miRNA seqencing for transcriptional profiles of blood specimens and NPC cells or NP69 cell

Extravasation is a key step in tumor metastasis. EBV plays a crucial role in nasopharyngeal carcinoma metastasis. However, the functions and molecular mechanisms of EBV during tumor cell extravasation remains unclear. Here, we showed that the expression of pyroptosis-associated proteins is greater in the endothelial cells of metastatic NPC tissues than in those of nontumor tissues Exosomes derived from NPC cells promoted endothelial cell pyroptosis, vascular permeability, and tumor cell extravasation. The above results suggest that EBV is involved in NPC exosome-induced endothelial pyroptosis. To validate this hypothesis, we treated HUVECs with exosomes derived from three pairs of NPC cell lines. Exosomes derived from the CNE2-EBV, TW03-EBV and NPC43 Vector significantly promote endothelial cell pyroptosis compared to that in the control group. These results suggest that EBV may promote endothelial cell pyroptosis by directly packaging viral components or by virus-induced cellular components.EBV produces non-coding RNAs, including a large family of BART miRNAs. To investigate the potential involvement of EBV BART miRNAs in this process, we induced the production of EBV virus using two lymphoma cell lines: Akata and B95-8. The above two kinds of viruses only share 10 common BART miRNAs. The viruses generated from the above two cell lines were used to infect HK1 cells. Subsequently, exosomes derived from infected HK-EBV cells were collected and used to treat HUVECs. There was no significant difference in endothelial cell pyroptosis between the two groups. This finding suggested that miRNAs shared by Akata- and B95-8- derived EBV might play a role in the regulation of endothelial cell pyroptosis. To investigate the miRNAs that regulate endothelial cell pyroptosis in exosomes, we sequenced serum exosomal miRNAs derived from EBV- healthy individuals, EBV+ healthy individuals, non-metastatic NPC patients and metastatic NPC patients. Moreover, we sequenced miRNAs in the exosomes of NP69 cells, NPC43 cells, C17 cells, and C666-1 cells. Compared to those in serum exosomes from the healthy individuals who were EBV- or EBV+, the levels of BART8-3p, BART2-5 and BART7-3p were significantly greater in serum exosomes from the patients with NPC, especially those with metastatic NPC. These miRNAs were also abundant in NPC cells

细胞外渗（Extravasation）是肿瘤转移的关键环节。EB病毒（Epstein-Barr Virus, EBV）在鼻咽癌（Nasopharyngeal Carcinoma, NPC）转移过程中发挥关键作用，但目前EBV在肿瘤细胞外渗过程中的功能与分子机制仍未明确。本研究发现，转移性鼻咽癌组织内皮细胞中细胞焦亡（Pyroptosis）相关蛋白的表达水平显著高于非肿瘤组织。鼻咽癌细胞来源的外泌体（Exosomes）可促进内皮细胞焦亡、升高血管通透性并促进肿瘤细胞外渗。上述结果提示，EBV参与了鼻咽癌外泌体诱导的内皮细胞焦亡过程。为验证该假说，我们使用三对鼻咽癌细胞系来源的外泌体处理人脐静脉内皮细胞（Human Umbilical Vein Endothelial Cells, HUVECs）。相较于对照组，CNE2-EBV、TW03-EBV及NPC43 Vector细胞来源的外泌体可显著促进内皮细胞焦亡。上述结果表明，EBV可能通过直接包装病毒组分或病毒诱导的细胞组分，促进内皮细胞焦亡。EBV可产生包括BART微小RNA（microRNA, miRNA）大家族在内的非编码RNA。为探究EBV BART miRNA在该过程中的潜在作用，我们使用两种淋巴瘤细胞系Akata与B95-8诱导制备EBV病毒。上述两种病毒仅共享10种共同的BART miRNA。我们使用这两种细胞系来源的病毒感染HK1细胞，随后收集感染后的HK-EBV细胞来源的外泌体并处理HUVECs。两组内皮细胞的焦亡水平无显著差异。该结果提示，Akata与B95-8来源的EBV所共有的miRNA可能参与调控内皮细胞焦亡。为筛选外泌体中调控内皮细胞焦亡的miRNA，我们对EBV阴性健康个体、EBV阳性健康个体、非转移性鼻咽癌患者及转移性鼻咽癌患者的血清外泌体miRNA进行测序。此外，我们还对NP69细胞、NPC43细胞、C17细胞及C666-1细胞的外泌体miRNA进行测序。相较于EBV阴性或阳性健康个体的血清外泌体，鼻咽癌患者（尤其是转移性鼻咽癌患者）的血清外泌体中BART8-3p、BART2-5及BART7-3p的表达水平显著升高。上述miRNA在鼻咽癌细胞中同样富集表达。