Fig. 5

lncMMPA interacts with miR-548 s and mediates ALDH1A3 expression in HCC cells. A Sequence alignment of miR-548 s with lncMMPA and lncMMPA mutant (Top). Dual-luciferase reporter assays of pGLO-vector and pGLO-lncMMPA (bottom). B Dual-luciferase reporter assays of pGLO-lncMMPA mutant (pGLO-lncMMPA mut). C MS2-RIP followed by qRT-PCR to detect miR-548 s (lower panel) that endogenously related with lncMMPA. lncMMPA-miR-548 s mutant was employed as a negative control. RNA level of miR-548 s was shown as a ratio relative to the expression level in control sample. A schematic outline of the MS2-RIP strategy, which is employed for the identification of lncRNA associated endogenous microRNAs is shown (upper panel). D Anti-AGO2 RIP was conducted in Hep3B cells with transient overexpression of miR-548 s, and the expression level of lncMMPA associated with AGO2 was shown as a ratio relative to the expression level in miR NC transfected Hep3B cells. E, F Hep3B cells transfected with indicated microRNA mimics were co-cultured with MDMs-Control or MDMs-lncMMPA in Transwell systems for 6 days. These Hep3B cells were then harvested for the specified experiments. E Lactate production. F Cell proliferation. G, H Hep3B cells transfected with indicated microRNA mimics were treated with exosomes from MDMs-Control or MDMs-lncMMPA for 48 h and then harvested for the specified experiments. G Lactate production. H Cell proliferation. I, J Hep3B cells were injected orthotopically into the right flank of BALB/c nude mice (n = 6 per group). The mice were also given an intratumoral injection of the indicated exosomes from MDMs and miR-548 s mimics adenovirus every 3 d. I Representative images of tumors in the xenografts. J Tumor growth curves. K Sequence alignment of miR-548 s with ALDH1A3 and ALDH1A3 mutant (Top). Dual-luciferase reporter assays of pGLO-vector, pGLO-ALDH1A3 and pGLO-ALDH1A3 mutant (pGLO-ALDH1A3 mut) (bottom). L The RNA level of ALDH1A3 in Hep3B cells treated with the indicated microRNA inhibitors or mimics of miR-548 s was normalized to that of actin gene and shown as a ratio relative to the expression level in Hep3B cells with miR NC transfection. M The protein level of ALDH1A3 was detected in Hep3B cells treated with the indicated microRNA inhibitors or mimics of miR-548 s. N The RNA level of ALDH1A3 in Hep3B cells treated with the microRNA mimics of miR-548 s and lncMMPA as indicated was normalized to that of actin gene and shown as a ratio relative to the expression level in Hep3B cells with miR NC transfection