miRNA经缝隙连接在肿瘤细胞间传递的规律及对其抗肿瘤作用的影响研究

MicroRNAs (miRNAs) have been considered as a hopeful novel therapeutic tool to target disease genes for treatment, especially for cancer. However, a considerable problem restricting the clinical application of miRNAs is that only partial cells in a tumor population can be transfected by current delivery systems for miRNAs. Low transfection rate means not every tumor cell is transfected and not all cells are killed, resulting recurrence of the tumor. Although cell based delivery of miRNA/siNRA via gap junction channels have offered a new transfer system for gene therapy, it still not ensure all cells in a tumor are transfected. A number of laboratories have shown that gap junction channels composed of Cx43 are permeable to siRNA and miRNA with minor diameters of~1.0 nm and that delivery of siRNA/miRNA from a source cell to a recipient cell, through Cx43 gap junctions can affect the function of a target gene. Our studies also demonstrated that gap junction enhanced the anti-tumor effect of miR-124 in human glioma cell by tranferred miR-124 mimics as "death signal" to arrest the cycle at G0/G1 phase. "Bystander" effect of functional gap junctions enhances the anti-tumor effect of miRNAs, suggesting that augmentation and/or recovery of GJIC in tumor cells provides a new guiding strategy to tumor cure, also offers a further feasibility for widespread use of gene therapy in clinic. At present, there are still unknown that what kinds of miRNA can pass through gap junction, and what kinds of connexin can mediate the miRNA transporting. In order to answer the question, we will firstly compare the difference of connexin in transferring tumor suppressors-miRNAs by TSF. And then we will investigate the permeability and selectivity properties of gap junction for tumor suppressors-miRNAs in human cancer cell with nature expressing connexin. Finally we will observe the influence of gap juction on miRNA anti-tumor effect in vitro and in vivo. Our works will provide a clue to develop new method to enhance the miRNA diffusion between adjacent cells, and supply a new guiding strategy to tumor cure with miRNA.

特定的miRNA能抑制肿瘤生长，有望用于治疗肿瘤。但是目前的方法只能将miRNA导入部分肿瘤细胞；而未导入miRNA的肿瘤细胞可能引起肿瘤复发。研究发现，某些miRNA可通过细胞缝隙连接（GJ）传递到毗邻细胞；我们的研究证实在U87细胞，GJ能增强miR-124的抑瘤作用；这表明GJ可能使miRNA扩散到更多的肿瘤细胞而增强其抗肿瘤作用。但至今还不清楚哪些miRNA可以通过GJ传递，以及不同的GJ对不同miRNA的通透性有何差异。本研究拟先用我们独创的TSF技术，在体外比较不同连接蛋白（Cx）通道对有抑瘤作用的miRNA的通透性差异；再在天然表达Cx的人肿瘤细胞，分析这些miRNA通过GJ传递的规律；最后在肿瘤细胞和动物移植瘤模型上，观察改变GJ对miRNA抗肿瘤作用的影响。本研究将为寻找能有效增加miRNA在肿瘤细胞间扩散的方法提供依据，并为miRNA在肿瘤治疗中的应用提供新的理论基础。

特定的miRNA能抑制肿瘤生长，有望用于治疗肿瘤。但是目前的方法只能将miRNA导入部分肿瘤细胞；而未导入miRNA的肿瘤细胞可能引起肿瘤复发。研究发现，某些miRNA可通过细胞缝隙连接（GJ）传递到毗邻细胞；但至今还不清楚哪些miRNA可以通过GJ传递，以及不同的GJ对不同miRNA的通透性有何差异。. 本项目首先用TSF技术，在体外比较了不同Cx通道（Cx43、Cx32、Cx26）对不同长度miRNA的通透性。结果发现，不同长度的miRNA均可以通过Cx43通道，但不能通过Cx32或Cx26通道。在此基础上，我们在天然表达Cx43的人胶质瘤细胞（U87）以及转染并稳定表达Cx32或Cx37的人宫颈癌HeLa细胞上，研究了不同Cx组成的GJ对miRNA的通透性。我们首次发现，不同Cx组成的GJ通道对miRNA的通透性不同。其中，由Cx43组成的GJ对miRNA的通透性最大；几乎所有长度的miRNA（18-27个核苷酸）均可通过由Cx43组成的GJ通道或半通道，而不同长度的miRNA均不能通过由Cx32或Cx37组成的GJ通道或半通道。 . 最后，我们在U87细胞上，首次发现，增强Cx43组成的GJ功能，可以通过增强miR-34a和miR-124在细胞间的传递，进而增加miR-34a和miR-124抑制肿瘤细胞增殖、诱导细胞周期阻滞的作用；而改变由Cx32或Cx37组成的GJ功能，不影响miR-34a抑制肿瘤细胞增殖、诱导细胞周阻滞的作用。在接种U87细胞的动物移植瘤模型上，抑制由Cx43组成的GJ同样可以显著减弱miR-124抑制肿瘤增殖的作用。 . 本研究将为寻找能有效增加miRNA在肿瘤细胞间扩散的方法提供依据，并为miRNA在肿瘤治疗中的应用提供新的理论基础。