敲除 PDGF-D 的 HepG2 外泌体对肝癌中正常成纤维细胞向肿瘤相关成纤维细胞转化的影响

IF 3.3 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Yan-Yan Wu, Liu-Shen-Yan Yu, Han-Yu Zhou, Jun-Chao Xue
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引用次数: 0

摘要

背景:众所周知,肝癌介导的成纤维细胞转化为癌症相关成纤维细胞(CAFs)有利于肝癌的发展。然而,具体机制尚不清楚。方法:用血小板衍生生长因子-d (shPDGF-D)载体短发夹RNA (shRNA)处理人肝癌(HepG2)细胞,采用超离心分离细胞分泌的外泌体,采用纳米颗粒跟踪分析、透射电镜和western blot分析鉴定。将外泌体与小鼠原代成纤维细胞共培养,分别通过细胞计数试剂盒-8 (CCK-8)、免疫荧光、流式细胞术、创面愈合、实时逆转录- pcr和western blotting检测成纤维细胞的活性、增殖、细胞周期、迁移、上皮-间质转化- (EMT-)和CAF标志物相关蛋白表达水平。将共培养成纤维细胞与HepG2细胞混合后皮下注射到小鼠体内构建动物模型。检测异种移植物肿瘤的大小和重量,以及上皮-间质转化- (EMT-)、血管生成-和CAFs标志物相关蛋白的表达。结果:外泌体抑制成纤维细胞的增殖、迁移、EMT,诱导细胞周期阻滞,降低α-SMA、FAP、MMP-9、VEGF的表达。在体内,sh-PDGF-D抑制肿瘤生长,降低CD31、vimentin、α-SMA、FAP、MMP9、VEGF的表达,促进E-cadherin的表达。结论:转染shPDGF-D的HepG2细胞衍生的外泌体阻止正常成纤维细胞向CAFs转化,从而抑制肝癌血管生成和EMT。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Effect of HepG2-Derived Exosome with PDGF-D Knockdown on Transformation of Normal Fibroblasts into Tumor-Associated Fibroblasts in Liver Cancer.

Background: It is known that the transformation of liver cancer-mediated fibroblasts into cancer-related fibroblasts (CAFs) is beneficial to the development of liver cancer. However, the specific mechanism is still unclear.

Methods: Human hepatocarcinoma (HepG2) cells were treated with short hairpin RNA (shRNA) of platelet-derived growth factor-D (shPDGF-D) vector, and the exosomes secreted by the cells were separated using ultracentrifugation and identified by using nanoparticle tracking analysis, transmission electron microscope, and western blot analysis. Exosomes were co-cultured with mouse primary fibroblasts, and then the activity, proliferation, cell cycle, migration, epithelial-mesenchymal transition- (EMT-) and CAF marker-related protein expression levels of fibroblasts were determined by cell counting kit-8 (CCK-8), immunofluorescence, flow cytometry, wound healing, real-time reverse transcription-PCR, and western blotting assays, respectively. Co-cultured fibroblasts were mixed with HepG2 cells and injected subcutaneously into mice to construct animal models. The size and weight of xenograft tumor and the expression of epithelial-mesenchymal transition- (EMT-), angiogenesis- and CAFs marker-related proteins were detected.

Results: The exosomes inhibited the proliferation, migration, EMT, and induced cell cycle arrest, as well as decreased the expression of α-SMA, FAP, MMP-9, and VEGF in fibroblasts. In vivo, sh-PDGF-D inhibited tumor growth, reduced the expressions of CD31, vimentin, α-SMA, FAP, MMP9, and VEGF, and promoted the expression of E-cadherin.

Conclusions: Exosomes derived from HepG2 cells transfected with shPDGF-D prevent normal fibroblasts from transforming into CAFs, thus inhibiting angiogenesis and EMT of liver cancer.

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