[摘 要] 目的: 探讨宫颈癌（CC）细胞SiHa源性外泌体hsa-miR-29c-3p在CC血管生成中的作用。方法∶收集2019年1月至 2021年12月在衡阳市中心医院妇科就诊的45例宫颈鳞状细胞癌（CSCC）患者的癌组织标本和 15例正常宫颈组织标本。常规 培养SiHa细胞和人脐静脉内皮细胞（HUVEC），用 Lipofectamine 2000 将 hsa-miR-29c-3p 、miRNA-NC 、si-hsa-miR-29c-3p和 si-miRNA-NC 转 染 至 SiHa 细 胞 中 ，记 为 miRNA-NC 组 、hsa-miR-29c-3p 组 、si-miRNA-NC 组 和 si-hsa-miR-29c-3p 组 。 用 Lipofectamine 2000将mimic-NC、miR-29c-3p-mimic、pCMV-NC、pCMV-含AAA结构域的ATPase家族蛋白2B（ATAD2B）载体分 别转染 HUVEC，记为mimic-NC组、miR-29c-3p-mimic组、pCMV-NC组、pCMV-ATAD2B组和 pCMV-ATAD2B + miR-29c-3p-mimic 组。原位杂交（ISH）法检测CSCC组织中hsa-miR-29c-3p的表达，免疫组化（IHC）法检测CSCC组织和移植瘤组织中的CD31阳 性血管。分离纯化SiHa、C33a细胞来源的外泌体，用透射电镜技术和WB法对其表征进行鉴定及进行HUVEC摄取实验。qPCR 法检测SiHa、C33a细胞和外泌体中hsa-miR-29c-3p和ATAD2B mRNA的表达。成管试验、Transwell小室实验和划痕愈合实验检 测外泌体对HUVEC成管和迁移能力的影响。双萤光素酶报告基因实验验证hsa-miR-29c-3p与ATAD2B的靶向结合关系，移植 瘤实验检测各组SiHa细胞来源外泌体对移植瘤生长和血管增生的影响。结果: hsa-miR-29c-3p在CSCC组织中呈高表达且与其 微血管密度（MVD）正相关（均 P < 0.05）；SiHa、C33a 细胞来源的外泌体完全符合典型外泌体形态和蛋白表达表征；在体外 HUVEC摄取SiHa、C33a细胞来源的外泌体和其包含的 hsa-miR-29c-3p；SiHa 细胞来源的外泌体hsa-miR-29c-3p可在体外促进 HUVEC 的成管和迁移能力（均P < 0.05）；SiHa细胞来源的外泌体hsa-miR-29c-3p可促进移植瘤生长和血管增生；hsa-miR-29c-3p可与 ATAD2B基因直接结合并调节其表达（均P < 0.05）。过表达ATAD2B可逆转hsa-miR-29c-3p对HUVEC的成管、迁移和划痕愈合 能力的促进作用（均P < 0.05）。结论: SiHa细胞源性外泌体hsa-miR-29c-3p通过靶向ATAD2B调控CSCC组织中的血管生成。 外泌体hsa-miR-29c-3p可能是CC诊疗的潜在标志物和治疗靶点。

[Abstract] Objective: To investigate the role of SiHa cell-derived exosomal hsa-miR-29c-3p in the angiogenesis of cervical cancer (CC). Methods: Cancer tissue specimens from 45 patients with cervical squamous cell carcinoma (CSCC) and normal cervical tissue specimens from 15 controls were collected from Department of Gynecology, Hengyang Central Hospital from January 2019 to December 2021. CSCC SiHa cells and human umbilical vein endothelial cells （HUVECs）were routinely cultured. miRNA-NC, hsa-miR-29c-3p, si-miRNA-NC, and si-hsa-miR-29c-3p were transfected into SiHa cells with Lipofectamine 2000, grouped as miRNA-NC group, hsa-miR-29c-3p group, si-miRNA-NC group and si-hsa-miR-29c-3p group, respectively. HUVECs were transfected with mimic-NC, miR-29c-3p-mimic, pCMV-NC, and pCMV-ATAD2B (ATPase family protein 2B with AAA domain) using Lipofectamine 2000, grouped as the mimic-NC group, miR-29c-3p-mimic group, pCMV-NC group, pCMV-ATAD2B group, and pCMV-ATAD2B + miR-29c-3p-mimic group. The expression of hsa-miR-29c-3p in CSCC tissues was detected by in situ hybridization (ISH), and CD31-positive blood vessels in CSCC tissues and xenograft tissues were detected by immunohistochemistry (IHC). Exosomes from SiHa and C33a cells were isolated and characterized using transmission electron microscopy (TEM) and western blotting (WB). The uptake of exosomes by HUVECs was examined. The expression of hsa-miR-29c-3p and ATAD2B mRNA in SiHa and C33a cells, as well as in their derived exosomes, was detected using qPCR. Tube-forming assay, Transwell assay, and scratch healing assay were performed to detect the effect of exosomes on the ability of HUVEC migration and tube formation. Dual luciferase reporter gene assay verified the interaction between hsa-miR-29c-3p and ATAD2B. Xenograft experiments examined the effects of SiHa cell-derived exosomes on transplanted tumor growth and angiogenesis in each group. Results: hsa-miR-29c-3p was highly expressed in CSCC tissues and was positively correlated with microvessel density (MVD) (all P < 0.05). Exosomes derived from SiHa and C33a cells exhibited typical exosomal morphology and protein expression patterns. Exosomal hsa-miR-29c-3p from SiHa and C33a cells were efficiently taken up by HUVECs in vitro. The SiHa cell-derived exosomal hsa-miR-29c-3p promoted not only the tube-forming and migration of HUVECs in vitro but also the xenograft growth and angiogenesis in vivo (all P < 0.05). hsa-miR-29c-3p directly targeted ATAD2B and regulated its expression (P < 0.05). Overexpression of ATAD2B reversed the promotive effect of hsa-miR-29c-3p on tube-formation, migration, and scratch-healing in HUVECs (all P < 0.05). Conclusion: SiHa cell-derived exosomal hsa-miR-29c-3p regulates angiogenesis in CSCC tissues by targeting ATAD2B. Exosomal hsa-miR-29c-3p may be a potential diagnostic marker and therapeutic target for CC diagnosis and treatment.

衡阳市指导性计划科研项目（No. 202222035651）