胃癌外泌体MiR-221靶向PTEN促进肿瘤相关成纤维细胞的形成

Gastric cancer exosomal miR-221 targets PTEN to promote the formation of tumor-associat⁃ed fibroblasts

目的:探究胃癌外泌体miR-221对肿瘤相关成纤维细胞的影响及机制。方法:提取并鉴定人胃黏膜细胞GES-1、人胃癌MGC-803、MKN45细胞分泌的外泌体(GES-1 exo、MGC-803 exo、MKN45 exo),qRT-PCR检测miR-221在GES-1、MGC-803、MKN45及其外泌体中的表达,将10μg/mL GES-1 exo、MGC-803 exo、MKN45 exo以及等体积的PBS与人成纤维细胞(HKF)共孵育后,qRT-PCR检测HKF细胞中MMP-9、α-SMA、Fibronectin、Vimentin、IL-1β、IL-6表达,ELISA检测细胞上清中IL-1β、IL-6的浓度。双荧光素酶报告基因实验检测miR-221与PTEN的靶向关系,将10μg/mL miR-NC exo和miR-221 exo与HKF共孵育,将miR-221 exo与HKF共孵育后转染PTEN质粒(miR-221 exo+PTEN),然后检测上述HKF细胞中MMP-9、α-SMA、Fibronectin、Vimentin、IL-1β、IL-6表达以及细胞上清中IL-1β、IL-6的浓度。结果:本研究所提取的外泌体符合其结构和生物学特征,miR-221在MGC-803、MKN45和MGC-803 exo、MKN45 exo中高表达。与PBS组比较,MGC-803 exo组和MKN45 exo组HKF细胞中MMP-9、α-SMA、Fibronectin、Vimentin、IL-1β、IL-6表达显著上调(均P<0.001),细胞上清中IL-1β和IL-6的浓度显著增加(均P<0.001),双荧光素酶报告基因结果显示PTEN为miR-221的靶基因。与miR-NC exo组比较,miR-221 exo组HKF细胞中MMP-9、α-SMA、Fibronectin、Vimentin、IL-1β、IL-6表达显著上调(均P<0.001),细胞上清中IL-1β和IL-6的浓度显著增加(均P<0.001);与miR-221 exo组比较,miR-221 exo+PTEN组HKF细胞中MMP-9、α-SMA、Fibronectin、Vimentin、IL-1β、IL-6表达显著下调(均P<0.001),细胞上清中IL-1β和IL-6的浓度显著降低(P<0.001)。结论:胃癌外泌体miR-221抑制PTEN表达而促进肿瘤相关成纤维细胞的生成。

Objective:To explore the effect and mechanism of gastric cancer exosomal miR-221 on tumor-associated fibroblasts.Methods:The exosomes(exo)secreted by human gastric mucosa fibroblasts GES-1,MGC-803 and MKN45(GES-1 exo,MGC-803 exo and MKN45 exo)were extracted and identified.The expressions of miR-221 in GES-1,MGC-803,MKN45 and their exosomes were detected by qRT-PCR.10μg/mL GES-1 exo,MGC-803 exo,MKN45 exo,or equal volume PBS was co-incubated with human fibroblasts(HKF),then the expressions of MMP-9,α-SMA,Fibronectin,Vimentin,IL-1β,and IL-6 were detected by qRT-PCR.The concentrations of IL-6 and IL-1βin cell supernatant were detected by ELISA.Double luciferase reporter gene assay was used to verify the targeting relationship between miR-221 and PTEN.HKF cells were co-cultured with 10μg/mL exo derived from miR-221-overexpressing MGC-803 cells.Afterwards,HKF cells were transfected with a plasmid containing PTEN gene.The expressions of MMP-9,α-SMA,Fibronectin,Vimentin,IL-1βand IL-6,and the concentrations of IL-6 and IL-1βwere measured as well.Results:Our prepared exo were consistent with the structure and biological characteristics of exosomes.miR-221 was over-expressed in MGC-803 cells,MKN45 cells,MGC-803 exo and MKN45 exo.Compared with PBS control,the expressions of MMP-9,α-SMA,Fibronectin,Vimentin,IL-1β,and IL-6 in HKF cells treated with MGC-803 exo or MKN45 exo were significantly up-regulated,and the concentrations of IL-6 and IL-1βin cell supernatants were significantly increased(P<0.001).PTEN was a target gene of miR-221.The expressions of MMP-9,α-SMA,Fibronectin,Vimentin,IL-1β,and IL-6 in HKF cells treated with miR-221 exo were significantly up-regulated compared with miR-NC exo treated cells,and the concentrations of IL-6 and IL-1βin cell supernatant were significantly increased(P<0.001).Moreover,PTEN over-expression reversed the promotive effects of miR-221 on the MMP-9,α-SMA,Fibronectin,Vimentin,IL-1β,and IL-6 mRNA levels and cell supernatant IL-6 and IL-1βcontents(P<0.001).Conclusion:Gastric cancer exosomal miR-221 inhibits the expression of PTEN and promotes the generation of tumor-associated fibroblasts.