Long Non-coding RNA AC122108.1 Promotes Lung Adenocarcinoma Brain Metastasis And Progression Through The Wnt/β-catenin Pathway By Directly Binding To Aldolase A

Backgroud:Lung cancer has the highest mortality rate among malignant tumors,accounting for 13%of tumor deaths.There are 1.8 million new lung cancer patients every year.One of the most common distant metastases of lung cancer is the brain.10%to 25%of firstdiagnosed patients have brain metastases.40%-50%of patients develop brain metastases during treatment.The quality of life of patients with NSCLC brain metastases is low,and the clinical prognosis is poor.The median survival（OS）is 3 to 6 month s.In recent years,the incidence of lung cancer has continued to rise.At the same time,with the continuous development of imaging technology and diagnosis and treatment technology,the survival time of patients has been effectively extended,which has led to the incidence and diagnosis of lung cancer brain metastasis.Lung adenocarcinoma belongs to non-small cell lung cancer and is one of the most common lung cancers.Its incidence is about 20%-30%of primary lung cancer.In recent years,as more and more tumor-targeted drugs targeting different gene loci have been used in clinical treatments,it has been found that lncRNA is involved in epigenetic,transcription and posttranscriptional regulation and other levels of regulation of gene expression.It is a major potential target for tumor treatment,and the treatment of lung cancer with brain metastasis has shown a huge turning point.With the development of molecular biology and epigenetics,the role of long non-coding RNA（lncRNA）in the occurrence and evolution of diseases has attracted more and more attention.lncRNA refers to non-coding RNA with a transcript longer than 200 nt,which can account for 80%of the genome transcript.Its functions are extensive and complex,involving almost all biological processes such as embryonic development,genetic imprinting,inflammatory stress,and tumors.A number of studies have confirmed that a variety of specific lncRNAs play an important role in the occurrence of lung cancer and the progression of brain metastasis,but most of the specific mechanisms of action are still unclear.For the first time,our study found that long-chain non-coding RNA-AC122108.1 is highly expressed in lung adenocarcinoma brain metastasis tissues and cells,but the current research on it is almost blank.In this study,we aimed to verify the expression level of lncRNA-AC 122108.1 in lung adenocarcinoma and lung adenocarcinoma brain metastasis and its biological functions,and explore the molecular mechanism of lncRNA-AC 122108.1 through ALDOA.The clinical diagnosis and prognosis of patients with lung adenocarcinoma brain metastasis provide potential new molecular markers,and at the same time lay an experimental and theoretical foundation for the targeted therapy and drug development of lung adenocarcinoma brain metastasis.As one of the most critical rate-limiting enzymes in glycolysis,Fructose bisphosphate aldolase A（ALDOA）is one of the most critical rate-limiting enzymes in glycolysis,and previous studies have focused on its role in the metabolism and energy supply mode of tumor cells.However,with the deepening of research,more and more recent evidence shows that:in the malignant biological behavior of various tumors,including lung cancer,pancreatic cancer,bladder cancer,and cholangiocarcinoma,the key enzyme of glycolysis ALDOA can also be independently performed.Signal molecules participate in tumor-related signal pathways,thereby changing the biological characteristics of tumor cell proliferation,migration and invasion.In tumors such as kidney cancer and colorectal cancer,ALDOA has been proven to destroy the β-catenin phosphorylation complex in the cytoplasm through the canonical Wnt signaling pathway,block its degradation,and ultimately lead to β-catenin cytoplasm,accumulate and translocate into the nucleus,express Wnt-related target genes,and finally achieve the effect of regulating tumor occurrence and progression.ObjectiveWhole transcriptome sequencing was used to screen out the differential genes and long non-coding RNAs of lung adenocarcinoma brain metastases and lung adenocarcinoma cell lines to find new potential targets for lung adenocarcinoma