Molecular Mechanism Of Long Non-coding RNA In Energy Metabolism Reprogramming Of Non-small Cell Lung Cancer

Background and ObjectiveNon-small cell lung cancer(NSCLC)is the most common and fatal disease in the world.The research on molecular mechanism of NSCLC progression will help to explore safety and effective biomarkers or therapeutic targets for diagnosis or treatment of lung cancer.Long non-codingRNAs(lncRNAs)are a class of transcripts longer than200 nucleotides without protein-coding function.LncRNAs emerge as a significant regulator responsible for various biological processes by regulating the expression of target genes from transcriptional,post-transcriptional and epigenetic levels.In the research field of NSCLC,lncRNAs have attracted wide concern and some abnormally expressed lncRNAs are considered as potential biomarkers or therapeutic targets for NSCLC.Metabolic reprogramming,known as aerobic glycolysis or the Warburg effect,has been identified as one of the hallmarks of cancer recent years.This metabolic shift,which relies on glycolysis rather than mitochondrial oxidative phosphorylation,is a key pathway to produce energy for cancer cells and thus fuels tumor progression.Increasing evidences have pointed out that cancer-related lncRNAs play an important role in metabolic reprogramming and promoting malignant phenotypes.In this study,abnormally expressed lncRNA in NSCLC was detected,the regulatory effect on metabolic reprogramming and their molecular mechanism was explored,which may provide a possibility for the diagnosis,treatment and intervention of NSCLC.Part 1 LINC01123,a c-Myc activated long non-codingRNA,promotes proliferation and aerobic glycolysis of non-small cell lung cancer through miR-199a-5p/c-Myc axis.Results: 1.364 differentially expressed genes were identified inRNA-seq assay and LINC01123 was one of the most overexpressed lncRNAs.2.Further validation in expanded NSCLC cohorts confirmed that LINC01123 was upregulated in 92 paired NSCLC tissues and associated with poor survival.3.Functional assays showed that LINC01123 promoted NSCLC cell proliferation and aerobic glycolysis.4.Mechanistic investigations revealed that LINC01123 was a direct transcriptional target of c-Myc.5.Meanwhile,LINC01123 increased c-Myc mRNA expression by sponging miR-199a-5p.6.In addition,rescue experiments showed that LINC01123 functioned as an oncogene depending on miR-199a-5p and c-Myc.Conclusion: Since LINC01123 is upregulated in NSCLC,correlates with prognosis and controls proliferation and aerobic glycolysis by a positive feedback loop with cMyc,it is expected to be a potential biomarker and therapeutic target for NSCLC.Part 2 Hypoxia-induced lncRNA-AC020978 promotes proliferation and glycolytic metabolism of non-small cell lung cancer through PKM2/HIF-1? axis.Results:1.The present study indicated that AC020978 was frequently upregulated in NSCLC,significantly correlated with advanced TNM stage and poor clinical outcomes,representing as an independent prognostic predictor.2.Functional assays revealed AC020978's role in promoting cell proliferation and glycolytic metabolism.3.AC020978 was a hypoxia-inducible lncRNA and directly transactivated by HIF-1?.4.Mechanistic investigations identified that AC020978 directly interacted with PKM2 and enhanced PKM2 protein stability.5.This study also uncovered an AC020978-mediated mechanism of promoting the nuclear translocation of PKM2 and regulating PKM2-enhanced HIF-1? transcription activity.Conclusions: Together,these data provided evidence that AC020978 conferred an aggressive phenotype to NSCLC and was a poor prognosticator.Targeting AC020978 might be an effective therapeutic strategy for NSCLC.Part 3 Materials and methods1.Next-generationRNA sequencing assay was performed to identify the differentially expressedRNAs between NSCLC tissues and their adjacent normal lung tissues.2.LINC01123 and AC020978 expression in NSCLC tissues was measured by real-time PCR and fluorescence in situ hybridization(FISH)assay.3.The survival analysis was evaluated by the kaplan-meier method and tested by the log-rank test.4.CCK-8,Ed U and clone formation experiments confirmed the role of LINC01123 and AC020978 in cell proliferation.The biological functions of LINC01123 and AC020978 in energy metabolism were tested by 18F-FDG uptake,lactate release,Seahorse detection of mitochondrial respiration and glycolysis.Subcutaneous neoplasia in nude mice and 18F-FDG PET/CT imaging were performed to verify the results in vitro.5.The transcription of LINC01123 and AC020978 was explored by bioinformatics analysis,dual-luciferase reporter assay and Chromatin immunoprecipitation(Ch IP)assay.6.RNA immunoprecipitation(RIP)and luciferase analyses were used to confirm the predicted competitive endogenousRNAs(ceRNAs)mechanisms between LINC01123 and c-Myc.7.RNA pull-down,mass spectrometry andRNA immunoprecipitation(RIP)assays were used to identify the potential mechanism of AC020978.Western blotting,in situ proximity ligation assay(PLA),and co-immunoprecipitation(co-IP)were performed to reveal the potential mechanism of AC020978.