Carolina Oliveira Gigek

Epigenetic mechanisms in gastric cancer

Cancer is considered one of the major health issues worldwide, and gastric cancer accounted for 8% of total cases and 10% of total deaths in 2008. Gastric cancer is considered an age-related disease, and the total number of newly diagnosed cases has been increasing as a result of the higher life expectancy. Therefore, the basic mechanisms underlying gastric tumorigenesis is worth investigation. This review provides an overview of the epigenetic mechanisms, such as DNA methylation, histone modifications, chromatin remodeling complex and miRNA, involved in gastric cancer. As the studies in gastric cancer continue, the mapping of an epigenome code is not far for this disease. In conclusion, an epigenetic therapy might appear in the not too distant future.

Promoter methylation analysis of SIRT3, SMARCA5, HTERT and CDH1 genes in aging and Alzheimer's disease.

Longevity related genes were investigated concerning promoter methylation. SIRT3, SMARCA5, HTERT and CDH1 promoters were analyzed in peripheral blood in relation to gender, age and Alzheimers disease (AD). Methylation Specific PCR assay (MSP) was used. There were no significant differences in methylation frequencies of SIRT3, SMARCA5 and CDH1 among young, elderly and AD groups (p> 0.05), showing no association with aging or AD. On the other hand, HTERT methylation frequency was associated with the aging process, in that AD patients differed from elderly controls (p=0.0086), probably due to telomere and immune dysfunctions involved in AD pathogenesis.

DNA and histone methylation in gastric carcinogenesis

Epigenetic alterations contribute significantly to the development and progression of gastric cancer, one of the leading causes of cancer death worldwide. Epigenetics refers to the number of modifications of the chromatin structure that affect gene expression without altering the primary sequence of DNA, and these changes lead to transcriptional activation or silencing of the gene. Over the years, the study of epigenetic processes has increased, and novel therapeutic approaches that target DNA methylation and histone modifications have emerged. A greater understanding of epigenetics and the therapeutic potential of manipulating these processes is necessary for gastric cancer treatment. Here, we review recent research on the effects of aberrant DNA and histone methylation on the onset and progression of gastric tumors and the development of compounds that target enzymes that regulate the epigenome.

Disruption of a Large Intergenic Noncoding RNA in Subjects with Neurodevelopmental Disabilities

Large intergenic noncoding (linc) RNAs represent a newly described class of ribonucleic acid whose importance in human disease remains undefined. We identified a severely developmentally delayed 16-year-old female with karyotype 46,XX,t(2;11)(p25.1;p15.1)dn in the absence of clinically significant copy number variants (CNVs). DNA capture followed by next-generation sequencing of the translocation breakpoints revealed disruption of a single noncoding gene on chromosome 2, LINC00299, whose RNA product is expressed in all tissues measured, but most abundantly in brain. Among a series of additional, unrelated subjects referred for clinical diagnostic testing who showed CNV affecting this locus, we identified four with exon-crossing deletions in association with neurodevelopmental abnormalities. No disruption of the LINC00299 coding sequence was seen in almost 14,000 control subjects. Together, these subjects with disruption of LINC00299 implicate this particular noncoding RNA in brain development and raise the possibility that, as a class, abnormalities of lincRNAs may play a significant role in human developmental disorders.

hTERT methylation and expression in gastric cancer

Gastric cancer is the second most prevalent cause of cancer death worldwide. DNA methylation is a common event in gastric carcinogenesis. hTERT seems to be the rate-limiting determinant of telomerase activation, which is responsible for stability and life span. hTERT hypermethylation has been associated with telomerase expression. In the present study, we investigated the promoter methylation status and hTERT protein expression in gastric cancer and normal mucosa samples. One hundred and nine gastric cancer and 53 normal mucosa samples were investigated through methylation-specific PCR. Immunohistochemistry was analysed using peroxidase in 55 gastric cancer and 18 normal gastric mucosa samples. This is the first study evaluating hTERT methylation status in gastric carcinogenesis. We did not observe hTERT protein expression in normal gastric mucosa. Moreover, hTERT expression was observed in 80% of tumours and was associated with gastric cancer (p < 0.0001). Partial methylation was the most frequent pattern in gastric samples, even in normal mucosa. The frequency of specimens presenting hypermethylation was significantly higher in tumours than in normal mucosa samples (p = 0.0002), although the presence of hypermethylated promoter was not associated with a higher frequency of hTERT expression. A low correlation between hTERT protein expression and methylation was verified in gastric cancer samples. There was a clear difference in the frequency of hTERT expression and methylation within tumoral and non-tumoral tissues. Methylation status and telomerase expression may be useful for the diagnosis of gastric cancer and may have an impact on the anti-telomerase strategy for cancer therapy.

Reference genes for quantitative RT-PCR data in gastric tissues and cell lines.

