Amplified genes are not necessarily over expressed, they can be unchanged or down regulated in cervical cancer cell lines [expression array data]

Several copy number altered regions (CNA) have been identified in the genome of cervical cancer, especially amplifications of 3q and 5p. However, the contribution of those alterations to cervical carcinogenesis is still a matter of debate, since genome-wide, there is a lack of correlation between CNAs and gene expression. In this study, we investigated whether the CNAs in cell lines (CaLo, CasKi, HeLa, SiHa), at a gene-by-gene level, are related to changes in gene expression. On average 19.2% of the whole genome of cell lines had CNA. However, only 2.4% consisted of minimal recurrent regions (MRR), common to all cell lines. Whereas 3q had just some sparse common gains (13%), 5p was entirely duplicated recurrently. Genome-wide, only 11% of genes located in CNAs changed gene expression. In contrast, the rate increased over 3 fold times in MRRs. Chr 5p was confirmed entirely amplified by FISH. In spite of this, at most 32.9% of the explored genes in 5p (n=202) were de-regulated. In 3q, the rate was just 11.8%. Even in 3q26, which had five MRRs and 38.7% of SNPs was gained recurrently, the rate rose slightly to 13.6% (10 out of 73). Interestingly, up to 16% of de-regulated genes in 5p and 80% in 3q26 were down-regulated, suggesting additional factors are involved in gene repression. The de-regulated genes in 3q and 5p were found in clusters, suggesting local chromatin factors may also influence gene expression. In regions amplified discontinuously, the rate of down-regulated genes rose steadily as the number of amplified SNPs increased (p<0.01, Spearman's correlation). This suggests partial gene amplification as a mechanism of silencing gene expression. Additional genes were identified up- or down-regulated in 5p and 3q, which could be involved in cervical carcinogenesis, especially implicated in apoptosis. Those include CLPTM1L, AHRR, PDCD6 and DAP in 5p and TNFSF10 and ECT2 in 3q. Overall, the gene expression and copy number profiles suggest other factors, like epigenetic or chromatin domains, may influence gene expression within the entirely amplified genome segments. Further studies are needed to elucidate how these mechanisms regulate gene expression. We analyzed four cervical cancer cell lines and normal exocervix tissue from 10 healthy female subjects using the Affymetrix Human Exon 1.0 ST platform. Array data was processed by Affymetrix Exon Array Computational Tool. Biological replicates were performed for the cell lines. biological replicate: Calo-a, Calo-b, Calo-c biological replicate: Caski-a, Caski-b, Caski-c biological replicate: Hela-a, Hela-b, Hela-c biological replicate: Siha-a, Siha-b, Siha-c

现已在宫颈癌基因组中鉴定出多个拷贝数变异区域（copy number altered regions, CNA），尤以3q和5p区域的扩增最为显著。然而，此类变异在宫颈癌发生发展中的作用仍存在争议，因为全基因组水平上，拷贝数变异与基因表达之间缺乏相关性。本研究旨在探究细胞系（CaLo、CasKi、HeLa、SiHa）中逐基因水平的拷贝数变异是否与基因表达变化相关。

平均而言，这些细胞系的全基因组中有19.2%存在拷贝数变异，但其中仅2.4%为所有细胞系共有的最小重复区域（minimal recurrent regions, MRR）。3q区域仅存在少量散在的共有的拷贝数增加（占比13%），而5p区域则全程发生重复性扩增。

全基因组范围内，位于拷贝数变异区域的基因中仅有11%发生了表达改变。与之形成对比的是，最小重复区域内的基因表达改变率提升了3倍以上。通过荧光原位杂交（fluorescence in situ hybridization, FISH）验证，5p区域确实发生了全程扩增。尽管如此，在5p区域所检测的202个基因中，至多仅有32.9%的基因出现表达失调。在3q区域，这一比例仅为11.8%。即便在3q26区域——该区域存在5个最小重复区域，且38.7%的单核苷酸多态性（single nucleotide polymorphism, SNP）发生了重复性增加——其基因表达失调率也仅小幅升至13.6%（73个基因中有10个）。

有趣的是，5p区域中多达16%的失调基因以及3q26区域中80%的失调基因均为表达下调，这提示存在额外的基因沉默相关调控因素。3q和5p区域的失调基因呈簇状分布，表明局部染色质环境也可能对基因表达产生影响。在非连续性扩增的区域中，随着扩增的单核苷酸多态性数量增加，下调基因的比例稳步上升（p<0.01，斯皮尔曼相关性分析），这提示部分基因扩增可能是一种基因表达沉默的机制。

在5p和3q区域中，还鉴定出了其他存在表达上调或下调的基因，这些基因可能参与宫颈癌发生发展，尤其与细胞凋亡过程相关。其中5p区域涉及CLPTM1L、AHRR、PDCD6和DAP，3q区域涉及TNFSF10和ECT2。总体而言，基因表达与拷贝数谱分析结果提示，表观遗传或染色质结构域等其他因素，可能会对全程扩增的基因组片段内的基因表达产生调控作用。后续仍需开展进一步研究，以阐明这些机制如何调控基因表达。

本研究采用Affymetrix人类外显子1.0 ST芯片平台，对4株宫颈癌细胞系以及10名健康女性受试者的正常宫颈外口组织进行检测。芯片数据通过Affymetrix外显子芯片计算工具进行处理。所有细胞系均设置生物学重复：
Calo组：Calo-a、Calo-b、Calo-c
Caski组：Caski-a、Caski-b、Caski-c
HeLa组：HeLa-a、HeLa-b、HeLa-c
SiHa组：SiHa-a、SiHa-b、SiHa-c