Comprehensive screening of genes resistant to an anticancer drug in esophageal squamous cell carcinoma.
Comprehensive screening of genes resistant to an anticancer drug in esophageal squamous cell carcinoma.
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DOI：
10.3892/ijo.2015.3085
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发表时间：
2015-09
影响因子：
5.2
通讯作者：
Kitagawa Y
中科院分区：
文献类型：
--
作者：
Tsutsui M;Kawakubo H;Hayashida T;Fukuda K;Nakamura R;Takahashi T;Wada N;Saikawa Y;Omori T;Takeuchi H;Kitagawa Y
化疗耐药是食管癌治疗中的一个主要问题。耐药性可能是由肿瘤的遗传变化介导的;因此，基因突变的鉴定可能会导致更好的治疗结果。我们使用了一种新的方法，涉及转座子筛选和鉴定耐药基因。转座子是从基因上的一个位置移动到另一个位置的DNA序列。设计了一种修饰的piggyBac转座子作为插入诱变剂，并加入巨细胞病毒（CMV）启动子序列以诱导强转录。当转座子插入到某个基因的上游时，该基因将被过度表达，而当插入或基因内插入时，它将被下调。在建立转座子标记的细胞库后，我们用顺铂（CDDP）处理来自食管鳞状细胞癌（ESCC）[东北食管（TE）]的细胞系。我们对抗性菌落进行了splinkerette PCR和TOPO克隆。对细菌菌落进行测序，并使用下一代测序来鉴定作为CDDP抗性候选基因的过表达/下调序列。我们建立了4个转座子标记细胞的细胞系，TE 4、5、9和15。我们用CDDP处理两个相对有活力的细胞系TE 4和TE 15。我们从8个抗性菌落中鉴定出37个候选基因。8个基因过度表达，29个基因下调。在这些基因中，Janus激酶2（JAK 2）与骨髓增生性肿瘤的进展有关。我们确定了37个候选基因负责CDDP耐药的两个细胞系来自ESCC细胞。该方法价格低廉，相对简单，并且能够在基因组中引入激活和失活突变，从而允许鉴定耐药基因。
Drug resistance to chemotherapy is a major issue in esophageal cancer management. Drug resistance may be mediated by genetic changes in the tumor; therefore, the identification of gene mutations may lead to better therapeutic outcomes. We used a novel method involving transposons to screen and identify drug-resistant genes. Transposons are DNA sequences that move from one location on the gene to another. A modified piggyBac transposon was designed as an insertion mutagen, and a cytomegalovirus (CMV) promoter sequence was added to induce strong transcription. When the transposon is inserted to the upstream of a certain gene, the gene will be overexpressed while when intserted down or intragenically, it will be downregulated. After establishing a transposon-tagged cell library, we treated cell lines derived from esophageal squamous cell carcinomas (ESCC) [Tohoku esophagus (TE)] with cisplatin (CDDP). We performed splinkerette PCR and TOPO cloning on the resistant colonies. Bacterial colonies were sequenced, and next-generation sequencing was used to identify the overexpressed/downregulated sequences as candidate genes for CDDP resistance. We established 4 cell lines of transposon-tagged cells, TE4, 5, 9 and 15. We treated the two relatively viable cell lines, TE4 and TE15, with CDDP. We identified 37 candidate genes from 8 resistant colonies. Eight genes were overexpressed whilst 29 were downregulated. Among these genes was Janus kinase 2 (JAK2) that is implicated in the progression of myeloproliferative neoplasms. We identified 37 candidate genes responsible for CDDP resistance in the two cell lines derived from ESCC cells. The method is inexpensive, relatively simple, and capable of introducing activating and de-activating mutations in the genome, allowing for drug-resistant genes to be identified.