Comparison of normalized signal intensities of various miRNAs in the exsosomes isolated by Exoquick or ultracentrifugation

MicroRNAs (miRNAs), small (18–22 nucleotides) non-coding RNAs, suppress the translation of target mRNAs by binding to their 3′ untranslated region. Evidence suggests that miRNAs are key regulators of several cellular processes, including angiogenesis. Recent findings indicate that Secreted miRNAs enclosed in exosomes also play an important role in cell–cell communication. To elucidate whether miRNAs secreted from neoplastic cells transfer into endothelial cells and are functionally active in the recipient cells, we investigated the interaction of a leukemia cell line and human umbilical vein endothelial cells (HUVECs). The culture medium was collected and centrifuged at 3000 × g for 15 min. The supernatant was filtered through a 0.22-µm PVDF filter (Millipore). The appropriate volume of Exoquick Exosome Precipitation Solution (System Biosciences, Mountain View, CA, USA) was added to the filtered culture medium and mixed well by inverting. After refrigeration for 12 h, the mixture was centrifuged at 1,500 × g for 30 min and all supernatant was removed by aspiration. Exosome pellets were resuspended with 500 µl of the serum-free AIM V medium (Invitrogen). K562 cells or K562/Cy3-miR-92a cells (K562 cells transfected with Cy3-labeled pre-miR-92a) were pre-cultured in serum-free AIM V medium (Invitrogen). The cells were cultured in 200 ml of AIM V medium (twenty 100 mm culture dishes). For exosome preparation, the culture media were collected and centrifuged at 2,000 × g for 10 min at room temperature, and the supernatant was centrifuged again at 12,000 × g for 30 min at room temperature, then the supernatant was ultracentrifuged at 110,000 × g for 70 min at 4 ºC. The pellets were washed with PBS, after ultracentrifugation, they were used for Microarray analysis.

微RNA（MicroRNAs, miRNAs）是一类长度为18~22个核苷酸的非编码RNA，可通过结合靶mRNA的3'非翻译区抑制其翻译过程。现有研究表明，miRNAs是包括血管生成在内的多种细胞进程的关键调控因子。近期研究发现，包裹于外泌体中的分泌型miRNAs在细胞间通讯中亦发挥重要作用。为阐明肿瘤细胞分泌的miRNAs能否转移至内皮细胞并在受体细胞中行使功能活性，本研究针对某白血病细胞系与人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVEC）的相互作用展开了探究。

首先收集培养液并以3000×g离心15分钟，将上清液通过0.22 μm PVDF滤膜（Millipore）过滤。向过滤后的培养液中加入适量体积的Exoquick外泌体沉淀溶液（System Biosciences，美国加利福尼亚州山景城），通过倒置操作充分混匀。4℃冷藏12小时后，将混合液以1500×g离心30分钟，吸弃全部上清液。使用500 μl无血清AIM V培养基（Invitrogen）重悬得到的外泌体沉淀。

将K562细胞或K562/Cy3-miR-92a细胞（即转染了Cy3标记的pre-miR-92a的K562细胞）置于无血清AIM V培养基（Invitrogen）中预培养。随后将细胞接种于200 ml AIM V培养基中培养（共使用20个100 mm培养皿）。外泌体提取流程如下：收集培养液并于室温下以2000×g离心10分钟，将上清液于室温下再次以12000×g离心30分钟，随后将上清液在4℃下以110000×g超速离心70分钟。超速离心得到的沉淀用磷酸盐缓冲液（PBS）洗涤后，用于微阵列分析（Microarray analysis）。