Supplementary Material for: Exploration of extracellular vesicle long non-coding RNAs in serum of patients with cervical cancer

Cervical cancer (CC) is the fourth most common cancer type and a leading cause of cancer-related deaths in women worldwide. Its underlying molecular mechanisms are unclear. Cancer cell-derived extracellular vesicles (EVs) are involved in cancer development and progression by delivering regulatory factors, including microRNAs and long non-coding RNAs (lncRNAs). Here, we identified the EV lncRNA expression profiles associated with different developmental stages of CC using next-generation sequencing. EVs from the serum of patients with stages I–III CC and healthy donors were characterized using EV marker immunoblotting and transmission electron microscopy. The EV concentration increases with progression of the disease. Most particles had a 100–250 nm diameter, and their sizes were similar in all groups. We identified many lncRNAs that were uniquely and differentially expressed (DE) in patients with different stages of CC. The pathway analysis results indicated that the upregulated DE EV lncRNAs abundant in stages I and II were associated with cell proliferation and inflammatory and cancer progression pathways, respectively. LINC00941, LINC01910, LINC02454, and DSG2-AS1 were highly expressed, suggesting poor overall survival of CC patients. Interestingly, DSG2-AS1 was associated with human papillomavirus infection pathway through AKT3, DLG1, and COL6A2 genes. This is the first study that reports the levels of EVs and their lncRNA contents change during cancer development, demonstrating the existence of a unique vesicle-mediated cell-to-cell communication network underlying cancer progression.

宫颈癌（Cervical cancer, CC）是全球女性第四大常见癌症类型，亦是癌症相关死亡的主要诱因之一，其潜在分子机制尚未阐明。癌细胞衍生的细胞外囊泡（extracellular vesicles, EVs）可通过递送包括微小RNA（microRNAs）及长链非编码RNA（long non-coding RNAs, lncRNAs）在内的调控因子，参与癌症的发生与进展过程。本研究借助下一代测序技术，鉴定出与宫颈癌不同临床分期相关的EV lncRNA表达谱。研究采集了Ⅰ~Ⅲ期宫颈癌患者及健康供者的血清来源EVs，并通过EV标志物免疫印迹及透射电子显微镜对其进行表征。结果显示，EV浓度随疾病进展逐渐升高；所有组别囊泡的直径多介于100~250 nm之间，尺寸无显著差异。本研究鉴定出大量在不同分期宫颈癌患者中呈特异性及差异性表达（differentially expressed, DE）的lncRNAs。通路分析结果表明，在Ⅰ期和Ⅱ期宫颈癌患者中富集的上调差异性表达EV lncRNAs，分别与细胞增殖、炎症反应及癌症进展通路相关。LINC00941、LINC01910、LINC02454及DSG2-AS1呈现高表达，提示宫颈癌患者总体生存期较差。值得注意的是，DSG2-AS1可通过AKT3、DLG1及COL6A2基因参与人乳头瘤病毒（human papillomavirus, HPV）感染通路。本研究首次报道了EV水平及其lncRNA含量随癌症发展发生动态变化，证实了囊泡介导的细胞间通讯网络在癌症进展过程中存在独特调控机制。