The Function And Mechanism Of Long Non Coding RNA SLCO4A1-AS1-202 In Promoting Lung Adenocarcinoma Progression

BackgroundLung cancer is currently the malignant tumor with the highest mortality in the world.In China,the mortality rate of lung cancer ranks first among all malignant tumors.According to histological morphology,lung cancer can be divided into small cell lung cancer(SCLC)and non-small cell lung cancer(Non-Small Cell Lung Cancer,NSCLC),of which NSCLC accounts for approximately 85%of all lung cancer cases.According to the pathological features,non-small cell lung cancer can be further divided into subtypes including adenocarcinoma,squamous cell carcinoma,adensquamous carcinoma,and large cell carcinoma,among which lung adenocarcinoma(LUAD)represents the most common pathological subtype of NSCLC.Although new advancement has been made in the clinical diagnosis and treatment of lung adenocarcinoma,the overall poor prognosis of lung adenocarcinoma patients has not been significantly improved.Therefore,identifying the key molecules and specific mechanisms that regulate the development and progression of lung adenocarcinoma is of important clinical significance for the diagnosis and treatment of lung adenocarcinoma.Identifying the biological characteristics that are ubiquitous in tumor cells but different from normal cells,and specifically intervening against these characteristics has been a key to improving the efficacy of tumor treatment.Cancer is also recognized as a metabolic disease.Unlike normal cells,which mainly obtain adenosine triphosphate(ATP)through mitochondrial oxidative phosphorylation,tumor cells obtain ATP mainly from glycolysis.In view of this metabolic feature of tumor cells,the glucose metabolism pathway has become an important potential target for tumor therapy.However,the molecular mechanism via which cancer cells promote their malignant behavior by regulating glucose metabolism pathways still remains unclear.Therefore,it is particularly important to study the mechanism of metabolic reprogramming of tumor cells and its relationship with the development and progression of malignant tumors.Not only can these studies elucidate new mechanisms of tumorigenesis from the perspective of metabolic abnormalities,but also they may provide new strategies for NSCLC diagnosis,prevention and therapy through utilizing,intervening and correcting abnormal cellular metabolism.Aberrant expression of genes plays an important role in the occurrence and development of lung adenocarcinoma.However,most of the current research focuses on the analysis of protein-coding genes that only account for 2%of human genome,while the nonprotein-coding sequences accounting for 98%of the genome sequence,especially the function of long non-coding RNA(lncRNA)in the development of adenocarcinomas remain poorly understood.The role of lncRNAs in the progression of lung adenocarcinoma needs to be further explored,and whether they may function via regulating tumor glucose metabolism,remains unknown.In the contexts as above described,in-depth investigation of the role of lncRNA in the glucose metabolism of lung adenocarcinoma and the specific molecular mechanism will help to further understand the molecular mechanisms underlying the development and progression of lung adenocarcinoma and to find new potential therapeutic targets.Based on the above research background,the following scientific questions are proposed:Are there lncRNAs that affect the malignant phenotype of lung adenocarcinoma by regulating glycolysis?What is the specific molecular mechanism by which it works?Methods1.Screening of long non-coding RNAs related to glycolysis in LUAD cells(1)The Cancer Genome Atlas(TCGA)public database was used to screen the expression of lncRNAs highly expressed in LUAD;(2)The TCGA database was used to comparatively analyze the difference in the expression of target lncRNA in adjacent normal tissue and lung adenocarcinoma tissue and the correlation between the expression level of target lncRNA and survival of lung adenocarcinoma patients;(3)Gene Set Enrichment Analysis(GSEA)was used to analyze the correlation between LUAD samples with high expression of target lncRNA and glycolysis;(4)Quantitive real-time polymerase chain reaction(qRT-PCR)was used to detect the expression changes of the target lncRNA in lung adenocarcinoma cells treated with complete medium and glucose-deficient medium;(5)Multi-parameter Analysis System was used to detect the changes of glucose and lactate content in the medium overexpressing the target lncRNA.2.Characterization of the target lncRNA(1)Rapid-amplification of cDNA ends(RACE)experiment was used to acquire the full-length sequences of the target lncRNA,and Western blot(WB)experiment was employed to examine the protein coding capacity of the target