Exosomes from FaDu cells treated with miR-21 inhibitor reversed induction of CAF-like features in hBMSCs.
To investigate the role of miR-21 in the transition of hBMSCs to CAFs, we collected the exosomes from FaDu cell culture system, which were treated with miR-21 inhibitor resulting in miR-21 gene knock-out. Normal hBMSCs were co-cultured with collected exosomes and the expression of the CAF-related markers was evaluated on day 7, 14, 21, 28, 35, 42 respectively. As shown in Fig. 4a, qRT-PCR results confirmed down-regulation of miR-21 level in collected exosomes or FaDu cells with miR-21 inhibited. Western blot results indicated that both the CAF-related proteins in hBMSCs were significantly decreased in miR-21 inhibitor-treated group compared to control (Fig. 4b).Furthermore, qRT-PCR results confirmed down-regulation of α-SMA and FAP mRNA level in hBMSCs in the miR-21 inhibitor-treated group compared to control (Fig. 4c,d), indicating the limited transition from hBMSCs to CAFs which consistent with the result of microscopic observation (Fig. 4e). Microscopic observation results showed that the number of CAF-like cells significantly decreased in miR-21 inhibitor-treated group compared to control. Flow cytometry results showed that the cell proportion of G1 phase in miR-21 inhibitor-treated group significantly increased, while the cell proportion of G2 phase significantly decreased compared to the control (Fig. 4f-h).