Data Availability StatementAll datasets generated for this research are contained in the manuscript/supplementary data files. dual luciferase reporter assay. NSCLC cell proliferation, apoptosis, and colony development were analyzed using MTT, movement cytometry, and colony development assays, respectively. It had Amentoflavone been discovered that AFAP1-Seeing that1 appearance was upregulated in NSCLC cells and tissue. Furthermore, AFAP1-AS1 destined to and downregulated the appearance of miR-139-5p, that was low in NSCLC tissues. Knockdown of AFAP1-AS1 and overexpression of miR-139-5p inhibited NSCLC cell proliferation, colony formation and chemotherapy resistance and increased cell apoptosis. Additionally, AFAP1-AS1 upregulates RRM2 expression via sponging miR-139-5p. Furthermore, AFAP1-AS1 enhanced NSCLC cell proliferation and chemotherapy resistance through upregulation of RRM2 by inhibiting miR-139-5p expression. Moreover, RRM2 promoted cellular chemotherapy resistance by activating EGFR/AKT. Finally, knockdown of AFAP1-AS1 significantly suppressed tumor growth and chemoresistance in nude mice. In conclusion, AFAP1-AS1 promoted chemotherapy resistance by supressing miR-139-5p expression and promoting RRM2/EGFR/AKT signaling pathway in NSCLC cells. Tukey’s honestly significant difference (HSD) test. = 20) and the chemotherapy non-response group (= 24). (D) AFAP1-AS1 expression in lung cancer cells analyzed by RT- PCR. The results shown as means S.D. #< 0.05 compared with BEAS-2B cells. AFAP1-AS1 Inhibits miR-139-5p Expression The potential binding sites between AFAP1-AS1 and miR-139-5p were predicted based on bioinformatic analysis (Physique 2A). The dual luciferase reporter assay demonstrated that this miR-139-5p mimic significantly reduced the luciferase activity of cells transfected with AFAP1-AS1 WT as well as that of cells transfected with the AFAP1-AS1 mutated type AFAP1-AS1 Mut2 (Physique 2B). However, the miR-139-5p mimic failed to suppress the luciferase activity of cells transfected with the Amentoflavone other AFAP1-AS1 mutated type Mut1, suggesting that miR-139-5p may bind to more than one site around the AFAP1-AS1 Mut1 construct (Physique 2B). We found that the level of miR-139-5p was lower in patients in the chemotherapy non-response group than in the chemotherapy response group (Physique 2C), and miR-139-5p was decreased in lung cancer cell lines compared with BEAS-2B cells (Physique 2D). Furthermore, transfection with siRNA targeting AFAP1-AS1 reduced AFAP1-AS1 expression (Figures 2E,F) and upregulated miR-139-5p expression (Figures 2G,H) in A549 and SPCA-1 cells. In contrast, pcDNA-AFAP1-AS1-mediated overexpression of AFAP1-AS1 reduced the miR-139-5p level in H1975 and PC-9 cells (Figures 2I,J). AFAP1-AS1 expression was significantly elevated in anti-Ago2 (Protein argonaute-2)-incubated A549 cells (Physique 2K), and AFAP1-AS1 could directly bind to miR-139-5p (Physique 2L). There was a negative correlation between AFAP1-AS1 and miR-139-5p expression in NSCLC cells (Physique 2M). These findings indicated that AFAP-AS1 was a sponge of miR-139-5p. Open in a separate window Physique 2 AFAP1-AS1 supresses miR-139-5p expression. (A) The potential binding sites between AFAP1-AS1 and miR-139-5p predicted by bioinformatics. AFAP1-AS1 Mut1 represents the mutation of the first two binding sites, and AFAP1-AS1 Mut2 represents the mutation of the latter two binding sites. (B) A dual luciferase reporter assay on cells transfected with AFAP1-AS1 WT, AFAP1-AS1 Mut1, and AFAP1-AS1 Mut2. Data proven as means S.D. #< 0.05 weighed against the pre-NC-transfected examples. (C) RT-PCR in the miR-139-5p appearance in chemoresistant tissue. Data proven as means S.D. #< 0.05 weighed against chemoresponsive tissues. (D) RT-PCR in the miR-139-5p appearance in tumor cells. Amentoflavone Data proven as means S.D. &< 0.05 weighed against BEAS-2B cells. (ECH) RT-PCR on the result of AFAP1-AS1 knockdown on miR-139-5p mRNA appearance. Data proven as means S.D. #< 0.05 weighed against the scramble-transfected group. (I,J) The result of AFAP1-AS1 overexpression on miR-139-5p mRNA appearance examined by RT- PCR. Data proven as means S.D. #< 0.05 weighed against the pcDNA-transfected group. (K) Cell lysate incubated with an anti-Ago2 antibody for RIP, as well as the AFAP1-AS1 articles discovered by RT- Rabbit Polyclonal to FZD9 PCR. Data proven as means S.D. #< 0.05 weighed against the IgG control group. (L) Cell lysate incubated with Bio-AFAP1-AS1 for RIP, as well as the enrichment of miR-139-5p discovered by RT- PCR. Data proven as means S.D. #< 0.05 weighed against Bio-control group. (M) The appearance of AFAP1-AS1 and miR-139-5p adversely correlated in NSCLC tissue. = ?0.7686 and 0.0001. Suppression of AFAP1-AS1 or Overexpression.