Fig. 6

MiR-124 directly binds to AKT2 3’UTR region and thus inhibits LUAD cell proliferation, migration and invasion. a Authoritative bioinformatic databases (miRDB, miRWalk, miRTarBase and TargetScan) were used to predict the putative miRNAs targeting AKT2. b After transfection with miR-124 mimics and miR-NC (left) or miR-124 inhibitor or inhibitor-NC (right) in A549 and H1299 cell lines, the mRNA and protein levels of AKT2 were analyzed by western blot and qRT-PCR assays. c Computational algorithms predict that AKT2 3’UTR region harbors a putative miR-124 binding site (upper). Reporter vector containing wild type AKT2 3’UTR fragment was constructed, and different mutation was then introduced into the potential miR-124 targeting site to obtain mutant type 3′UTR. The generated luciferase reporter plasmids were co-transfected with negative control (miR-NC) or miR-124 mimics into A549 and H1299 cells. The relative firefly luciferase activity was determined and normalized to the Renilla luciferase activity (bottom). d qRT-PCR analysis of miR-124 expression in 45 paired NSCLC tissues and adjacent normal tissues. e Kaplan-meier analysis was used to detect the correlation between miR-124 and AKT2 expression in 45 paired NSCLC tissues and adjacent normal tissues. miR-124 and AKT2 mRNA levels are normalized against U6 and β-actin, respectively. X and y axes represent the log₁₀ transformed T/N expression ratios of miR-124 and AKT2 mRNA, respectively. f and g CCK8 (f) and colony formation (g) assays were used to determine the proliferation abilities of miR-124 mimics or negative control (Si-NC) transfected A549 and H1299 cells. h and i miR-124 overexpressed A549 and H1299 cells were allowed to migrate through an 8-μm pore or invade through matrigel-coated membrane in transwells. Migratory and invasive cells were stained and counted in at least three light microscopic fields. Representative images (h) and migratory or invasive cell numbers (i) were shown. Scale bar, 2 mm. j and k Flow cytometry cell cycle analysis of A549 and H1299 cell lines after miR-124 overexpression (left). The distribution (%) of G0/G1, S and G2/M phases are shown in the histograms (right). l and m Indicated EMT-related mRNAs (l) and proteins (m) were analyzed by qRT-PCR and western blot in miR-124 overexpressed A549 and H1299 cells. Each analysis was performed in triplicate and Values are represented as means ± SD.*P < 0.05 **P < 0.01 ***P < 0.001