These results indicated that CCAT1/miR-130a-3p axis contributed DDP resistance of NSCLC cells by targeting SOX4
These results indicated that CCAT1/miR-130a-3p axis contributed DDP resistance of NSCLC cells by targeting SOX4. In conclusion, our study demonstrated the important role of CCAT1-miR-130a-3p-SOX4 regulatory network in DDP resistance of NSCLC cells, providing potential targets to overcome DDP resistance and enhance efficacy of chemotherapy during the treatment of NSCLC. Disclosure Statement No potential conflicts of interest were disclosed. Acknowledgments This work was supported by Science and Technology Research Key Project in Henan Province Department of Education (16A320034). Authors’ Contribution Baoli Hu, Haifeng Zhang and Li Li designed the experiment. counting kits-8 (CCK-8) assay and western blot, respectively. Results: CCAT1 and SOX4 were up-regulated, and miR-130a-3p was down-regulated in DDP-resistant NSCLC cells compared with AWD 131-138 their parental NSCLC cells. CCAT1 directly interacted with miR-130a-3p and negatively regulated miR-130a-3p expression. CCAT1 contributed to DDP resistance of A549/DDP cells by down-regulating miR-130a-3p. miR-130a-3p was found KL-1 to directly target SOX4 to suppress its expression. SOX4 knockdown reversed miR-130a-3p-inhibition-induced increase of DDP resistance and ABCG2 expression in NSCLC cells. Exogenous expression of SOX4 abrogated CCAT1-knockdown-mediated decrease of DDP resistance and ABCG2 expression in DDP-resistant NSCLC cells. Conclusion: CCAT1/miR-130a-3p axis enhanced DDP resistance of NSCLC cells by targeting SOX4, providing potential targets to overcome DDP resistance and improve efficacy of chemotherapy for patients with NSCLC. < 0.05. CCAT1 enhanced DDP resistance of NSCLC cells Accumulating evidence has indicated that numerous lncRNAs contain motif with complementary sequence to miRNAs and have an inhibitory effect AWD 131-138 on the expression and activity of miRNAs. Therefore, we predicted AWD 131-138 the potential miRNAs which could interact with CCAT1 by the online software starBase v2.0. By searching this database, we discovered that CCAT1 contained potential binding sites complementary to miR-130a-3p, as displayed in Fig.?2A. To confirm the direct binding between CCAT1 and miR-130a-3p, luciferase reporter assay was performed. The results demonstrated that cotransfection with pGL3-CCAT1-WT and miR mimic resulted in an obvious suppression of luciferase activity (Fig.?2B) while cotransfection with pGL3-CCAT1-WT and miR inhibitor led to a marked increase of luciferase activity in A549/DDP cells (Fig.?2C), but the luciferase activity in A549/DDP cells cotransfected with pGL3-CCAT1-MUT and miR mimic or miR inhibitor was not significantly changed, suggesting that CCAT1 could interact with miR-130a-3p. Furthermore, miRNAs are known to exert their gene silencing functions through forming RNA-induced silencing complex (RISC) containing Ago2.12 To explore the endogenetic mutual effect between CCAT1 AWD 131-138 and miR-130a-3p, RIP assay was performed on A549/DDP cell extracts using antibody against Ago2. RNA levels in immunoprecipitates were examined by qRT-PCR. As expected, CCAT1 and miR-130a-3p were both significantly enriched in the Ago2 pellets relative to control IgG immunoprecipitates (Fig.?2D), suggesting that CCAT1 and miR-130a-3p might be in the same RISC complex. To investigate the authentic effect of CCAT1 on the expression of miR-130a-3p, qRT-PCR was conducted to determine the expression of miR-130a-3p in A549, H1299, A549/DDP and H1299/DDP cells transfected with pcDAN-CCAT1, si-CCAT1 or respective controls. The transfection efficiency was verified by qRT-PCR and pcDAN-CCAT1 transfection significantly upregulated CCAT1 level in A549 and H1299 cells while si-CCAT1 treatment dramatically repressed CCAT1 expression in A549/DDP and H1299/DDP cells (Fig.?2E and ?andF).F). As illustrated in Fig.?2G and ?andH,H, the expression of miR-130a-3p was strikingly lower in pcDAN-CCAT1-transfected A549 and H1299 cells but remarkably higher in si-CCAT1-transfected A549/DDP and H1299/DDP cells. Collectively, these results revealed that CCAT1 directly targeted miR-130a-3p to suppress its expression. Open in a separate window Figure 2. miR-130a-3p is a direct target of CCAT1. (A) The predicted wild-type or mutated binding sites between CCAT1 and miR-130a-3p. (B and C) Luciferase reporter assay was performed to measure luciferase activity in A549/DDP cells cotransfected with pGL3-CCAT1-WT or pGL3-CCAT1-MUT and miR mimic, miR inhibitor, miR Con or inhibitor Con. (D) RIP assay was performed on A549/DDP cell extracts using antibody against Ago2. CCAT1 and miR-130C3p levels were evaluated by qRT-PCR. A549 and H1299 cells were were transfected with CCAT1 or vector, and A549/DDP and H1299/DDP cells were transfected with si-CCAT1 or siRNA Con, then qRT-PCR was carried out to detect the expression levels of CCAT1 (E and F) and miR-130a-3p (G and H). *< 0.05. CCAT1 contributed to DDP resistance of NSCLC cells by downregulating miR-130a-3p Since a previous study reported that CCAT1 was involved in the development of docetaxel resistance in lung adenocarcinoma cells11 we further explored the effect of CCAT1 on DDP resistance in NSCLC cells. IC50 values after DDP treatment have always been.