记录细胞力学微环境的合成生物学基因线路

肿瘤的发生通常伴随基质硬度的增加，如乳腺癌。良性肿瘤组织的弹性模量为1.9~3.7 kPa，高于正常组织的1.1~1.8 kPa。Piezo1作为第一个满足严格定义的哺乳动物机械力敏感的Ca²⁺离子通道，可被多种类型的机械刺激激活，但将其作为哺乳动物细胞力学方面感受元件还未见报道。NFAT作为一种对免疫细胞功能、发育、干细胞分化和肿瘤进展至关重要的钙依赖性转录因子，主要由脉冲形式的钙信号实现激活。基于从特定启动子pNFAT启动转录，Ca²⁺-NFAT信号通路能够实现细胞的外界刺激可与信号表达直接相关。我们设计了定量诱导表达Piezo1-BFP及稳定表达Ca²⁺信号下游pNFAT-EGFP基因线路，并探究这一基因线路能否作为探测肿瘤硬度的工具。我们将基因线路构建在CHO^WT和CHO^Piezo1-/-细胞中，进行2D/3D培养。在没有DOX诱导Piezo1表达的情况下，NFAT-EGFP在2D培养的CHO^WT中有很高的基底值，在3D培养中表达量较2D培养低，在CHO^piezo1-/-中2D/3D培养时均不表达。Piezo1-NFAT-CHO^Piezo1-/-细胞2D培养条件时，细胞受力均一，Piezo1-EGFP表达量与NFAT-EGFP表达量呈线性相关，随DOX浓度的升高而升高。并且细胞培养在PDMS上2D培养Piezo1-BFP表达量较塑料培养板2D培养低，表明Piezo1的表达量与基质硬度有关。Piezo1-NFAT-CHO^Piezo1-/-细胞3D培养时，Piezo1-EGFP与NFAT-EGFP表达量之间的线性关系被破环。超低吸附3D培养，细胞受到连续不均匀的力，NFAT-EGFP峰图呈连续弥散的形态；1%Agarose 凹槽3D培养，外围细胞受到的力与内细胞团受到的不同，NFAT-EGFP的表达量呈双峰，体现出Piezo1-NFAT基因线路对细胞受力的响应。综上，我们认为Piezo1-NFAT基因线路具有监测体内组织力学的变化的潜力。

Tumors are often accompanied by an increase in matrix stiffness, as in breast cancer. The elastic modulus of benign tumor tissue was 1.9~3.7 kPa, which is higher than 1.1~1.8 kPa of normal tissue. Piezo1 is the first mechanism-sensitive Ca²⁺ channel protein in mammals. It can be activated by many types of mechanical stimuli, but its role as a mechanical sensing element in the mammalian cell has not been reported. NFAT is a calcium-dependent transcription factor that is critical for immune cell function, development, stem cell differentiation, and tumor progression. Its gene expression is selected by calcium signals in the form of pulses. Based on the specific promoter pNFAT, the Ca²⁺-NFAT signaling pathway can record a signal that is directly related to cellular stimulation. We designed a gene circuit consisting of the DOX-induced and quantitative expression of Piezo1-BFP and the stable expression of Ca²⁺ signal downstream to investigate whether this gene circuit can be used as a tool to detect tumor stiffness. We constructed the gene circuits in CHO^WT and CHO^Piezo1-/- cells for 2D/3D culture. In the absence of DOX, which induced Piezo1 expression, NFAT-EGFP had a high baseline in CHO^WT cells and a lower expression in the 3D culture than in the 2D culture and was not expressed in the 2D/3D culture of CHO^piezo1-/- cells. Piezo1-NFAT-CHO^Piezo1-/- cells were tensed uniformly during 2D culture, and the expression of Piezo1-EGFP was linear dependent on the expression of NFAT-EGFP, which increased with the increase of DOX concentration. The expression of Piezo1-BFP in 2D culture on PDMS was lower than that in 2D culture on plastic plates, indicating that the expression of Piezo1 was related to matrix stiffness. With Piezo1-NFAT-CHO^Piezo1-/- cell 3D culture, the linear relationship between Piezo1-EGFP and the expression of NFAT-EGFP was broken. In a 3D culture with an Ultra-low attachment microplate, cells were subjected to continuous non-uniform force, and the NFAT-EGFP showed a continuous dispersion peak pattern. In the 1%Agarose groove 3D culture, the force of the peripheral cells was different from that of the inner cell mass. The expression of NFAT-EGFP was bimodal. The Piezo1-NFAT gene circuit in the cells had different responses. In conclusion, we think the Piezo1-NFAT gene circuit has the potential to monitor the changes in internal tissue mechanics.

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