Genetic and epigenetic silencing of the beclin 1 gene in sporadic breast tumors.
BACKGROUND
Beclin 1, an important autophagy-related protein in human cells, is involved in cell death and cell survival. Beclin 1 mapped to human chromosome 17q21. It is widely expressed in normal mammary epithelial cells. Although down-regulated expression with mono-allelic deletions of beclin 1 gene was frequently observed in breast tumors, whether there was other regulatory mechanism of beclin 1 was to be investigated. We studied the expression of beclin 1 and explored the possible regulatory mechanisms on its expression in breast tumors.


METHODS
20 pairs of tumors and adjacent normal tissues from patients with sporadic breast invasive ductal cancer (IDCs) were collected. The mRNA expression of beclin 1 was detected by real-time quantitative RT-PCR. Loss of heterozygosity (LOH) was determined by real-time quantitative PCR and microsatellite methods. The protein expression of beclin 1, p53, BRCA1 and BRCA2 was assessed by immunohistochemistry. CpG islands in 5' genomic region of beclin 1 gene were identified using MethylPrimer Program. Sodium bisulfite sequencing was used in examining the methylation status of each CpG island.


RESULTS
Decreased beclin 1 mRNA expression was detected in 70% of the breast tumors, and the protein levels were co-related to the mRNA levels. Expression of beclin 1 mRNA was demonstrated to be much higher in the BRCA1 positive tumors than that in the BRCA1 negative ones. Loss of heterozygosity was detected in more than 45% of the breast tumors, and a dense cluster of CpG islands was found from the 5' end to the intron 2 of the beclin 1 gene. Methylation analysis showed that the promoter and the intron 2 of beclin 1 were aberrantly methylated in the tumors with decreased expression.


CONCLUSIONS
These data indicated that LOH and aberrant DNA methylation might be the possible reasons of the decreased expression of beclin 1 in the breast tumors. The findings here shed some new light on the regulatory mechanisms of beclin 1 in breast cancer.
Institute of Biomedical Informatics, School of Medicine, Tsinghua University, Beijing, PR China.
Zidong
Z
Li
Bo
B
Chen
Yiqing
Y
Wu
Feng
F
Jin
Yongjing
Y
Xia
Xiangjun
X
Liu
D000328:Adult; D000368:Aged; D051017:Apoptosis Regulatory Proteins / Q000096:biosynthesis / Q000235:genetics*; D019313:BRCA1 Protein / Q000096:biosynthesis / Q000235:genetics; D024682:BRCA2 Protein / Q000096:biosynthesis / Q000235:genetics; D000071186:Beclin-1; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D018899:CpG Islands; D019175:DNA Methylation; D015536:Down-Regulation; D044127:Epigenesis, Genetic; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D007438:Introns; D019656:Loss of Heterozygosity; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D008875:Middle Aged; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000235:genetics
D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; C118024:BLID protein, human; D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D024682:BRCA2 Protein; C551750:BRCA2 protein, human; D000071186:Beclin-1; D008565:Membrane Proteins; D012333:RNA, Messenger; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53