基因编码的信号增强型荧光成像系统用于细胞和组织中miRNAs的原位检测

MicroRNAs (miRNAs) have been shown to play important roles in a broad range of biological processes including development, cellular differentiation, proliferation and apoptosis. It be found has close relationship with tumor occurrence or inhibition. Effective and reliable detection methods are vital to study the function of miRNAs in controlling the network of living cells.. Conventional detection methods, such as northern blot, real-time PCR or microarray, have been used to assess miRNAs expression. However, these techniques require the fixation or lysis of cells, and thus cannot be used to study the dynamic function of miRNAs in living cells. Although there are many noninvasive methods to be established monitoring the miRNA biogenesis and function in vivo, there are still many disadvantages cannot be ignored, such as the reporter gene imaging is a kind of signal-off system, which we cannot judge the real reason of the signal attenuation, as well as the molecular beacon is hardly to further test the dynamic changes of miRNAs in the process of cell differentiation and regulation. As an alternative strategy，the protein-based systems such as Tet-on system have been exploit to simulate the signal-on behaviors. This transcription regulatory proteins serve as the synthetic inducible promoters, whose immunogenicity is potentially harmful to organisms, thus limit the broader applications and flexibility of the systems.. Here, we first designed the riboswitch-based gene coded EGFP fluorescent imaging method for miRNAs detection in mammalian cells. In this riboswitch, miRNAs responsed stem–loop structure recombined with the internal ribosome entry site (IRES)sequence ,which constructed by inserting into the upstream of reporter genes (EGFP or Fluc) . The riboswitch is promoted only in the presence of the target miRNAs (miR-23a), while is inhibited in the absence of them. Through ribosome binding or releasing of IRES, the concentration variation of intracellular target miRNAs can be changed with fluorescence intensity of reporter genes, which can be monitored by confocal microscopy or Flow Cytometer, avoiding the disadvantage of molecular beacon or protein-based or signal-off report gene expression systems. The system will be further studied in mammalian tissues and it is hoped to reveal more clues about the roles miRNAs play in organism development. .This work highlights the power of visualized real-time and in situ signal-on reporter genes fluorescence imaging method for mature miRNAs detection in eukaryotic animal cells or tissues. and reveals the general rule or intrinsic mechanism for construction of report gene fluorescent sensors in vivo. The work provides a new method for in suit detection of dynamic activities of miRNAs in cells and tissues. This technique is applicable to a broad range of pathways for tracking miRNA levels during changes in biological processes.

本研究利用人工核糖开关可通过构象变化调控下游基因表达的机理，及真核细胞RNA核糖体内部结合位点（IRES）特性，构建了基于miR-23a浓度变化的荧光增强型成像系统。当胞内无靶miRNAs时，IRES与anti-IRES形成互补区域封闭核糖体与RNA的结合，抑制报告基因表达；反之，胞内有靶miRNAs时，互补区茎环结构打开，释放anti-IRES封闭区，荧光成像系统激活。将此系统稳定整合至细胞基因组中，可通过报告基因荧光的变化，对HeLa细胞、P19细胞分化不同阶段中靶miRNAs实现原位实时动态的可视化检测。进一步将此系统导入活体动物组织中，运用于生物发育过程中动态检测组织中miRNAs的变化，可望揭示出其在生物发生调控网络中的分布和作用。本研究建立miRNAs响应的基因编码的荧光增强型的核开关，为实现在活细胞及活体组织中无创动态、实时原位的miRNAs生物分子荧光成像提供思路。

本研究在真核动物细胞中建立miRNAs响应的基因编码的荧光增强型的核开关，利用细胞内待检测目标 miR-23a 与开关的茎环环状区的互补结合，RNA双链干扰剪切暴露核糖体结合位点kozak序列的封闭作用，从而激活下游报告基因 EGFP 的表达，开关系统对miRNA有较好的特异性和识别的有效性，可以实现细胞内miRNA浓度与EGFP 表达量相对应。Hela细胞中优化序列S4‘-TC3.6对EGFP的表达可以提高4倍以上。稳定表达S4‘-TC3.6转染细胞系p19，人工诱导控制其分化，通过EGFP表达变化检测该细胞在的分化过程中的体内miRNAs的变化。证明在细胞分化不同阶段，其有不同，在加入维甲酸6天时，EGFP表达最高可达到2.3倍的提高。这与我们体外检测miRNA-23a的浓度提高水平基本一致。通过此荧光成像技术实现活细胞内miRNAs 浓度的实时原位的荧光可视化检测，并动态观察及分析miRNAs在生物发生中的 变化及作用，为更加方便和深入了解研究细胞的转录后miRNAs调控机理提供重要手段。