Abstract
BackgroundCisplatin is the first-line chemotherapeutic drug for non-small cell lung cancer (NSCLC), and emerging evidences suggests that targeting circular RNAs (circRNAs) is an effective strategy to increase cisplatin-sensitivity in NSCLC, but the detailed mechanisms are still not fully delineated.MethodsCell proliferation, viability and apoptosis were examined by using the cell counting kit-8 (CCK-8) assay, trypan blue staining assay and Annexin V-FITC/PI double staining assay, respectively. The expression levels of cancer associated genes were measured by using the Real-Time qPCR and Western Blot analysis at transcriptional and translated levels. Dual-luciferase reporter gene system assay was conducted to validated the targeting sites among hsa_circRNA_103809, miR-377-3p and 3′ untranslated region (3’UTR) of GOT1 mRNA. The expression status, including expression levels and localization, were determined by immunohistochemistry (IHC) assay in mice tumor tissues.ResultsHere we identified a novel hsa_circRNA_103809/miR-377-3p/GOT1 signaling cascade which contributes to cisplatin-resistance in NSCLC in vitro and in vivo. Mechanistically, parental cisplatin-sensitive NSCLC (CS-NSCLC) cells were subjected to continuous low-dose cisplatin treatment to generate cisplatin-resistant NSCLC (CR-NSCLC) cells, and we found that hsa_circRNA_103809 and GOT1 were upregulated, while miR-377-3p was downregulated in CR-NSCLC cells but not in CS-NSCLC cells. In addition, hsa_circRNA_103809 sponged miR-337-3p to upregulate GOT1 in CS-NSCLC cells, and knock-down of hsa_circRNA_103809 enhanced the inhibiting effects of cisplatin on cell proliferation and viability, and induced cell apoptosis in CR-NSCLC cells, which were reversed by downregulating miR-377-3p and overexpressing GOT1. Consistently, overexpression of hsa_circRNA_103809 increased cisplatin-resistance in CS-NSCLC cells by regulating the miR-377-3p/GOT1 axis. Finally, silencing of hsa_circRNA_103809 aggravated the inhibiting effects of cisplatin treatment on NSCLC cell growth in vivo.ConclusionsAnalysis of data suggested that targeting the hsa_circRNA_103809/miR-377-3p/GOT1 pathway increased susceptibility of CR-NSCLC cells to cisplatin, and this study provided novel targets to improve the therapeutic efficacy of cisplatin for NSCLC treatment in clinic.
Highlights
Cisplatin is the first-line chemotherapeutic drug for non-small cell lung cancer (NSCLC), and emerging evidences suggests that targeting circular RNAs is an effective strategy to increase cisplatin-sensitivity in NSCLC, but the detailed mechanisms are still not fully delineated
Vectors transfection The overexpression and downregulation vectors for hsa_circRNA_103809 and Glutamate oxaloacetate transaminase 1 (GOT1), and miR-377-3p mimic and inhibitor were designed and synthesized by Sangon Biotech (Shanghai, China), and the above vectors were delivered into CS-NSCLC and CR-NSCLC cells to manipulate genes expressions by using the commercial Lipofectamine 2000 reagent (Invitrogen, USA), based on the experimental protocols provided by the producer
The expression patterns of hsa_circRNA_103809, miR-3773p and GOT1 in CS-NSCLC and CR-NSCLC cells The CS-NSCLC cell lines (A549, H1299 and Calu-3) were subjected to continuous low-dose cisplatin treatment to generate CR-NSCLC cells (A549/DDP, H1299/DDP and Calu-3/DDP), which simulated the realistic conditions of cisplatin-resistance in NSCLC patients in vitro
Summary
Cisplatin is the first-line chemotherapeutic drug for non-small cell lung cancer (NSCLC), and emerging evidences suggests that targeting circular RNAs (circRNAs) is an effective strategy to increase cisplatin-sensitivity in NSCLC, but the detailed mechanisms are still not fully delineated. Non-small cell lung cancer (NSCLC) is a common malignancy with high morbidity and mortality, which has increased in incidence in the last decades [1, 2]. The chemotherapeutic drug cisplatin is currently used as first-line treatment for NSCLC [13, 14]. Recent evidence show that continuous long-term stimulation of NSCLC cells by cisplatin caused alteration of multiple cancer associated Circular RNAs (circRNAs), resulting in a decrease in the effectiveness of the drug [13, 14]. To date, the role of hsa_circRNA_103809 in drug resistance in cancer remains largely unknown and important to investigate
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