 2002. 158(4): p. 647-57.

8. Shang, J. and A.S. Pena, Multidisciplinary approach to understand the

pathogenesis of gastric cancer. World J Gastroenterol, 2005. 11(27): p. 4131-9.

9. Gigek, C.O., et al., hTERT methylation and expression in gastric cancer.

Biomarkers, 2009. 14(8): p. 630-6.

10. Lima, E.M., et al., Study of Methylation Pattern of de Novo DNA

Methytransferase Genes and its Correlation with DNA Methylation Pattern of RUNX3 in

Individuals with Gastric Cancer from Northern Region of Brazil. Int J Morphol, 2007.

25(4): p. 817-824.

11. Leal, M.F., et al., Promoter hypermethylation of CDH1, FHIT, MTAP and PLAGL1

in gastric adenocarcinoma in individuals from Northern Brazil. World J Gastroenterol,

2007. 13(18): p. 2568-74.

12. Lima, E.M., et al., Methylation status of ANAPC1, CDKN2A and TP53 promoter

genes in individuals with gastric cancer. Braz J Med Biol Res, 2008. 41(6): p. 539-43.

13. Moura Lima, E., et al., DNA mismatch repair gene methylation in gastric cancer

in individuals from northern Brazil. Biocell, 2008. 32(3): p. 237-43.

14. Guimaraes, A.C., et al., Interrelationships among chromosome aneuploidy,

promoter hypermethylation, and protein expression of the CDKN2A gene in individuals


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from northern Brazil with gastric adenocarcinoma. Cancer Genet Cytogenet, 2007.

179(1): p. 45-51.

15. Gigek, C.O., et al., Insulin-like growth factor binding protein-3 gene methylation

and protein expression in gastric adenocarcinoma. Growth Horm IGF Res, 2010.

16. Li, L.C. and R. Dahiya, MethPrimer: designing primers for methylation PCRs.

Bioinformatics, 2002. 18(11): p. 1427-31.

17. Bellizzi, D., et al., Characterization of a bidirectional promoter shared between

two human genes related to aging: SIRT3 and PSMD13. Genomics, 2007. 89(1): p.

143-50.

18. Batista dos Santos, S.E., et al., Differential contribution of indigenous men and

women to the formation of an urban population in the Amazon region as revealed by

mtDNA and Y-DNA. Am J Phys Anthropol, 1999. 109(2): p. 175-80.

19. Lauren, P., The Two Histological Main Types of Gastric Carcinoma: Diffuse and

So-Called Intestinal-Type Carcinoma. an Attempt at a Histo-Clinical Classification. Acta

Pathol Microbiol Scand, 1965. 64: p. 31-49.

20. Sobin, L.H. and C.H. Wittekind, TNM: Classification of malignant tumours. 6th

ed2002, New York: Wiley-Liss

21. Herman, J.G., et al., Methylation-specific PCR: a novel PCR assay for

methylation status of CpG islands. Proc Natl Acad Sci U S A, 1996. 93(18): p. 9821-6.

22. Silva, P.N., et al., Promoter methylation analysis of SIRT3, SMARCA5, HTERT

and CDH1 genes in aging and Alzheimer's disease. J Alzheimers Dis, 2008. 13(2): p.

173-6.

23. Cha, E.J., et al., Expression of DBC1 and SIRT1 is associated with poor

prognosis of gastric carcinoma. Clin Cancer Res, 2009. 15(13): p. 4453-9.

24. Huffman, D.M., et al., SIRT1 is significantly elevated in mouse and human

prostate cancer. Cancer Res, 2007. 67(14): p. 6612-8.

25. Stunkel, W., et al., Function of the SIRT1 protein deacetylase in cancer.

Biotechnol J, 2007. 2(11): p. 1360-8.

26. Khayat, A.S., et al., Interrelationship between TP53 gene deletion, protein

expression and chromosome 17 aneusomy in gastric adenocarcinoma. BMC

Gastroenterol, 2009. 9(1): p. 55.


Artigos

27. Yuan, J., K. Minter-Dykhouse, and Z. Lou, A c-Myc-SIRT1 feedback loop

regulates cell growth and transformation. J Cell Biol, 2009. 185(2): p. 203-11.

28. Calcagno, D.Q., et al., Aneuploidy of chromosome 8 and C-MYC amplification in

individuals from northern Brazil with gastric adenocarcinoma. Anticancer Res, 2005.

25(6B): p. 4069-74.