AIM To evaluate the suitability of reference genes in gastric tissue samples and cell lines. METHODS The suitability of genes ACTB, B2M, GAPDH, RPL29, and 18S rRNA was assessed in 21 matched pairs of neoplastic and adjacent non-neoplastic gastric tissues from patients with gastric adenocarcinoma, 27 normal gastric tissues from patients without cancer, and 4 cell lines using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). The ranking of the best single and combination of reference genes was determined by NormFinder, geNorm™, BestKeeper, and DataAssist™. In addition, GenEx software was used to determine the optimal number of reference genes. To validate the results, the mRNA expression of a target gene, DNMT1, was quantified using the different reference gene combinations suggested by the various software packages for normalization. RESULTS ACTB was the best reference gene for all gastric tissues, cell lines and all gastric tissues plus cell lines. GAPDH + B2M or ACTB + B2M was the best combination of reference genes for all the gastric tissues. On the other hand, ACTB + B2M was the best combination for all the cell lines tested and was also the best combination for analyses involving all the gastric tissues plus cell lines. According to the GenEx software, 2 or 3 genes were the optimal number of references genes for all the gastric tissues. The relative quantification of DNMT1 showed similar patterns when normalized by each combination of reference genes. The level of expression of DNMT1 in neoplastic, adjacent non-neoplastic and normal gastric tissues did not differ when these samples were normalized using GAPDH + B2M (P = 0.32), ACTB + B2M (P = 0.61), or GAPDH + B2M + ACTB (P = 0.44). CONCLUSION GAPDH + B2M or ACTB + B2M is the best combination of reference gene for all the gastric tissues, and ACTB + B2M is the best combination for the cell lines tested.

SMARCA5 Methylation and Expression in Gastric Cancer

Here, we first evaluated SMARCA5 expression and promoter DNA methylation in gastric carcinogenesis. Immunohistochemistry and methylation-specific PCR were analyzed in 19 and 48 normal mucosa and in 52 and 92 gastric cancer samples, respectively. We observed higher immunoreactivity of SMARCA5 in gastric cancer samples than in normal mucosa. Moreover, SMARCA5 promoter methylation was associated with the absence of protein expression. Our findings suggest that SMARCA5 has a potential role in proliferation and malignancy in gastric carcinogenesis.

Insulin-like growth factor binding protein-3 gene methylation and protein expression in gastric adenocarcinoma.

OBJECTIVE The aim of this study was to evaluate IGFBP-3 protein expression, its correlation with gene promoter methylation pattern in gastric carcinogenesis and with clinicopathological characteristics. DESIGN Forty-three normal gastric mucosa and 94 adenocarcinoma samples were investigated through methylation specific PCR, after bisulfite modification. Immunohistochemistry was analyzed using peroxidase in 54 gastric cancer and 20 normal gastric mucosa samples. RESULTS IGFBP-3 expression was higher in tumor samples than in normal mucosa (p<0.0001). Intestinal type presented a higher frequency of protein expression than diffuse type (p=0.0412). Methylation frequency of IGFBP-3 promoter in gastric samples revealed, respectively, 95.7% and 97.7% in neoplastic and non-neoplastic samples. The frequency of IGFBP-3 methylation did not differ between tumor and normal samples (95.7% versus 97.7%, p=0.7810). We did not observe a significant correlation between IGFBP-3 promoter methylation and protein expression. CONCLUSION In summary, our study did not observe any influence of IGFBP-3 promoter methylation on protein expression. Moreover we propose that IGFBP-3 immunostaining in gastric tissue may be a useful marker for malignancy.

Role of miRNAs and their potential to be useful as diagnostic and prognostic biomarkers in gastric cancer.

Alterations in epigenetic control of gene expression play an important role in many diseases, including gastric cancer. Many studies have identified a large number of upregulated oncogenic miRNAs and downregulated tumour-suppressor miRNAs in this type of cancer. In this review, we provide an overview of the role of miRNAs, pointing to their potential to be useful as diagnostic and/or prognostic biomarkers in gastric cancer. Moreover, we discuss the influence of polymorphisms and epigenetic modifications on miRNA activity.

Reduced mRNA expression levels of MBD2 and MBD3 in gastric carcinogenesis.

Aberrant methylation has been reported in several neoplasias, including gastric cancer. The methyl-CpG-binding domain (MBD) family proteins have been implicated in the chromatin remodeling process, leading to the modulation of gene expression. To evaluate the role of MBD2 and MBD3 in gastric carcinogenesis and the possible association with clinicopathological characteristics, we assessed the mRNA levels and promoter methylation patterns in gastric tissues. In this study, MBD2 and MBD3 mRNA levels were determined by RT-qPCR in 28 neoplastic and adjacent nonneoplastic and 27 gastritis and non-gastritis samples. The promoter methylation status was determined by bisulfite sequencing, and we found reduced MBD2 and MBD3 levels in the neoplastic samples compared with the other groups. Moreover, a strong correlation between the MBD2 and MBD3 expression levels was observed in each set of paired samples. Our data also showed that the neoplastic tissues exhibited higher MBD2 promoter methylation than the other groups. Interestingly, the non-gastritis group was the only one with positive methylation in the MBD3 promoter region. Furthermore, a weak correlation between gene expression and methylation was observed. Therefore, our data suggest that DNA methylation plays a minor role in the regulation of MBD2 and MBD3 expression, and the presence of methylation at CpGs that interact with transcription factor complexes might also be involved in the modulation of these genes. Moreover, reduced mRNA expression of MBD2 and MBD3 is implicated in gastric carcinogenesis, and thus, further investigations about these genes should be conducted for a better understanding of the role of abnormal methylation involved in this neoplasia.