人侵袭性乳腺癌中Claudin 1转录变体的鉴定
Identification of Claudin 1 Transcript Variants in Human Invasive Breast Cancer.
作者信息
Blanchard Anne A, Zelinski Teresa, Xie Jiuyong, Cooper Steven, Penner Carla, Leygue Etienne, Myal Yvonne
机构信息
Department of Pathology, University of Manitoba, Winnipeg, Manitoba, Canada.
Department of Physiology and Pathophysiology, University of Manitoba, Winnipeg, Manitoba, Canada.
出版信息
PLoS One. 2016 Sep 20;11(9):e0163387. doi: 10.1371/journal.pone.0163387. eCollection 2016.
BACKGROUND
The claudin 1 tight junction protein, solely responsible for the barrier function of epithelial cells, is frequently down regulated in invasive human breast cancer. The underlying mechanism is largely unknown, and no obvious mutations in the claudin 1 gene (CLDN1) have been identified to date in breast cancer. Since many genes have been shown to undergo deregulation through splicing and mis-splicing events in cancer, the current study was undertaken to investigate the occurrence of transcript variants for CLDN1 in human invasive breast cancer.
METHODS
RT-PCR analysis of CLDN1 transcripts was conducted on RNA isolated from 12 human invasive breast tumors. The PCR products from each tumor were resolved by agarose gel electrophoresis, cloned and sequenced. Genomic DNA was also isolated from each of the 12 tumors and amplified using PCR CLDN1 specific primers. Sanger sequencing and single nucleotide polymorphism (SNP) analyses were conducted.
RESULTS
A number of CLDN1 transcript variants were identified in these breast tumors. All variants were shorter than the classical CLDN1 transcript. Sequence analysis of the PCR products revealed several splice variants, primarily in exon 1 of CLDN1; resulting in truncated proteins. One variant, V1, resulted in a premature stop codon and thus likely led to nonsense mediated decay. Interestingly, another transcript variant, V2, was not detected in normal breast tissue samples. Further, sequence analysis of the tumor genomic DNA revealed SNPs in 3 of the 4 coding exons, including a rare missense SNP (rs140846629) in exon 2 which represents an Ala124Thr substitution. To our knowledge this is the first report of CLDN1 transcript variants in human invasive breast cancer. These studies suggest that alternate splicing may also be a mechanism by which claudin 1 is down regulated at both the mRNA and protein levels in invasive breast cancer and may provide novel insights into how CLDN1 is reduced or silenced in human breast cancer.
背景
紧密连接蛋白claudin 1单独负责上皮细胞的屏障功能，在侵袭性人类乳腺癌中经常下调。其潜在机制很大程度上未知，迄今为止在乳腺癌中尚未发现claudin 1基因（CLDN1）有明显突变。由于许多基因已被证明在癌症中通过剪接和错配剪接事件发生失调，因此进行了本研究以调查CLDN1转录变体在人类侵袭性乳腺癌中的发生情况。
方法
对从12个人类侵袭性乳腺肿瘤中分离的RNA进行CLDN1转录本的逆转录聚合酶链反应（RT-PCR）分析。每个肿瘤的PCR产物通过琼脂糖凝胶电泳分离、克隆并测序。还从这12个肿瘤中的每一个中分离基因组DNA，并使用CLDN1特异性引物进行PCR扩增。进行桑格测序和单核苷酸多态性（SNP）分析。
结果
在这些乳腺肿瘤中鉴定出许多CLDN1转录变体。所有变体都比经典的CLDN1转录本短。PCR产物的序列分析揭示了几种剪接变体，主要在CLDN1的外显子1中；导致截短的蛋白质。一种变体V1导致过早的终止密码子，因此可能导致无义介导的衰变。有趣的是，另一种转录变体V2在正常乳腺组织样本中未检测到。此外，肿瘤基因组DNA的序列分析揭示了4个编码外显子中的3个存在SNP，包括外显子2中一个罕见的错义SNP（rs140846629），它代表Ala124Thr替代。据我们所知，这是人类侵袭性乳腺癌中CLDN1转录变体的首次报道。这些研究表明，可变剪接也可能是claudin 1在侵袭性乳腺癌的mRNA和蛋白质水平下调的一种机制，并可能为CLDN1在人类乳腺癌中如何减少或沉默提供新的见解。
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