29. Calcagno, D.Q., et al., Interrelationship between chromosome 8 aneuploidy, C-

MYC amplification and increased expression in individuals from northern Brazil with

gastric adenocarcinoma. World J Gastroenterol, 2006. 12(38): p. 6207-11.

30. Costa Raiol, L.C., et al., Interrelationship between MYC gene numerical

aberrations and protein expression in individuals from northern Brazil with early gastric

adenocarcinoma. Cancer Genet Cytogenet, 2008. 181(1): p. 31-5.

31. Esteller, M., CpG island hypermethylation and tumor suppressor genes: a

booming present, a brighter future. Oncogene, 2002. 21(35): p. 5427-40.

32. Nosho, K., et al., SIRT1 histone deacetylase expression is associated with

microsatellite instability and CpG island methylator phenotype in colorectal cancer. Mod

Pathol, 2009. 22(7): p. 922-32.

33. Leung, W.K., et al., Concurrent hypermethylation of multiple tumor-related genes

in gastric carcinoma and adjacent normal tissues. Cancer, 2001. 91(12): p. 2294-301.

34. Jacobs, K.M., et al., SIRT3 interacts with the daf-16 homolog FOXO3a in the

mitochondria, as well as increases FOXO3a dependent gene expression. Int J Biol Sci,

2008. 4(5): p. 291-9.

35. Tsamandas, A.C., et al., The potential role of Bcl-2 expression, apoptosis and

cell proliferation (Ki-67 expression) in cases of gastric carcinoma and correlation with

classic prognostic factors and patient outcome. Anticancer Res, 2009. 29(2): p. 703-9.

36. Ito, Y. and K. Miyazono, RUNX transcription factors as key targets of TGF-beta

superfamily signaling. Curr Opin Genet Dev, 2003. 13(1): p. 43-7.

37. Hanai, J., et al., Interaction and functional cooperation of PEBP2/CBF with

Smads. Synergistic induction of the immunoglobulin germline Calpha promoter. J Biol

Chem, 1999. 274(44): p. 31577-82.

38. Schumacker, P.T., A tumor suppressor SIRTainty. Cancer Cell, 2010. 17(1): p. 5-

6.


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Table 1: Clinicopathological and immunohistochemistry results of gastric tissue samples, n (%).

SIRT1 SIRT3

Variable Total Positive Negative p value Total Positive Negative p value

Gender

Male

Female

49

20

40 (81.6%)

16 (80%)

9 (18.4%)

4 (20%)

p=0.9990

52

22

34 (65.4%)

12 (54.5%)

18 (34.6%)

10 (45.5%)

p=0.5375

Tissue

NGM

GC

18

52

10 (55.6%)

46 (88.5%)

8 (44.4%)

6 (11.5%)

p=0.0053*

20

54

16 (80%)

30 (55.5%)

4 (20%)

24 (44.5%)

p=0.0638

Onset

≤ 45y

> 45y

4

48

3 (75%)

43 (89.6%)

1 (25%)

5 (10.4%)

p=0.3938

5

49

2 (40%)

28 (57.1%)

3 (60%)

21 (42.9%)

p=0.6357

H. pylori

Present

Absent

39

13

34 (87.2%)

12 (92.3%)

5 (12.8%)

1 (7.7%)

p=1

40

14

25 (62.5%)

5 (35.7%)

15 (37.5%)

9 (64.3%)

p=0.1198

Laurén

Classification

Diffuse

Intestinal

27

25

25 (92.6%)

21 (84%)

2 (7.4%)

4 (16%)

p=0.4109

28

26

16 (57.1%)

14 (53.8%)

12 (42.9%)

12 (46.2%)

p=1

Tumor Location

Cardia

Noncardia

10

42

9 (90%)

37 (88.1%)

1 (10%)

5 (11.9%)

p=1

11

43

3 (27.3%)

27 (62.8%)

8 (72.7%)

16 (37.2%)

p=0.0459

Stage

I/II

III/IV

3

48

3 (100%)

42 (87.5%)

0

6 (12.5%)

p=1

4

50

3 (75%)

27 (54%)

1 (25%)

23 (46%)

p=0.6204

Lymph node

mestasis

p=1

p=0.6204


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Present

Absent

49

3

43 (87.8%)

3 (100%)

6 (12.2%)

0

50

4

27 (54%)

3 (75%)

23 (46%)

1 (25%)

Distant

metastasis

Present

Absent

Unkown

17

33

2

13 (76.5%)

31 (93.9%)

4 (23.5%)

2 (6.1%)

p=0.1624

17

34

10 (58.8%)

18 (52.9%)

7 (41.2%)

16 (47.1%)

p=0.7707

IHC: immunohistochemistry assay; NGM: normal gastric mucosa; GC: gastric cancer; *significant p value

after multiple comparison corrections.