lncRNA;(2)Nuclear cytoplasmic RNA extraction and qRT-PCR were used to detect the subcellular localization of the target lncRNA;(3)Fluorescence in situ hybridization(FISH)experiment was performed to detect the subcellular localization of the target lncRNA.3.Functional study of lncRNA SLCO4A1-AS1-202 in promoting the growth,invasion and metastasis of lung adenocarcinoma(1)LUAD cell lines(A549 and H1299)that could stably overexpress the target lncRNA and its encoded short peptide were established;(2)The effects of target lncRNA and its encoded short peptide on the proliferation and metastasis of lung adenocarcinoma cells were detected by plate clony formation assay,thiazole blue cell growth assay,wound healing assay,transwell migration and invasion assay;(3)The effect of target lncRNA on the growth of lung adenocarcinoma cells in vivo was detected by subcutaneous tumorigenesis experiments in nude mice.4.Molecular mechanism via which the target LncRNA promoting glucose metabolism in lung adenocarcinoma cells(1)RNA pull down combined with Western blotting experiments were performed to confirm that the lncRNA specifically binds PFKP and EGFR in the cytoplasm;(2)Co-IP assay of membrane proteins was used to verify that lncRNA SLCO4A1-AS1-202 enhances the interaction between PFKP and EGFR;(3)Western blot assay was used to detect the activation of the AKT pathway by lncRNA SLCO4A1-AS1-202;(4)Phosphofructokinase activity assay was used to detect the effect of target lncRNA on kinase activity of phosphofructokinase.Results1.Long noncoding RNA SLCO4A1-AS1-202 is upregulated in lung adenocarcinoma and is associated with lung cancer glycolysis(1)TCGA public database analysis showed that the expression of lncRNA SLCO4A1-AS1-202 in lung adenocarcinoma tissues was significantly higher than that in adjacent non-cancerous tissue,and the expression levels of lncRNA SLCO4A1-AS1-202 in lung adenocarcinoma patients positively correlated with poor prognosis of the patients;(2)GSEA analysis showed that the highly expressed lncRNA SLCO4A1-AS1202 positively correlated with glycolysis;(3)QRT-PCR results showed that compared with complete medium,glucosefree medium significantly upregulated the expression of lncRNA SLCO4A1-AS 1-202 in cultured lung adenocarcinoma cells;(4)Multi-parameter Analysis System showed that overexpression of lncRNA SLCO4A1-AS1-202 accelerates glucose uptake and increases lactate production.2.Characterization of lncRNA SLCO4A1-AS1-202(1)RACE experiments revealed that full-length SLCO4A1-AS 1-202 is a 1422nt polynucleotide and has a 3’-polyA tail structure,and the lncRNA SLCO4A1-AS 1-202 has the ability to encode short peptides;(2)Nuclear cytoplasmic RNA extraction and FISH showed that lncRNA SLCO4A1-AS 1-202 was distributed in the nucleus and cytoplasm of lung adenocarcinoma cells.3.Overexpressting lncRNA SLCO4A1-AS1-202 promotes the growth,migration and invasion of LUAD in vitro(1)Plate colony formation assay,thiazole blue cell growth assay,wound healing assay,and transwell invasion and migration assay showed that overexpression of lncRNA SLCO4A1-AS1-202 could promote the proliferation,invasion and migration of lung adenocarcinoma cells;(2)Subcutaneously xenotransplantation tumorigenesis experiment in nude mice showed that overexpressing lncRNA SLCO4A1-AS 1-202 promoted lung adenocarcinoma cells proliferation in vivo;(3)Plate colony formation assay,wound healing assay,and transwell invasion and migration assay showed that overexpression of IncRNA SLCO4A1-AS1-202 and lncRNA SLCO4A1-AS 1-202-mut promoted the growth,invasion and metastasis of lung adenocarcinoma cells,but overexpression of ORF3 alone had no such effects.4.Molecular mechanism via which lncRNA SLCO4A1-AS1-202 promotes glucose metabolism in lung adenocarcinoma cells(1)RNA pull-down combined with Western blotting experiments showed that lncRNA SLCO4A1-AS 1-202 specifically binds PFKP and EGFR in the cytoplasm;(2)IP experiments of membrane proteins showed that lncRNA SLCO4A1-AS1202 enhanced the interaction between PFKP and EGFR;(3)Western blot results showed that overexpression of lncRNA SLCO4A1-AS1202 could activate the AKT signaling pathway;(4)Phosphofructokinase activity assay showed that overexpressing lncRNA SLCO4A1-AS 1-202 increased phosphofructokinase kinase activity.Conclusions1.LncRNA SLCO4A1-AS 1-202 is up-regulated in LUAD and correlated with glycolysis in LUAD cells.2.LncRNA SLCO4A1-AS1-202 plays a role in promoting growth,invasion and metastasis in LUAD.3.LncRNA SLCO4A1-AS1-202 promotes LUAD cell growth,invasion and metastasis independent of its encoded short peptide(s).4.LncRNA SLCO4A1-AS 1-202 activates the Akt signaling pathway by promoting the interaction between PFKP and EGFR in the cytoplasm,resulting in the increase of phosphofructokinase activity and promoting glycolysis.