癌相关成纤维细胞来源的外泌体miR-625-3p对卵巢癌细胞SKOV3迁移和侵袭能力的影响及其机制  

Cancer-associated fibroblast-derived exosome miR-625-3p promotes migration and invasion of SKOV3 ovarian cancer cells by targeting SCAI expression

作　　者：宋永祯 王菲 刘忠杰 王静 SONG Yongzhen;WANG Fei;LIU Zhongjie;WANG Jing(Department of Gynecology,Hengshui People's Hospital,Hengshui 053000,Hebei,China)

机构地区：[1]衡水市人民医院妇科,衡水053000

出　　处：《医学研究与战创伤救治》2023年第12期1250-1256,共7页Journal of Medical Research & Combat Trauma Care

基　　金：衡水市科技计划项目(2019014060Z)。

摘　　要：目的探讨癌相关成纤维细胞(CAFs)分泌的外泌体(exos)miR-625-3p对卵巢癌细胞SKOV3迁移和侵袭能力的影响及其机制。方法选取2021年5月至12月于衡水市人民医院接受手术的卵巢癌患者的癌组织和邻近的非癌组织(5对)。从卵巢癌组织和相邻的正常组织中分离出CAFs和正常成纤维细胞(NFs)。采用Western blot评估NFs和CAFs中α-SMA和vimentin的蛋白表达,利用外泌体提取试剂盒从NFs和CAFs培养基中获得NFs-/CAFs-exos。通过透射电子显微镜(TEM)、纳米颗粒跟踪分析(NTA)和Western blot评估NFs-/CAFs-exos的形状、粒径以及表面marker表达。实时荧光定量PCR(qRT-PCR)和Western blot检测CFAs、CAFs-exos和SKOV3中miR-625-3p和癌细胞侵袭抑制因子(SCAI)的表达。Transwell试验检测细胞迁移和侵袭能力。双荧光素酶报告基因实验确定miR-625-3p和SCAI的相互作用。最后,通过功能挽救实验确定SCAI对SKOV3细胞迁移和侵袭能力的影响。结果Western blot结果显示,α-SMA和vimentin在NFs和CFAs中阳性表达。TEM观察下,NFs-/CAFs-exos均呈杯状形态,粒径大约100 nm,且CD63、HSP70、CD81在NFs-/CAFs-exos中阳性表达。与NFs相比,CAFs中α-SMA高表达(P<0.05)。与NFs-exo相比,CAFs-exo能够上调SKOV3细胞中miR-625-3p表达并且在功能上促进细胞迁移和侵袭(P<0.05)。然而,抑制CAFs-exos miR-625-3p表达能够显著降低SKOV3细胞的迁移和侵袭能力(P<0.05)。报告基因显示,miR-625-3p能够直接靶向SCAI mRNA的3′-UTR区。挽救实验显示,过表达SCAI可有效逆转CAFs-exo miR-625-3p对卵巢癌细胞迁移能力的影响。结论CAF-exos miR-625-3p能够通过抑制SCAI表达促进卵巢癌细胞的迁移和侵袭。Objective To investigate the effect of cancer-associated fibroblasts(CAFs)exosome(exos)miR-625-3p on the migration and invasion of ovarian cancer(OC)cell line SKOV3 and its molecular mechanism.Methods Firstly,CAFs and normal fibroblasts(NFs)were isolated from OC tissues and adjacent normal tissues.The expression ofα-SMA and vimentin were examined by Westernblottig in order to mark the characteristic of NFs-and CAFsexos.Then,NFs-and CAFs-exos were isolated from the culture medium using Exosome Extraction Kit.NFs-and CAFs-exso were identified by Transmission electron microscopy(TEM),Nanoparticle tracking analysis(NTA)and Western blotting.The relative expression levels of miR-625-3p in CAFs,CAFs-exos,and SKOV3 cells were determined by quantitative real-time PCR(qRT-PCR).Tran-swell assay was used to assess the roles of CAFs-exosmiR-625-3p on the cell migration and invasion.The interaction between miR-625-3p and SCAI was verified by dual luciferase reporter gene assay.Finally,the roles of SCAI on the cell migration and invasion of SKOV3 cells were determined by rescue experiment.Results Western blotting showedα-SMA and vimentin were positively expressed in NFs and CFAs.TEM disclosed a cup-shaped structure for NFs-/CAFs-exos.Also,most of the exos were around 100 nm in size.The relative protein expression of CD63、HSP70、CD81 was increased in CAFs-exos compared to CAFs.Besides,CAFs-exos significantly upregulated the miR-625-3p expression in SKOV3 cells,and promoted the malignant phenotype of SKOV3 cells,especially in migration and invasion.Moreover,the silence of miR-625-3p in CAFs-exos repressed the migration and invasion in SKOV3 cells.This result from the report gene assay implied SCAI mRNA was the direct target gene of miR-625-3p.Importantly,SCA I overexpression effectively reversed the effect of miR-625-3p on the migration and invasion of OC cells.Conclusion CAF-exosmiR-625-3p might promote the migration and invasion of ovarian cancer cell line SKOV3 by inhibiting SCAI expression.

关 键 词：卵巢癌 SKOV3 癌相关成纤维细胞 外泌体 miR-625-3p 癌细胞侵袭抑制因子 

分 类 号：R737.31[医药卫生—肿瘤]