Figure 1.

MiR-301a and miR-301b are hypoxia-responsive miRNAs that enhance autophagy and radioresistance of prostate cancer cells. (A, B) PC-3, DU145, and LNCaP cells were cultured in hypoxia for 36 h. The expression of miR-301a (A) and miR-301b (B) were determined by qRT-PCR (n=3). (C, D) LNCaP cells were subjected to hypoxia for up to 72 h and the expression of miR-301a (C) and miR-301b (D) at indicated time points were determined by qRT-PCR. (E, F) LNCaP cells were transfected with HIF1-α expression vector or the negative control. (E) Western blot analysis was performed 72 h after the transfection to detect the expression of HIF1-α. (F) QRT-PCR analysis was performed to detect the expression of miR-301a and miR-301b. (G) LNCaP cells with miR-301a or miR-301b overexpression or with knockdown of both miR-301a and miR-301b were exposed to hypoxia (1%) for 36 h. LNCaP cells without any transfection were treated with 3-MA treatment (5 mM) 1 h before hypoxia and then exposed to hypoxia (1%) for 36 h. Autophagy was visualized by detecting LC3B and p62 via Western blot. (H) LNCaP cells with stable GFP-LC3 expression were transfected with miR-301a mimics, miR-301b mimics, or co-transfected with miR-301a and miR-301b inhibitors, then the cells were subjected to hypoxia for 36 h. GFP-LC3 puncta accumulation was then observed under confocal microscopy. (I) Survival fraction of LNCaP cells with or without miR-301a or miR-301b overexpression after irradiation. (J) Flow cytometry analysis of apoptotic LNCaP cells with or without miR-301a or miR-301b overexpression 24 h after irradiation. * p<0.05, ** p<0.01, *** p<0.001.