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Table 2: Clinicopathological and methylation frequency in gastric tissue samples, n (%).

SIRT1 SIRT3

Variable Total M

n (%)

U

n (%) p value Total

M

n (%)

U

n (%) p value

Gender

Male

Female

109

53

10 (9.2%)

8 (15.1%)

99 (90.8%)

45 (84.9%)

p=0.3906

58

30

19 (32.8%)

11 (36.7%)

39 (67.2%)

19 (63.3%)

p=0.8970

Tissue

NGM

GC

53

109

6 (11.3%)

12 (11.1%)

47 (88.7%)

97 (88.9%)

p=0.7810

26

62

8 (30.8%)

22 (35.5%)

18 (69.2%)

40 (64.5%)

p=0.8066

Onset

≤ 45y

> 45y

15

94

3 (20%)

9 (9.6%)

12 (80%)

85 (90.4%)

p=0.3664

6

56

2 (33.3%)

20 (35.7%)

4 (66.7%)

36 (64.3%)

p=1

H. pylori

Present

Absent

74

35

8 (10.8%)

4 (11.4%)

66 (89.2%)

31 (88.6%)

p=0.9984

39

23

13 (33.3%)

9 (39.1%)

26 (66.7%)

14 (60.9%)

p=0.7845

Laurén

Classification

Diffuse

Intestinal

58

51

7 (12.1%)

5 (9.8%)

51 (87.9%)

46 (90.2%)

p=0.7674

34

28

11 (32.4%)

11 (39.3%)

23 (67.6%)

17 (60.7%)

p=0.5770

Tumor Location

Cardia

Noncardia

22

81

2 (9.1%)

10 (12.3%)

20 (90.9%)

71 (87.7%)

p=1

11

50

7 (63.6%)

14 (28%)

4 (36.4%)

36 (72%)

p=0.0363

Stage

I/II

III/IV

15

94

0

12 (12.8%)

15 (100%)

82 (87.2%)

p=0.2121

7

55

3 (42.9%)

28 (50.9%)

4 (57.1%)

12 (21.8%)

p=0.6906

Lymph node


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mestasis

Present

Absent

95

14

11 (11.6%)

1 (7.1%)

84 (88.4%)

13 (92.9%)

p=1

57

5

21 (36.8%)

1 (20%)

36 (63.2%)

4 (80%)

p=0.6473

Distant metastasis

Present

Absent

Unkown

29

63

17

3 (10.3%)

7 (11.1%)

26 (89.7%)

56 (88.9%)

p=1

16

42

4

6 (37.5%)

15 (35.7%)

10 (62.5%)

27 (64.3%)

p=1

IHC: immunohistochemistry assay; NGM: normal gastric mucosa; GC: gastric cancer; *significant p value

after multiple comparison corrections.


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Table 3: SIRT1 and SIRT3 promoter methylation and protein expression in tumor and normal gastric samples.

SIRT1 SIRT3

IHC

MSP

Positive Negative p Positive Negative p

Tumor M

U

M

U

M

U

4 (66.7%) 2 (33.3%) p=0.1827 5 (33.3%) 10 (66.7%) p=0.0047*

42 (93.3%) 3 (6.7%) 20 (83.3%) 4 (16.7%)

Normal 3 (100%) 0 (0%) p=0.2452 2 (66.7%) 1 (33.3%) p=0.8206

6 (42.9%) 8 (57.1%) 9 (81.8%) 2 (18.2%)

Total 7 (77.8%) 2 (22.2%) p=0.8409 7 (38.9%) 11 (61.1%) p=0.0033*

48 (81.4%) 11 (18.6%) 29 (82.9%) 6 (17.1%)

IHC: immunohistochemistry assay; MSP: methylation specific PCR; M: methylated; U: unmethylated; *significant p value after multiple comparison

corrections.


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Figure 1: Immunohistochemical detection of SIRT1 antibody. a) and b) negative and

positive immunostaining of normal gastric tissue, respectively; c) and d) diffuse and

intestinal type gastric adenocarcinoma samples. Magnification: 40x.


