Next Generation Sequencing of circRNAs in bladder cancer derived ITGA6+EVs

EVs served as crucial mediators preferably achieve their biological effect in recipient cells to create a supportive pre-metastatic niche via delivering distinct biomolecules, among which circular RNAs (circRNAs) with high stability and abundance in tumor derived EVs were under extensive exploration. Moreover, it is well-established that circRNAs play a significant role in BCa lymphatic metastasis. To explore the crucial circRNAs in BCa derived ITGA6+EVs, the ITGA6+EVs from total UM-UC-3-EVs were isolated by affinity capture using anti-ITGA6 magnetic beads and the circRNA expression profile was investigated using next-generation sequencing (NGS). We isolated ITGA6+EVs from total UM-UC-3-EVs by affinity capture using anti-ITGA6 magnetic beads and degrades the abundant linear RNAs with RNase R. Then, each sample was amplified and transcribed into fluorescent cRNA utilizing a random priming method. Double-stranded cDNA fragments are subjected to end-repair and A-tailing. After amplification by PCR, the reaction system and program for circularization are configured and set up. The concentration and specific activity of circRNAs were measured by DNBSEQ-G400.

细胞外囊泡（Extracellular Vesicles, EVs）作为关键介导因子，可通过向受体细胞递送特异性生物分子，在受体细胞中发挥生物学效应，进而构建促转移前微环境；其中，肿瘤来源EVs中稳定性佳、丰度高的环状RNA（circular RNAs, circRNAs）正受到广泛研究。此外，已有研究证实环状RNA在膀胱癌（Bladder Cancer, BCa）淋巴转移中发挥重要作用。为探究膀胱癌来源的整合素α6阳性细胞外囊泡（ITGA6+ Extracellular Vesicles, ITGA6+EVs）中的关键环状RNA，本研究通过抗ITGA6磁珠亲和捕获法从总UM-UC-3细胞来源EVs中分离得到ITGA6+EVs，并采用下一代测序（NGS）分析其环状RNA表达谱。本研究通过该亲和捕获法分离目标囊泡后，利用核糖核酸酶R（RNase R）降解丰度较高的线性RNA。随后，采用随机引物法对每份样本进行扩增并转录为荧光标记互补RNA（fluorescent cRNA）。将双链互补DNA片段进行末端修复并加A尾。经聚合酶链式反应（PCR）扩增后，配置并搭建环化反应体系与反应程序。采用DNBSEQ-G400平台测定环状RNA的浓度与比活性。