Senchenko Vera N, Kisseljova Natalia P, Ivanova Tatyana A, Dmitriev Alexey A, Krasnov George S, Kudryavtseva Anna V, Panasenko Grigory V, Tsitrin Evgeny B, Lerman Michael I, Kisseljov Fyodor L, Kashuba Vladimir I, Zabarovsky Eugene R
Engelhardt Institute of Molecular Biology; Russian Academy of Sciences; Moscow, Russia.
N.N. Blokhin Russian Cancer Research Center; Russian Academy of Medical Sciences; Moscow, Russia.
Epigenetics. 2013 Apr;8(4):409-20. doi: 10.4161/epi.24233. Epub 2013 Mar 11.
Genetic and epigenetic alterations in cervical carcinomas were investigated using NotI-microarrays containing 180 cloned sequences flanking all NotI-sites associated with genes on chromosome 3. In total, 48 paired normal/tumor DNA samples, specifically enriched in NotI-sites, were hybridized to NotI-microarrays. Thirty genes, including tumor suppressors or candidates (for example, VHL, RBSP3/CTDSPL, ITGA9, LRRC3B, ALDH1L1, EPHB1) and genes previously unknown as cancer-associated (ABHD5, C3orf77, PRL32, LOC285375, FGD5 and others), showed methylation/deletion in 21-44% of tumors. The genes were more frequently altered in squamous cell carcinomas (SCC) than in adenocarcinomas (ADC, p<0.01). A set of seven potential markers (LRRN1, PRICKLE2, VHL, BHLHE40, RBSP3, CGGBP1 and SOX14) is promising for discrimination of ADC and SCC. Alterations of more than 20 genes simultaneously were revealed in 23% of SCC. Bisulfite sequencing analysis confirmed methylation as a frequent event in SCC. High down-regulation frequency was shown for RBSP3, ITGA9, VILL, APRG1/C3orf35 and RASSF1 (isoform A) genes (3p21.3 locus) in SCC. Both frequency and extent of RASSF1A and RBSP3 mRNA level decrease were more pronounced in tumors with lymph node metastases compared with non-metastatic ones (p ≤ 0.05). We confirmed by bisulfite sequencing that RASSF1 promoter methylation was a rare event in SCC and, for the first time, demonstrated RASSF1A down-regulation at both the mRNA and protein levels without promoter methylation in tumors of this histological type. Thus, our data revealed novel tumor suppressor candidates located on chromosome 3 and a frequent loss of epigenetic stability of 3p21.3 locus in combination with down-regulation of genes in cervical cancer.
利用NotI微阵列研究宫颈癌中的基因和表观遗传改变，该微阵列包含180个克隆序列，这些序列位于与3号染色体上基因相关的所有NotI位点两侧。总共48对正常/肿瘤DNA样本（特别富集NotI位点）与NotI微阵列进行杂交。30个基因，包括肿瘤抑制基因或候选基因（例如，VHL、RBSP3/CTDSPL、ITGA9、LRRC3B、ALDH1L1、EPHB1）以及先前未知与癌症相关的基因（ABHD5、C3orf77、PRL32、LOC285375、FGD5等），在21% - 44%的肿瘤中显示出甲基化/缺失。这些基因在鳞状细胞癌（SCC）中比在腺癌（ADC）中更频繁地发生改变（p<0.01）。一组七个潜在标志物（LRRN1、PRICKLE2、VHL、BHLHE40、RBSP3、CGGBP1和SOX14）有望用于区分ADC和SCC。在23%的SCC中发现同时有超过20个基因发生改变。亚硫酸氢盐测序分析证实甲基化在SCC中是常见事件。SCC中RBSP3、ITGA9、VILL、APRG1/C3orf35和RASSF1（异构体A）基因（3p21.3位点）显示出高下调频率。与无淋巴结转移的肿瘤相比，有淋巴结转移的肿瘤中RASSF1A和RBSP3 mRNA水平降低的频率和程度更明显（p≤0.05）。我们通过亚硫酸氢盐测序证实RASSF1启动子甲基化在SCC中是罕见事件，并且首次证明在这种组织学类型的肿瘤中，RASSF1A在mRNA和蛋白质水平均下调，而无启动子甲基化。因此，我们的数据揭示了位于3号染色体上的新的肿瘤抑制候选基因，以及宫颈癌中3p21.3位点频繁的表观遗传稳定性丧失并伴有基因下调。
