The Study On The Mechanism And Molecular Imaging Of Long Non-coding RNA AP000695.2 Promoting Malignant Progression In Lung Adenocarcinoma Via TEAD1 Upregulation

Objective:The morbidity and mortality of lung cancer are in the forefront of malignant tumors,and lung adenocarcinoma（LUAD）is the most common pathological type.Although the diagnosis and treatment of lung adenocarcinoma have made great progress,the overall prognosis is still unsatisfactory.Therefore,it is of great significance and concern to deeply study the progress of lung adenocarcinoma and provide help for individualized treatment and prognosis evaluation of lung adenocarcinoma patients.TEA domain transcription factor 1（TEAD1）,as the key regulator of Hippo signaling pathway,plays an important biological role in the malignant progression of lung cancer,and participates in cell proliferation,invasion,migration and other development processes.Tumors tend to generate energy by glycolytic pathway,thus adapting to the environment with limited nutrition.At present,the regulation of Hippo signaling pathway on glycolysis of lung adenocarcinoma is in urgent need of exploration,which has important diagnostic value.Long non-coding RNAs（lnc RNAs）could be involved in many important biological functions,such as proliferation,apoptosis,metastasis and cell metabolism.The interaction between lnc RNAs and cell metabolism is closely related to the progress of tumor,so it is of great significance to explore the mechanism of this correlation.AP000695.2 is an unverified lnc RNA.Bioinformatics analysis results showed that the expression of AP000695.2 was significantly correlated with the expression of glucose metabolism-related genes and TEAD1,but the specific regulatory mechanism of AP000695.2 in lung adenocarcinoma remains unclear.This study aims to explore the expression of AP000695.2and its target gene TEAD1 in lung adenocarcinoma and its regulation of glycolysis,proliferation and migration,determine the molecular mechanism involved and provide a new research target for anti-tumor energy metabolism therapy.Methods:1.79 paraffin-embedded specimens of primary lung adenocarcinoma and 40corresponding adjacent tissues were selected,then immunohistochemistry（IHC）staining was used.The relationship between the expression of TEAD1 and glycolysis in lung adenocarcinoma was studied by analyzing the correlation between the expression of glycolysis-related enzymes such as glucose transporter 1（GLUT1）,hexokinase 2（HK2）,pyruvate kinase isozymes M2（PKM2）and lactate dehydrogenase A（LDHA）and semi-quantitative indicators of glucose metabolism in F-18-fluorodeoxyglucose(¹⁸F-FDG)positron emission tomography/computed tomography（PET/CT）imaging,including the maximum standard uptake value（SUVmax）,tumor metabolic volume（MTV）and total lesion glycolysis（TLG）.To analyze the relationship between the expression of TEAD1 in lung adenocarcinoma and the clinical features of patients,including age,gender,lymph node metastasis,tumor diameter and clinical stage.Then,the correlation between TEAD1and glycolysis indexes in different groups of patients was further analyzed.2.R project was used to analyze the transcriptome data and clinical information of patients with LUAD in the database of The Cancer Genome Atlas（TCGA）,and predict lnc RNA—AP000695.2which could participate in glycolysis of LUAD.q PCR detected the expression of AP000695.2 in lung cancer cells and detected its localization by FISH experiment.The expression changes of AP000695.2 in different glucose environments were detected by q PCR experiments.After transfecting the over-expression plasmid and si RNA to change its expression,its effects on cell glycolysis(Western blot,¹⁸F-FDG uptake experiment,lactic acid detection experiment),proliferation（CCK-8 assay,Ed U experiment）,migration（Transwell experiment,wound healing assay）,reactive oxygen species level and epithelial-mesenchymal transition（EMT）process（Western blot）were detected.The subcutaneous transplanted tumor model in nude mice was established,the volume of transplanted tumor was dynamically monitored,and the molecular imaging of ¹⁸F-FDG micro-PET was evaluated.3 Bioinformatics analysis predicted the correlation between AP000695.2 and TEAD1,and verified the regulatory effect between them by Western blot.q PCR and Western blot were used to detect the regulatory effect of TEAD1 on GLUT1,and the regulatory mechanism was verified by dual-luciferase reporter assay.Rescue experiments detected the regulatory effect of AP000695.2 on cell glycolysis,proliferation and migration through TEAD1.Using bioinformatics software to predict mi R-335-3p which could target AP000695.2 and TEAD1 at the same time.The effect of changing the expression of AP000695.2 on mi R-335-3p and the effect of changing the expression of mi R-335-3p on TEAD1 were detected.The regulation mechanism was verified by dual-luciferase reporter assay.Results:1.IHC results indicated that there was a significant positive correlation between the expression of TEAD1 and glucose metabolism indexes（GLUT1,HK2,PKM2 and LDHA）in lung adenocarcinoma（r=0.7681,0.6629,0.4249,0.5674,P<0.001）.There was a significant positive correlation between TEAD1 expression level and PET/CT indexes（SUVmax,MTV and TLG）（r=0.2464,0.2542,0.3152,P<0.05）.The expression level of TEAD1 was closely correlated with lymph node metastasis and clinical stage（P<0.05）.The patients were divided into groups according to whether the age was less than 60 years old,genders,whether the tumor diameter was greater than 30mm,whether there was lymph node metastasis and clinical stages.The expression of TEAD1 was positively correlated with GLUT1,HK2,PKM2 and LDHA in each group.2.Bioinformatics analysis showed that the expression of AP000695.2 in LUAD was higher than that in normal tissues,and its high expression was closely related to poor prognosis.The expression of AP000695.2was significantly positively correlated with a variety of glycolysis-related genes.AP000695.2 is upregulated in many lung cancer cell lines and is localized in the cytoplasm and nucleus.The expression of AP000695.2 decreased in long-term glucose deprivation and increased in short-term stimulation.AP000695.2 could promote the expression of glycolysis and EMT-related indicators,FDG uptake and lactic acid production,inhibit the level of reactive oxygen species,and promote cell proliferation and migration.In vivo experiments showed that the SUVmax of ¹⁸F-FDG micro-PET imaging decreased after the down-regulation of AP000695.2,and the expression levels of various glycolysis-related proteins decreased.3.AP000695.2 promoted the expression of TEAD1,and TEAD1promoted the expression of GLUT1.Dual-luciferase reporter assay confirmed that TEAD1promoted GLUT1 transcription.It was confirmed that AP000695.2 could promote glycolysis,proliferation and migration of cells via TEAD1.AP000695.2 inhibited the expression of mi R-335-3p,and mi R-335-3p inhibited the expression of TEAD1.Dual-luciferase reporter assay verified that mi R-335-3p exists binding sites with TEAD1 and AP000695.2.Conclusion:1.The expression level of TEAD1 in lung adenocarcinoma was positively correlated with the expression of key factors of glycolysis,such as GLUT1,HK2,PKM2,LDHA and PET/CT indexes,including SUVmax,MTV and TLG.2.The expression of lnc RNA AP000695.2 is increased in lung adenocarcinoma tissues and cells.Experiments in vivo and in vitro showed that AP000695.2 could promote the glycolysis,proliferation and migration of cells.3.AP000695.2 could promote the proliferation,migration and glycolysis of lung adenocarcinoma through mi R-335-3p/TEAD1/GLUT1 pathway.AP000695.2 is expected to be a potential target of anti-tumor energy metabolism therapy.