Systems biology approach to transplant tolerance: proof of concept experiments using RNA interference (RNAi) to knock down hub genes in Jurkat and HeLa cells in vitro.
Systems biology approach to transplant tolerance: proof of concept experiments using RNA interference (RNAi) to knock down hub genes in Jurkat and HeLa cells in vitro.
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系统生物学方法的移植耐受性：使用RNA干扰（RNAi）在体外敲低概念实验的概念证明实验（RNAi）。
DOI：
10.1016/j.jss.2011.12.002
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发表时间：
2012-07
期刊：
影响因子：
--
通讯作者：
Khanna A
中科院分区：
文献类型：
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作者：
Lwin WW;Park K;Wauson M;Gao Q;Finn PW;Perkins D;Khanna A
系统生物学在研究复杂系统（如人类基因的功能相互联系）方面越来越重要。为了研究T细胞免疫反应中涉及的分子相互作用，我们使用物理基因-基因相互作用数据库，用R语言算法构建分子相互作用网络（互连）。这有助于鉴定高度互连的“枢纽”基因AT（1）P5 C1、IL 6ST、PRKCZ、MYC、FOS、JUN和MAPK 1。我们假设基因网络中这些枢纽基因的抑制将导致对T细胞的显著表型效应，并在体外对此进行了研究。然后使用Celloscape分析并可视化分子相互作用网络。Jurkat和HeLa细胞用针对所选中枢基因的siRNA转染。使用ATP发光和BrdU标记测量细胞增殖，其在活化后36、72和96小时测量。在T细胞刺激后，我们发现当使用siRNA转染敲低枢纽基因ATP 5C 1和PRKCZ时，ATP产生显著降低（P<0.05），而在siRNA转染的HeLa细胞中没有观察到ATP产生的差异。而MAPK 1、IL 6ST、ATP 5C 1、JUN和FOS基因敲除后，HeLa细胞的增殖能力明显下降（P<0.05）。在Jukat和HeLa细胞中，使用siRNA的靶向基因敲除显示Jurkat和HeLa细胞中的细胞增殖和ATP产生减少。然而，Jurkat T细胞和HELA细胞使用不同的枢纽基因来调节激活反应。该实验提供了应用T细胞中枢基因的siRNA敲低来评估其增殖能力和ATP产生的原理证明。这一新概念概述了一种系统生物学方法，以确定枢纽基因的靶向治疗。
Systems biology is gaining importance in studying complex systems such as the functional interconnections of human genes. To investigate the molecular interactions involved in T cell immune responses, we used databases of physical gene-gene interactions to constructed molecular interaction networks (interconnections) with R language algorithms. This helped to identify highly interconnected “hub” genes AT(1)P5C1, IL6ST, PRKCZ, MYC, FOS, JUN and MAPK1. We hypothesized that suppression of these hub genes in the gene network would result in significant phenotypic effects on T cells and examined this in-vitro. The molecular interaction networks were then analyzed and visualized with Cytoscape. Jurkat and HeLa cells were transfected with siRNA for the selected hub genes. Cell proliferation was measured using ATP luminescence and BrdU labeling, which were measured 36, 72 and 96 hours after activation. Following T cell stimulation, we found a significant decrease in ATP production (P<0.05) when the hub genes ATP5C1 and PRKCZ were knocked down using siRNA transfection, whereas no difference in ATP production was observed in siRNA transfected HeLa cells. However, HeLa cells showed a significant (P<0.05) decrease in cell proliferation when the genes MAPK1, IL6ST, ATP5C1, JUN and FOS were knocked down. In both Jukat and HeLa cells, targeted gene knockdown using siRNA showed decreased cell proliferation and ATP production in both Jurkat andHeLa cells. However, Jurkat T cells and HELA cells use different hub genes to regulate activation responses. This experiment provides proof of principle of applying siRNA knockdown of T cell hub genes to evaluate their proliferative capacity and ATP production. This novel concept outlines a systems biology approach to identify hub genes for targeted therapeutics.
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