The Function And Mechanism Of LncrRNA GLS-AS Mediated C-Myc/GLS Pathway In Pancreatic Cancer

ObjectivePancreatic ductal adenocarcinoma(PDAC)is a highly malignant tumor.Due to the the pathological features of high fibrosis and vascular deficiency of pancreatic cancer,as well the metabolic reprogramming in cancer cells,there is almost no effective treatment for pancreatic cancer.The aim of this study is to investigate how our newly discovered antisense long non-coding RNA(lncRNA GLS-AS)of glutaminase(GLS)can reprogram metabolism by regulating c-Myc/GLS signaling pathway under nutrition stress microenvironment in pancreatic cancer.Our study investigated the effect of dysregulated lncRNA GLS-AS on changing tumor growth and metastasis,and revealed its novel regulatory mechanisms,which may provide new insights and theoretical bases for the treatment of pancreatic cancer.MethodsDifferentially expressed of long non-coding RNAs(lncRNAs)in pancreatic cancer tissues were screened by next-generation sequencing.The antisense long non-coding RNA GLS-AS(AK123493.1)of glutaminase(GLS)was selected as a target of continued study.The expression of lncRNA GLS-AS and GLS mRNA was detected by real-time quantitative PCR.Northern blot analysis confirmed that lncRNA GLS-AS was expressed in pancreatic cancer tissues and cells.RNA fluorescence in situ hybridization(RNA-FISH)emerged the subcellular localization of lncRNA GLS-AS in pancreatic cancer cells.Surgical specimens of pancreatic cancer patients were obtained from Wuhan Union Hospital,including 30 pairs of cancer tissues and corresponding normal tissues adjacent to each other.Small interfering RNAs(siRNASs)or overexpression plasmid of lncRNA GLS-AS was transfected into pancreatic cancer cell lines BxPC-3 and PANC-1 cells.The proliferation activity of the transfected cells was detected by MTT assay and colony formation assay;the wound healing assay and Transwell assay were used to detect cell migration and invasion ability.Lentiviral was used to coate lncRNA GLS-AS small interfering RNA.The coated lentivirus was transfected into PANC-1 cells and used to xenograft model in nude mice to observe the growth and metastasis ability of the transfected cells in vivo.qPCR and Western Blot were used to detect the expression of lncRNA GLS-AS /GLS mRNA and GLS protein in pancreatic cancer tissues and in pancreatic cancer cells which transfected with small interfering RNA or overexpression plasmid of lncRNA GLS-AS.Simultaneously,co-staining of RNA in situ hybridization and GLS protein immunofluorescence to observe the co-localization of lncRNA GLS-AS RNA and GLS protein in pancreatic cancer tissues and pancreatic cancer cell lines.Specific RNA probes were designed for lncRNA GLS-AS and GLS precursor mRNA(GLS pre-mRNA).LncRNA GLS-AS and GLS mRNA were co-localized in pancreatic cancer cells using RNA fluorescence in situ hybridization.Fragments of different parts of lncRNA GLS-AS were synthesized by in vitro transcription technique and labeled with biotin,the biotin-labeled probes were used in RNA pulldown assay to investigate the position of lncRNA GLS-AS binding to GLS pre-mRNA.After ?-amanitin blocked RNA synthesis in pancreatic cancer cell lines,the effect of transfection of lncRNA GLS-AS small interfering RNA or overexpression plasmid on the stability of GLS pre-mRNA was observed.Co-immunoprecipitation(Co-IP)was used to demonstrate that ADAR1 binds to Dicer in pancreatic cancer cells.RNA-binding protein immunoprecipitation(RIP)demonstrates that the ADAR1/Dicer protein complex binds to the lncRNA GLS-AS /GLS pre-mRNA double-stranded RNA(dsRNA)complex.In the pancreatic cancer cells,the expression of lncRNA GLS-AS,GLS pre-mRNA and the stability of GLS pre-mRNA after transfected with siRNAs of ADAR1 or Dicer and then with treatment of ?-amanitin were detected by qPCR,respectively.In vitro simulation of pancreatic cancer tissue microenvironment,including hypoxia,acidity,low glucose and low glutamine,then the lncRNA GLS-AS level was measured by qPCR,to explore why lncRNA GLS-AS low expression in pancreatic cancer.RNA fluorescence in situ hybridization was used to verify the low expression of lncRNA GLS-AS in nutrition stress environments.qPCR and Western Blot were used to detect the expression of GLS mRNA and protein in low levels of glucose and glutamine environments.Western Blot was used to detect the expression of c-Myc protein in low levels of glucose or glutamine deprivation environments.Chromatin immunoprecipitation(ChIP)verified the binding sequences between c-Myc protein on lncRNA GLS-AS promoter.The dual luciferase reporter assay detected the effect of c-Myc protein on the transcriptional activity of the lncRNA GLS-AS promoter.The lentiviral vector of LncRNA GLS-AS(LV-GLS-AS)was transfected into cells,and the lentivirus with empty vector was used as a negative control(LV-NC)to construct xenograft model.The effect of lncRNA GLS-AS overexpression on tumor growth and metastasis was observed.ResultsLong non-coding RNA GLS-AS(AK123493.1)is downregulated in pancreatic cancer tissues and pancreatic cancer cell lines,and most of lncRNA GLS-AS is enriched in the nucleus.In vitro cell experiments and xenograft model in nude mouse,the results indicate that low expression of lncRNA GLS-AS promotes proliferation and invasion of pancreatic cancer cells.The low expression of lncRNA GLS-AS in pancreatic cancer tissues is negatively correlated with the expression of GLS.The low expression of lncRNA GLS-AS in pancreatic cancer cells promotes the growth and invasion of tumor cells mainly by increasing the level of GLS protein.LncRNA GLS-AS inhibits GLS expression through ADAR1/Dicer-dependent RNA silencing in pancreatic cancer cells.Nutrition stress leads to increased accumulation of c-Myc protein in pancreatic cancer cells,which is responsible for the downregulation of lncRNA GLS-AS.The novel mechanism is that c-Myc protein can bind to the specific promoter sequences of lncRNA GLS-AS gene and then inhibit transcriptional activity of lncRNA GLS-AS.ConclusionThe nutrient stress causes an increase of c-Myc in pancreatic cancer cells,and c-Myc inhibits the transcription of the long non-coding RNA GLS-AS(lncRNA GLS-AS)of glutaminase(GLS),further leading to increased expression of GLS protein.Collectively,our study suggests a novel lncRNA-mediated c-Myc/GLS pathway that serves as a metabolic target for the treatment of pancreatic cancer and promotes our understanding of the coupling of lncRNA in nutrition stress and tumorigenesis.