Figure 5.

CaMKII is a target gene of miR-214 in CSCs. Cultured CSCs were transfected with CaMKII overexpression cDNA with or without the 3′UTR for 48 h. Subsequently, the cells were transfected with miR-214 mimics, inhibitors, or negative control RNA for 48 h. The cells were harvested and subjected to RT-qPCR or Western blotting analysis after treatment with BMSC-exos collected under different conditions for 24 h and/or cultured with 100 μM H₂O₂ for 2 h. (a) RT-qPCR analysis of CaMKII expression in CSCs after different treatments. After overexpressing cDNA for CaMKII containing the 3′UTR (CaMKII3′) in CSCs, CaMKII3′ mRNA levels dramatically decreased in response to treatment with miR-21 mimics as demonstrated by RT-qPCR. However, miR-214 mimics had no effect on mRNA levels of CaMKII without the 3′UTR. (b-c) CaMKII protein levels were detected by immunoblotting. miR-214 mimics could significantly downregulate the expression of CaMKII with the 3′UTR at protein levels. However, miR-214 mimics had no effect on the protein levels of CaMKII without the 3′UTR. n = 3; ^∗P < 0.05 compared with the mimics + CaMKII3′ group. (d) RT-qPCR analysis of CaMKII expression in CSCs after different treatments. Compared with that in the normal group, the CaMKII mRNA level was significantly upregulated in the H₂O₂ group. Compared with H₂O₂, Hypoxic-exos or miR-214 mimics significantly suppressed CaMKII mRNA expression. (e-f) Western blotting was used to verify the effect of exosomal miR-214 on CaMKII expression in CSCs. CaMKII protein levels were dramatically decreased after Hypoxic-exo or mimic-exo (exosomes from miR-214-mimic-modified BMSCs) treatment relative to those with H₂O₂ treatment. However, compared with Hypoxic-exos, miR-214 inhibitors or inhibitor-exos upregulated CaMKII protein levels. n = 3; ^∗P < 0.05 compared with the H₂O₂ group; ^#P < 0.05 compared with the Hypoxic-exos group.