Figure 5. miR-224 modulated the SIRT3-AMPK-mTOR-HIF-1α axis by directly targeting SIRT3. (A) SIRT3 mRNA contained a binding site with miR-224 as predicted by Starbase (https://web.archive.org/web/20110222111721/http://starbase.sysu.edu.cn/). (B, C) Dual-luciferase reporter assay was carried out in the two NSCLC cells (A549 and H1299). (D) NSCLC cells (A549 and H1299) were transfected with miR-224 mimics and NC-miR, then the mRNA level of Sirt3 in the NSCLC cells was detected by RT-PCR. E-F. Cellular IF (E) and western blot (F) were conducted to detect Sirt3 expression in NSCLC cells. (G) The expression of AMPK/mTOR/HIF-1α in NSCLC cells was monitored by western blot. (H) The mRNA levels of VEGFA, ANGPT1, FLT1 and TIMP1 in NSCLC cells were compared by RT-PCR. (I) The expression of SIRT3/AMPK/mTOR/HIF-1α in HUVECs was detected by western blot. (J) The mRNA levels of VEGFA, ANGPT1, FLT1 and TIMP1 in HUVECs were determined by RT-PCR. NS represents P>0.05, *** represents P<0.001. N=3.