Effect of HepG2-Derived Exosome with PDGF-D Knockdown on Transformation of Normal Fibroblasts into Tumor-Associated...

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.

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.

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.

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