Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi-cho, Takamatsu, Kagawa 761-0395, Japan.
Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan.
Analyst. 2019 Aug 7;144(15):4613-4621. doi: 10.1039/c9an00725c. Epub 2019 Jun 26.
Lung cancer cells harbor various gene mutations in the mRNA sequence of the Epidermal Growth Factor Receptor (EGFR), especially the mutations of exon19del E746-A750, T790M, and L858R. This results in cancer progression and resistance to anticancer drugs (tyrosine kinase inhibitor; TKI). Therefore, the imaging analysis of EGFR mutations is required for the treatment planning for non-small cell lung cancers. This study focused on the imaging analysis of a single nucleotide substitute in EGFR mutated cancer cells. We developed three novel peptide nucleic acid (PNA)-DNA probes for recognizing and detecting the following three gene mutations in EGFR gene mutations. The PNA-DNA probes consist of fluorescein isothiocyanate (FITC) conjugated PNA as a detection probe and Dabcyl conjugated DNA as a quencher probe. The PNA-DNA probes were used to validate the feasibility for detecting three EGFR mutated sequences: exon19del E746-A750, T790M, and L858R. The three probes emitted fluorescent dose-dependent signals against three target DNA and RNA. Using the three PNA-DNA probes, we succeeded in distinguishing three kinds of lung-cancer cell lines (H1975, PC-9, and A549) which have different EGFR mutations by the fluorescence in situ hybridization (FISH) method.
肺癌细胞在表皮生长因子受体 (EGFR) 的 mRNA 序列中存在各种基因突变，特别是外显子 19del E746-A750、T790M 和 L858R 的突变。这导致癌症的进展和对抗癌药物（酪氨酸激酶抑制剂；TKI）的耐药性。因此，需要对 EGFR 突变进行成像分析，以制定非小细胞肺癌的治疗计划。本研究集中于 EGFR 突变癌细胞中单核苷酸替代的成像分析。我们开发了三种新型肽核酸（PNA）-DNA 探针，用于识别和检测 EGFR 基因突变中的以下三种基因突变。PNA-DNA 探针由荧光素异硫氰酸酯（FITC）缀合的 PNA 作为检测探针和 Dabcyl 缀合的 DNA 作为淬灭探针组成。PNA-DNA 探针用于验证检测三种 EGFR 突变序列：外显子 19del E746-A750、T790M 和 L858R 的可行性。三种探针针对三种靶 DNA 和 RNA 发出荧光剂量依赖性信号。使用这三种 PNA-DNA 探针，我们成功地通过荧光原位杂交（FISH）方法区分了具有不同 EGFR 突变的三种肺癌细胞系（H1975、PC-9 和 A549）。
