Inhibitory Effect And Mechanism Of CELF2 On Lipid Metabolism Of Lung Adenocarcinoma By Regulating SREBP-1

Background and purposeLung cancer is the most common cause of cancer-related death,and its death rate is the first among deaths caused by cancer worldwide.According to statistics,about 1.8million people are diagnosed with lung cancer each year,and about 1.6 million people die from lung cancer.Lung adenocarcinoma（LUAD）is the most frequently diagnosed histological subtype of lung cancer.The response rate of LUAD to standard therapies is low such as chemotherapy and radiotherapy.Therefore,a full understanding of the mechanisms related to the occurrence and development of LUAD may lead to the discovery of new therapeutic candidates to improve the clinical efficacy of LUAD.RNA binding proteins（RBPs）are proteins that regulate the expression of thousands of transcripts and can bind to double-or single-stranded RNA.At present,thousands of RBPs have been identified and studied,indicating that RBPs are universally expressed and are evolutionarily conserved.CELF2（CUGBP-and ETR-3-like family 2）is an RBP from the CELF protein family,which can regulate downstream effectors of different signaling pathways through protein-protein or protein-m RNA interaction.Sterol regulatory element binding proteins（SREBPs）can activate a series of enzymes required for the synthesis of triglycerides（TG）,fatty acids（FA）,endogenous cholesterol and phospholipids at the transcriptional level.Therefore,SREBPs are considered as the main regulators of cholesterol and lipogenesis.In mammals,there are three members of the SREBP family:SREBP-1a and SREBP-1c from the same gene（SREBF-1）and SREBP-2 from a single gene（SREBF-2）,but their functions are not the same.SREBP-1 mainly regulates genes related to the synthesis of triglycerides,fatty acids and phospholipids,while SREBP-2preferentially activates genes involved in the synthesis of cholesterol.Previously,we found that CELF2 was a new tumor suppressor gene in lung adenocarcinoma,which reduced the AKT signal transduction in LUAD by interacting with PREX2,thereby inhibiting the growth of LUAD cells.However,the effect of CELF2 on downstream genes after inhibiting AKT is still unclear.In this study,we first used RNA-Seq technology to analyze CELF2 overexpression stable cell lines,and found that CELF2 mainly affects lipid metabolism,which was verified by testing the content of triglyceride,lactic acid and intracellular lipid droplets.Secondly,we sequenced the CELF2 knockdown stable cell lines and MK2202 treated cells,and analyzed the three sets of sequencing results and found that there is only one overlapping gene,namely SREBP-1.It is speculated that SREBP1 may be a downstream target gene of CELF2.Then,after knockdown and overexpression of CELF2 and the treatment of lung cancer cells with MK2202,the expression of SREBP-1 was detected via Western blot and q RT-PCR to verify our conjecture.The q RT-PCR revealed that SREBP-1c may be the main subtype of SREBP-1 regulated by CELF2,which was further verified by the luciferase reporter gene experiment.Finally,an animal model of lung cancer induced by Kras^G12D was used to verify the tumor suppressor function of CELF2 and its effect on SREBP-1 and lipid metabolism.Methods1.Screening the downstream targets of CELF2 in lung cancer cellsIn lung cancer cells A549 and H1299,CELF2 overexpression and knockdown stable cell lines were constructed.At the same time,lung cancer cells were treated with AKT inhibitor MK2202,and all three groups were sent to transcriptomics for sequencing.The target of CELF2 was screened out from the sequencing data.Design primers for differentially expressed genes and verify RNA-Seq data by q RT-PCR technology.2.Exploring the effect of CELF2 on lipid metabolismIn the stable cell lines with overexpression and knockdown of CELF2,collect the cells and use the triglyceride content assay to detect the effect of CELF2 on the intracellular triglyceride level;use the lactic acid content assay to explore the effect of CELF2 on the intracellular lactic acid content;The stable cell line with overexpression and knockdown of CELF2 was seeded into a 6-well plate,and the effect of CELF2 on the number of intracellular lipid droplets was studied by the oil red O staining method.3.Exploring the regulation of CELF2 and AKT signaling pathways on SREBP-1and its subtypesIn lung cancer cells,after knocking down or overexpressing CELF2 and treating the cells with MK2202,Western blot and q RT-PCR were used to detect the expression changes of SREBP-1 and its subtypes SREBP-1aand SREBP-1c.4.Exploring the role of SREBP-1c in lipid metabolism regulated by CELF2In 293T cells,the sh CELF2 plasmid was transfected first,and then the p GL3-Basic-SREBP-1a/1c and p RL-CMV plasmids were cotransfected.The luciferase reporter gene experiment technology was used to study the transcriptional regulation of CELF2 on SREBP-1a and 1c.In the stable cells overexpressing CELF2,the SREBP-1c plasmid was overexpressed,and the cells were collected to detect the changes of triglycerides,lactate and lipid droplets in lung cancer cells.5.Exploring the anti-tumor effect of CELF2 in Kras^G12D-induced lung cancer animal modelsUsing the animal model of lung cancer induced by Kras G12D,the adeno-associated virus carrying Cre enzyme and CELF2 gene was delivered into the lungs of mice by the method of nose drops,and the HE staining and triglyceride content detection methods were used to study the effects of CELF2 on lung cancer progression and lipids metabolism.Results1.The results of RNA-Seq analysis revealed that CELF2 mainly affects lipid metabolism of lung cancer cells,and SREBP-1 may be a downstream target gene of CELF2.2.Knockdown of CELF2 can increase the content of triglycerides,lactic acid and the number of lipid droplets,while overexpression of CELF2 will reduce the content of triglycerides,lactic acid and the number of lipid droplets.3.Knockdown of CELF2 can promote the expression of SREBP-1 and its subtypes,while overexpression of CELF2 or treatment of cells with MK2202 will inhibit the expression of SREBP-1 and its subtypes.4.Knockdown of CELF2 can increase the promoter transcription activity of SREBP-1c,but not SREBP-1a;CELF2 affects lipid metabolism by regulating SREBP-1c.5.In vivo experiments showed that CELF2 overexpression can inhibit the development of lung cancer.ConclusionsCELF2 inhibits the transcription of SREBP-1c,thereby inhibiting the expression of SREBP-1;while the reduction of SREBP-1 expression inhibits lipid metabolism,thereby slowing down the occurrence and development of lung adenocarcinoma.