In their study, colony formation assay was performed to compare the radiation response in vitro. Xenograft mouse model was used to study the OL effects on radiation in vivo. Chromatin immunoprecipitation and luciferase reporter assays were performed to identify the relations among HIF1α, miR-519d and PDRG1, while miRNA microarray service was performed to look at differential miRNA expression. Stable HIF1α or PDRG1 overexpression, and miR-519d downregulation were performed to test the radiation response both in vitro and in vivo.
Results showed that OL strongly enhanced radiosensitivity of NPC cells both in vitro and in vivo. Chromatin immunoprecipitation and luciferase reporter assays suggested miR-519d was a direct target of HIF1α, and PDRG1 was a direct target of miR-519d. Overexpression of HIF1α or PDRG1, and downregulation of miR-519d abolished the radiation sensitizing effects of OL.
This study hereby demonstrates OL is a radiation sensitizing agent in NPC both in vivo and in vitro. OL treatment reduces the activity of HIF1α-miR-519d-PDRG1 pathway, which is essential to the radiosensitizing effects of OL.
a NPC cells HNE-1 and HONE-1 were incubated for 24 h with media containing 0 or 200 μM of OL, respectively, and then subjected to microRNA array analysis. 22 miRNAs upregulated in both HNE-1 and HONE-1 cells were shown. b to c mRNA levels of HIF1α (b), PDRG1 (c), as well as their protein levels (d) were analyzed in both HNE-1 and HONE-1 cells treated as in (a). Values were expressed as mean ± SD. * p < 0.05, ** p < 0.01, compared to 0 μM OL