Real-time PCR as a tool for quantitative analysis of PI-PLCbeta1 gene expression in myelodysplastic syndrome.
Real-time PCR as a tool for quantitative analysis of PI-PLCbeta1 gene expression in myelodysplastic syndrome.
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实时 PCR 作为骨髓增生异常综合征中 PI-PLCbeta1 基因表达定量分析的工具。
DOI：
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发表时间：
2006
影响因子：
5.4
通讯作者：
L. Cocco
中科院分区：
文献类型：
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作者：
M. Follo;C. Bosi;C. Finelli;R. Fiume;I. Faenza;G. Ramazzotti;G. Gaboardi;L. Manzoli;L. Cocco
phosphoinosiide -specific phospholipase C (PI-PLC) beta1是核信号转导的关键酶，参与许多细胞过程，如增殖和分化。特别是，PI-PLCbeta1基因在红细胞分化中的参与，使我们在高危骨髓增生异常综合征（MDS）患者中研究该基因。通过荧光原位杂交（FISH）分析，我们之前已经证明，在GTG带正常和致命结局的MDS患者中，PI-PLCbeta1基因经历了单等位基因和间质缺失。Real-time PCR具有灵敏度高、精密度好、动态范围大等特点，已成为基因表达定量测量的首选方法。在本研究中，我们对所有进行FISH分析的MDS患者进行了相对定量的实时聚合酶链反应（PCR）分析。此外，我们还评估了PI-PLCbeta1基因在健康供体和HL60细胞系上的表达，这有助于测试该技术的准确性，因为PI-PLCbeta1的表达水平很低。为了分析和量化PI-PLCbeta1基因的两种不同剪接变体（1a和1b）的水平，我们使用了TaqMan同种异构体特异性探针。我们已经看到，与预期的HL60细胞系相比，所有MDS患者的PI-PLCbeta1 mRNA水平较高，但与健康供者相比水平较低。此外，与PI-PLCbeta1a mRNA相比，MDS母细胞总是表达更高水平的PI-PLCbeta1b mRNA。我们的数据支持这样的观点，即PI-PLCbeta1基因的缺失确实导致了酶的表达减少。此外，剪接异构体1b仅为细胞核，与1a异构体相比，它似乎在某种程度上部分保存了下来，1a异构体是细胞核和细胞质，这暗示细胞核与细胞质的PI-PLC信号传导可能不平衡，这反过来可能影响MDS细胞周期的进展。
Phosphoinositide-specific phospholipase C (PI-PLC) beta1 is a key enzyme in nuclear signal transduction, and it is involved in many cellular processes, such as proliferation and differentiation. In particular, the involvement of the PI-PLCbeta1 gene in erythroid differentiation lead us to investigate this gene in patients affected by high-risk myelodysplastic syndrome (MDS). By using fluorescence in situ hybridization (FISH) analysis, we have previously evidenced that, in MDS patients with normal GTG banding and a fatal outcome, the PI-PLCbeta1 gene undergoes monoallelic and interstitial deletion. Real-time PCR is characterized by high sensitivity, excellent precision and large dynamic range, and has become the method of choice for quantitative gene expression measurements. In the present study, we have performed a relative quantification real-time polymerase chain reaction (PCR) analysis on all of the MDS patients tested for FISH analysis. Furthermore, we have evaluated the expression of the PI-PLCbeta1 gene on healthy donors and the HL60 cell line, which is useful for testing the accuracy of the technology because of its low expression of PI-PLCbeta1. To analyze and quantify the levels of the two different splicing variants of PI-PLCbeta1 gene (1a and 1b), we have used a TaqMan isoform specific probe. We have seen that all of the MDS patients have higher levels of the PI-PLCbeta1 mRNA compared to the HL60 cell line as expected, but lower levels compared to the healthy donors. Furthermore, MDS blasts always express higher levels of PI-PLCbeta1b mRNA compared to PI-PLCbeta1a mRNA. Our data support the contention that the deletion of the PI-PLCbeta1 gene is indeed responsible for a reduced expression of the enzyme. In addition, the splicing isoform 1b, which is only nuclear, seems to be somehow partially preserved compared to the 1a isoform, which is nuclear and cytoplasmatic, hinting at a possible imbalance of the nuclear versus cytoplasmatic PI-PLC signaling which, in turn, could affect the cell cycle progression of MDS blasts.