brain metastases.Functional experiments confirmed that lncRNA-AC122108.1 and ALDOA regulate the biological functions of lung adenocarcinoma and brain metastases cell proliferation,apoptosis,migration and invasion.To explore the mechanism of interaction between lncRNA-AC 122108.1 and ALDOA and lncRNA-AC 122108.1 to regulate the Wnt/β-catenin axis through ALDOA to promote the progression of lung adenocarcinoma brain metastasisMethodsLncRNA-AC 122108.1 was screened in lung adenocarcinoma and brain metastatic cell lines by high-throughput sequencing,and its expression was verified by real-time quantitative PCR（RT-qPCR）experiments using pathological tissues collected from clinical cases.Factor analysis predicts its value.The biological function of lncRNA-AC122108.1 in the regulation of lung adenocarcinoma and brain metastases cells was studied by CCK-8,cell clone formation,cell apoptosis,cell scratching and Trans well experiments.In vivo experimental verification was completed with tissue immunohistochemical experiments.Using fluorescence in situ hybridization technology to locate the intracellular distribution of lncRN A-AC122108.1,using RNA pull-down and RNA-binding protein immunoprecipitation and protein generalization techniques to verify that ALDOA is the binding protein of lncRNA-AC 122108.1,through RNA The 3D predictive modeling technology preliminarily predicts the three-dimensional space mechanism of the combination of the two.The ability of ALDOA to independently intervene in tumor progression through the identity of a non-glycolytic rate-limiting enzyme was verified using a 96-well cellular metabolic respiration dynamics analyzer.Co-immunoprecipitation（Co-Immunoprecipitation,Co-IP）,Western blot and immunofluorescence techniques were used to verify that 4 lncRNAAC122108.1 promoted the progression of lung adenocarcinoma brain metastases by binding to ALDOA through the Wnt/β-catenin axis.ResultLncRNA-AC 122108.1 was significantly highly expressed in lung adenocarcinoma and brain metastases cell lines and tissues,and was positively correlated with the TNM stage of lung cancer（P=0.003）and the expression of pathological Ki-67（P<0.001）.Univariate analysis（P≈0）and multivariate analysis（P=0.003）of the expression of lncRNA-AC 122108.1 suggested that it could be used as a potential target to predict the prognosis of patients.LncRNA-AC 122108.1 has biological functions such as promoting proliferation,migration and invasion,and inhibiting apoptosis in lung adenocarcinoma and brain metastases cells.After PC14/B knockdown lncRNA-AC 122108.1,it was implanted into the brain of nude mice.The tumor volume was significantly reduced（P<0.05）,the number was significantly reduced,and the survival cycle was prolonged.ALDOA can specifically bind to lncRNA-AC122108.1,and it is preliminarily predicted that the three-dimensional structure of RNA-protein binding formed by the two is involved in regulating the biological functions of PC 14 and PC14/B cells,promoting proliferation,migration and invasion.Inhibit apoptosis,etc.ALDOA may have the ability to independently intervene in the process of lung adenocarcinoma brain metastasis as a non-glycolytic rate-limiting enzyme（P<0.001）.Low expression of lncRNA-AC 122108.1 and ALDOA can lead to increased expression of phosphorylated β-catenin.Reducing the accumulation and translocation of β-catenin into the nucleus eventually leads to a significant decrease in the expression of Wnt pathway target genes such as LEF1,Vimentin,Cycline D1,c-Myc,and MMP7,thereby affecting the progression of brain metastases from lung adenocarcinoma.ConclusionAs an oncogene,LncRNA-AC 122108.1 is highly expressed in lung adenocarcinoma brain metastases cells and tissues,and its expression level is correlated with tumor TNM stage and Ki-67 expression.The expression level of LncRNA-AC122108.1 can be used as a prognostic indicator for lung adenocarcinoma and lung adenocarcinoma brain metastases to a certain extent.Functional studies confirmed that LncRNA-AC 122108.1,ALDOA could regulate the proliferation and EMT of lung adenocarcinoma brain metastases cells.LncRNA-AC 122108.1 can bind to ALDOA in lung adenocarcinoma brain metastatic cells and tissues,and promote the progression of lung adenocarcinoma brain metastases via the Wnt/β-catenin axis.