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Figure 2: Immunohistochemical detection of SIRT3 antibody. a) and b) negative and

positive immunostaining of normal gastric tissue, respectively; c) and d) diffuse and

intestinal type gastric adenocarcinoma samples. Magnification: 100x (c) and 40x (a,b

and d).

Figure 3: Methylation analysis by MSP. a) and b) SIRT1 and SIRT3 promoter showing

unmethylated bands of 112bp and 167bp, respectively. L: size marker; M: methylated;

U: unmethylated.


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4.2.3 SIRT1, IGFBP-3 and CAV1 promoter DNA methylation in aging process

Submetido como Research Letter à “Age and Ageing” em 04/03/11.

SIRT1, IGFBP-3 and CAV1 promoter DNA methylation in aging process

Running head: DNA methylation in aging

Carolina Oliveira Gigek1; Luara Carolina Frias Lisboa1; Patricia Natalia Oliveira Silva1;

Mariana Ferreira Leal1, Roger W. de Labio2; Spencer Luiz Marques Payão2; Marilia de

Arruda Cardoso Smith1 1 Genetics Division, Departament of Morphology and Genetics Universidade

Federal de São Paulo, SP, Brazil. 2 Genetics and Molecular Biology, Hemocentro, Faculdade de Medicina de Mar´ılia

(FAMEMA), Marília, SP, Brazil

Keywords: aging, DNA methylation, epigenetic

SIR - Nowadays, epigenetics is defined as the heritable changes in gene

activity and expression, and, as the stable, long-term alterations in the transcriptional

potential of a cell that are not necessarily heritable [1]. The most known epigenetic

modifications are DNA methylation and histone post-transcriptional modifications. DNA

methylation consists in an addition of a methyl group on the aromatic ring of 5-carbon

from the cytosine ring of a CpG dinucleotide. This dinucleotide is concentrated in

particular gene promoters, called CpG islands, where it can regulate gene’s expression,

often blocking transcription when methyl group is present [1].

The human genome is a fragile and highly conserved structure which

accumulates a wide range of damaging alterations with age, despite continuous

surveillance and repair [2]. The relationship between epigenetics and aging was

proposed many years ago, showing a decrease in the genomic global DNA methyation

in ageing [3]. In addition to this global hypomethylation, a number of specific loci have


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been described as becoming hypermethylated during the ageing process [1].

Interesting, this events are known epigenetic alterations in cancer [4], which suggests

the accumulation of epigenetic alterations during ageing may directly contribute to

malignant transformation.

We selected three genes, SIRT1, IGFBP-3 and CAV-1, in order to

evaluate DNA promoter methylation in the aging process. To our knowledge, this is a

novel study of DNA promoter methylation of these genes in aging process.

Material and Methods

1. Samples

Methylation status of SIRT1, IGFBP-3 and CAV-1 were evaluated in

peripheral blood samples from about 50 healthy older adults, with exclusion criteria for

depression, stroke, dementia and low cognitive levels using Mini-Mental State Exam

(MMSE), and 60 young adults controls. The mean age was 70.06 ± 9.644 years and

21.46 ± 2.482 years, respectively. All older individuals were selected at Neurology

Department of Universidade Federal de São Paulo – UNIFESP. The younger group was

formed by healthy volunteers also of UNIFESP.

2. DNA Extraction and Methylation Specific PCR (MSP)

DNA extraction was performed using the QIAamp DNA Blood Midi Kit

(Qiagen). To evaluate methylation status, genomic DNA (200 ng) of all samples

underwent bisulfite modification using EpiTect Bisulfite Kit (Qiagen, Germany) according

to the manufacturer’s instructions. Specific primers for MSP, located within genes’

promoter, are described in Table 1. Briefly, PCR reaction was carried out in a 25μL

volume with 200μmol/L of MgCl2, 100ng of DNA, 200 pmol/L of primers and 1.25 units

of Taq (LGC, Brazil). After initial denaturating for 5 min at 94ºC, 40 cycles at 94ºC for 45

s, at respectively temperature (see Supplementary data) for 45 s, and at 72ºC for 30 s,

samples were carried out, followed by a final extension for 5 min at 72ºC.

Results were scored when emerged a clear visible band on the

electrophoresis gel with the methylated and unmethylated primers [5] (Figure 1).

3. Statistical analyses

original article digestive and liver disease...regulação epigenética no envelhecimento e no...

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