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首页> 外文期刊>BMC Molecular Biology >qPCR in gastrointestinal stromal tumors: Evaluation of reference genes and expression analysis of KIT and the alternative receptor tyrosine kinases FLT3, CSF1-R, PDGFRB, MET and AXL
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qPCR in gastrointestinal stromal tumors: Evaluation of reference genes and expression analysis of KIT and the alternative receptor tyrosine kinases FLT3, CSF1-R, PDGFRB, MET and AXL

机译：胃肠道间质瘤中的qPCR：参考基因的评估以及KIT和其他受体酪氨酸激酶FLT3，CSF1-R，PDGFRB，MET和AXL的表达分析

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Background Gastrointestinal stromal tumors (GIST) represent the most common mesenchymal tumors of the gastrointestinal tract. About 85% carry an activating mutation in the KIT or PDGFRA gene. Approximately 10% of GIST are so-called wild type GIST (wt-GIST) without mutations in the hot spots. In the present study we evaluated appropriate reference genes for the expression analysis of formalin-fixed, paraffin-embedded and fresh frozen samples from gastrointestinal stromal tumors. We evaluated the gene expression of KIT as well as of the alternative receptor tyrosine kinase genes FLT3, CSF1-R, PDGFRB, AXL and MET by qPCR. wt-GIST were compared to samples with mutations in KIT exon 9 and 11 and PDGFRA exon 18 in order to evaluate whether overexpression of these alternative RTK might contribute to the pathogenesis of wt-GIST.Results Gene expression variability of the pooled cDNA samples is much lower than the single reverse transcription cDNA synthesis. By combining the lowest variability values of fixed and fresh tissue, the genes POLR2A, PPIA, RPLPO and TFRC were chosen for further analysis of the GIST samples. Overexpression of KIT compared to the corresponding normal tissue was detected in each GIST subgroup except in GIST with PDGFRA exon 18 mutation. Comparing our sample groups, no significant differences in the gene expression levels of FLT3, CSF1R and AXL were determined. An exception was the sample group with KIT exon 9 mutation. A significantly reduced expression of CSF1R, FLT3 and PDGFRB compared to the normal tissue was detected. GIST with mutations in KIT exon 9 and 11 and in PDGFRA exon 18 showed a significant PDGFRB downregulation.Conclusions As the variability of expression levels for the reference genes is very high comparing fresh frozen and formalin-fixed tissue there is a strong need for validation in each tissue type. None of the alternative receptor tyrosine kinases analyzed is associated with the pathogenesis of wild-type or mutated GIST. It remains to be clarified whether an autocrine or paracrine mechanism by overexpression of receptor tyrosine kinase ligands is responsible for the tumorigenesis of wt-GIST.
机译：背景技术胃肠道间质瘤（GIST）代表胃肠道最常见的间质肿瘤。大约85％的人在KIT或PDGFRA基因中带有激活突变。大约10％的GIST是所谓的野生型GIST（wt-GIST），热点没有突变。在本研究中，我们评估了合适的参考基因，用于胃肠道间质瘤中福尔马林固定，石蜡包埋和新鲜冷冻样品的表达分析。我们通过qPCR评估了KIT以及其他受体酪氨酸激酶基因FLT3，CSF1-R，PDGFRB，AXL和MET的基因表达。将wt-GIST与KIT外显子9和11以及PDGFRA外显子18突变的样品进行比较，以评估这些替代RTK的过表达是否可能导致wt-GIST的发病机理。结果合并的cDNA样品的基因表达变异性很大低于单反转录cDNA的合成。通过结合固定和新鲜组织的最低变异性值，选择基因POLR2A，PPIA，RPLPO和TFRC进行GIST样品的进一步分析。在每个GIST亚组中检测到与相应正常组织相比KIT的过表达，但带有PDGFRA外显子18突变的GIST除外。与我们的样本组相比，未确定FLT3，CSF1R和AXL的基因表达水平有显着差异。一个例外是具有KIT外显子9突变的样本组。与正常组织相比，CSF1R，FLT3和PDGFRB的表达明显降低。在KIT外显子9和11以及PDGFRA外显子18中发生突变的GIST显示出显着的PDGFRB下调。结论由于与新鲜冷冻和福尔马林固定的组织相比，参考基因的表达水平差异很大，因此强烈需要在每种组织类型。分析的替代受体酪氨酸激酶均与野生型或突变的GIST的发病机制无关。尚不清楚由受体酪氨酸激酶配体的过表达引起的自分泌或旁分泌机制是wt-GIST的肿瘤发生原因。

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