4B), and AEG-1 mRNA expression was increased in A549/5-FU cells compared with A549 cells (Fig. target gene of miR-124-5p, and its expression was increased in A549/5-FU cells compared with A549 cells. Additionally, the Beclabuvir upregulation of miR-124-5p was associated with lower expression levels of AEG-1 in A549/5-FU cells, compared with parental A549 cells. Moreover, the Dual-luciferase reporter assay confirmed the ability of miR-124-5p to bind directly to the 3-untranslated region of AEG-1 mRNA. Notably, the overexpression of AEG-1 reversed the ability of the miR-124-5p mimic to increase the sensitivity of A549/5-FU cells to 5-FU treatment. Additionally, a significant negative correlation between miR-124-5p expression and AEG-1 mRNA levels was detected in 40 pairs of NSCLC tissues and their corresponding adjacent paracancerous tissues. The results of the present study indicated that miR-124-5p may regulate the chemotherapeutic sensitivity of NSCLC cells, and may therefore represent a promising biomarker or therapeutic target for patients with NSCLC. luciferase. Knockdown and overexpression of AEG-1 Control small interfering (si)RNA (5-TTCTCCGAACGTGTCACGT-3) and AEG-1 siRNA (5-AACAGAAGAAGAAGAACCGGA-3) were purchased from Shanghai GenePharma Co., Ltd. Transient silencing was performed on AEG-1 cells; 50 nM AEG-1 siRNA was mixed with Lipofectamine? RNAiMax (Invitrogen; Thermo Fisher Scientific, Inc.) in serum-free DMEM for 5 min at room temperature and added to the A549 and A549/5-FU cells. The cells were used for further experimentation 72 h post-transfection. Beclabuvir Full length AEG-1 cDNA was amplified from A549 cDNA and cloned into a pcDNA3.1 vector (Addgene, Inc.) with PrimeSTAR? GXL DNA Polymerase (Takara Bio, Inc.). The thermocycling conditions were 30 cycles at 98C for 10 sec followed by 68C for 120 sec. To initiate Beclabuvir overexpression of AEG-1, 2 g pcDNA3.1-AEG-1 was incubated with Lipofectamine? 2000 in serum-free DMEM for 15 min at room temperature and subsequently added to the A549 and A549/5-FU cells. These cells were used for further experimentation 24 h after transfection. Statistical analysis The data were analyzed using GraphPad Prism software 6.0 (GraphPad Software, Inc.) and are expressed as the mean SD. Two-tailed paired Student’s t-test was used to evaluate statistical differences between two groups. Rabbit polyclonal to AMID One-way ANOVA followed by the Newman Keul’s post-hoc test was used for the analysis of three groups. Pearson’s correlation analysis was used to determine the correlation between the expression levels of miR-124-5p and AEG-1 in patient tissues. P<0.05 was considered to indicate a statistically significant difference. Results miR-124-5p inhibitor decreases Beclabuvir A549 and H1299 cell sensitivity to 5-FU miR-124-5p has previously been identified as a prognostic predictor for patients with NSCLC (25). As demonstrated in Fig. 1A, transfection with the miR-124-5p inhibitor decreased miR-124-5p expression in A549 cells. Inhibition of miR-124-5p significantly increased the 5-FU IC50 value (7.29 vs. 35.01 M) of A549 cells compared with the NC, suggesting decreased sensitivity of A549 cells to 5-FU (Fig. 1B). Similarly, in another NSCLC cell line H1299, downregulation of miR-124-5p significantly increased the 5-FU IC50 value (8.25 vs. 17.45 M) of H1299 cells compared with the NC (Fig. 1C and D). These results indicated that miR-124-5p may mediate 5-FU sensitivity in A549 and H1299 cells. Open in a separate window Figure 1. miR-124-5p increases 5-FU sensitivity in A549 and H1299 cells. (A) Transfection with a miR-124-5p inhibitor decreased miR-124-5p expression in A549 cells. (B) Inhibition of miR-124-5p desensitized A549 cells to 5-FU treatment. (C) Transfection with a miR-124-5p inhibitor decreased miR-124-5p expression in H1299 cells. (D) Inhibition of miR-124-5p reduced the sensitivity of H1299 cells to treatment with 5-FU. *P<0.05 and ***P<0.001. miR, microRNA; 5-FU, 5-fluorouracil; NC, negative control; IC50, half-maximal inhibitory concentration. miR-124-5p negatively regulates AEG-1 expression in NSCLC cells TargetScan was used to predict the potential target genes of miR-124-5p, which was determined to be complementary to the 3-UTR of AEG-1 mRNA, a known sensitizer of chemotherapy (21). This indicated that miR-124-5p may regulate AEG-1 expression (Fig. 2A). Beclabuvir In addition, in A549/5-FU cells, overexpression of miR-124-5p reduced AEG-1 mRNA expression (Fig. 2B). Western blot analysis revealed that AEG-1 protein expression was decreased following miR-124-5p overexpression in A549/5-FU cells, which was also demonstrated in H1299 cells (Fig. 2C and D). These results revealed that miR-124-5p negatively regulated the expression of AEG-1 in NSCLC cells. Open in a separate window Figure 2. miR-124-5p negatively regulates AEG-1 expression in A549 and H1299 cells. (A) The 3UTR of AEG-1 mRNA exhibited a binding site complementary to miR-124-5p. Two site mutations were introduced into the putative binding site to.