转移性宫颈癌细胞衍生的外泌体miR-21-5p促进宫颈癌侵袭及转移

目的 探讨转移性宫颈癌组织的旁分泌作用对肿瘤细胞扩散的影响。方法 选择22例转移性宫颈鳞癌患者的手术标本（癌组织和癌旁组织）和血清标本，以及22名健康体检者的血清标本，采用逆转录-实时荧光定量PCR（RT-qPCR）检测miR-21-5p在癌组织和癌旁组织中的表达水平;分别转染miR-21-5p模拟物/抑制剂到宫颈癌SiHa细胞，采用RT-qPCR、细胞集落形成实验、Transwell迁移实验、噻唑蓝（MTT）比色法、蛋白免疫印迹法、双荧光素酶报告分析差异;采用高速离心法提取血清外泌体;采用纳米颗粒跟踪分析（NTA）技术、蛋白免疫印迹法、透射电子显微镜（TEM）对外泌体进行表征鉴定;采用RT-qPCR 检测宫颈鳞癌患者和健康体检者血清外泌体中miR-21-5p的表达差异;采用受试者操作特征（ROC）曲线分析血清外泌体中miR-21-5p对肿瘤转移的诊断效能。结果 miR-21-5p在转移性宫颈鳞癌组织中表达上调;miR-21-5p过表达能促进宫颈鳞癌SiHa细胞集落形成以及细胞增殖与迁移（P均< 0.01）;双荧光素酶报告显示，miR-21-5p可以靶向快速发育生长因子同源蛋白2抗体（SPRY2），对SPYR2的表达水平进行调控;miR-21-5p模拟物转染的SiHa细胞中，磷酸化丝裂原和应激激活的蛋白激酶、磷酸化核糖体S6蛋白激酶、磷酸化原癌基因C-RAF、磷酸化丝裂原活化蛋白激酶（MAPK）激酶和磷酸化MAPK蛋白的表达均上调（P均< 0.05）。miR-21-5p在宫颈鳞癌患者的血清外泌体中上调（P < 0.05）。血清外泌体评估宫颈癌转移的ROC曲线下面积为0.9375。结论 转移性宫颈鳞癌患者组织中miR-21-5p通过外泌体传递促进肿瘤转移，其机制可能与靶向SPRY2/细胞外调节蛋白激酶/MAPK信号通路有关。

Exosomal miR-21-5p derived from metastatic cervical cancer cells promotes cervical cancer invasion and metastasis

Objective To explore the influence of paracrine function of recurring and metastatic cervical cancer tissues on the spread of cancer cells. Methods Surgical specimens （cancerous and para-cancerous tissues） were obtained from 22 patients with metastatic cervical squamous cancer. Serum specimens were collected from 22 patients with metastatic cervical squamous cancer and 22 healthy volunteers， respectively. The expression levels of miR-21-5p in the cancerous and para-cancerous tissues were determined by quantitative real-time PCR （RT-qPCR）. SiHa cell were transferred with miR-21-5p mimics/inhibitors， respectively. The differences between two groups were analyzed by RT-qPCR， colony formation test， Transwell migration test， MTT， western blotting and dual luciferase reporter assay. Serum exosomes were extracted by high-speed centrifugation. The exosome characterization was performed by nanoparticle tracking analysis （NTA）， western blotting and transmission electron microscopy （TEM）. The differences in the expression levels of miR-21-5p in serum exosomes between cervical squamous cancer patients and healthy volunteers were assessed by RT-qPCR. The diagnostic efficiency of miR-21-5p in serum exosomes was evaluated by the receiver operating characteristic （ROC） curve. Results The expression level of miR-21-5p was up-regulated in cervical cancerous tissues compared with para-cancerous tissues. And over-expressed miR-21-5p could significantly promote SiHa cell colony formation， cell proliferation and migration （all P <0.01）. Dual luciferase reporter assay showed that miR-21-5p could target SPYR2 and regulate the expression level of SPYR2. In SiHa cells transfected with miR-21-5p mimics， the expression levels of p-MSK， p-90RSK， p-C-RAF， p-MEK and p-MAPK proteins were significantly up-regulated （all P < 0.05）. The expression level of miR-21-5p was significantly up-regulated in serum exosomes of patients with cervical squamous cancer （P < 0.05）. The area under the ROC curve （AUC） of serum exosomes for the assessment of cervical cancer metastasis was 0.9375. Conclusions The transfer of miR-21-5p to exosomes in the tissues of patients with metastatic cervical squamous cancer promotes the spread of cancer cells. The mechanism may be related to the targeting of the SPRY2/ERK/MAPK signaling pathway.