LncRNA MALAT1 Regulates The Biological Characteristics And Glycolysis Of Non-small Cell Lung Cancer Cells Through The MiR-515-5p/TRIM65 Axis

Background:Lung cancer is one of the most common and deadly cancers in the world,and non-small cell lung cancer(NSCLC)accounts for about 85% of all lung cancer cases.Many patients with NSCLC are already in the advanced stage when they are diagnosed.At present,treatment options for patients with advanced and metastatic NSCLC are very limited.Therefore,there is an urgent need to discover biomarkers for early diagnosis and new therapeutic targets.Long non-coding RNA(lncRNA)is a type of non-coding RNA with a length of more than 200 nucleotides.It is well known that it can regulate the expression of various genes and participate in the occurrence and development of tumors.For example,lncRNA lung adenocarcinoma transcript 1 associated with metastasis(MALAT1)promotes the progression of NSCLC by regulating miRNA.However,the mechanism of MALAT1 in NSCLC has not yet been fully elucidated.Objectives:The purpose of this study is to explore the effect of MALAT1 on the biological characteristics and glycolysis of NSCLC cells,and to explore whether its molecular mechanism is related to the regulation of miR-515-5p/TRIM65,in order to provide an experimental basis for the diagnosis and treatment of NSCLC.Methods: 1.Use qRT-PCR technology to detect the expression of MALAT1 and LOXL1-AS1 in NSCLC tissues and cell lines,and transfect MALAT1 or LOXL1-AS1 si RNA into A549 and SPC-A-1 cells by liposome transfection technology.2.qRT-PCR technology to detect the transfection effect,MTT experiment,plate cloning experiment to detect cell proliferation and clone formation ability.3.Use flow cytometry and Annexin V-FITC/PI double staining to detect the influence of interference with MALAT1 or LOXL1-AS1 on the cell cycle and apoptosis of A549 and SPC-A-1.4.Use Transwell experiment,scratch experiment,Western blot detection and kits to detect the influence of interference with MALAT1 or LOXL1-AS1 on the invasion,migration,epithelial-mesenchymal transition and glycolysis of A549 and SPC-A-1 cells.5.Construct A549 and SPC-A-1 cell lines that both interfere with MALAT1 and overexpression of TRIM65.Through MTT experiment,clone formation,flow cytometry,Transwell and other experiments to analyze whether overexpression of TRIM65 can restore the interference of MALAT1 on the proliferation,cell cycle,apoptosis,invasion,migration,epithelial-mesenchymal transition and glycolysis of A549 and SPC-A-1 cells.6.The bioinformatics online database Starbase predicts the targeted binding sites between MALAT1 and miR-515-5p or miR-515-5p and TRIM65,using dual luciferase reporter gene experiment,RIP experiment and qRT-PCR technology to verify MALAT1 Target binding relationship with miR-515-5p,miR-515-5p and TRIM65.7.Using qRT-PCR and Western blot to detect the expression of miR-515-5p and TRIM65 in NSCLC tissues and cells,and analyze the correlation of MALAT1,miR-515-5p and TRIM65 in NSCLC tissues.8.Detect whether MALAT1 can regulate the expression of TRIM65 through miR-515-5p in NSCLC cells by Western blot.9.Xenograft tumor experiments in nude mice confirmed that lncRNA MALAT1 is involved in regulating the progression of NSCLC through the miR-515-5p/TRIM65 axis.Results: 1.MALAT1 and LOXL1-AS1 are highly expressed in NSCLC tissues and cell lines.Transfection of MALAT1 or LOXL1-AS1 si RNA can effectively interfere with the expression of MALAT1 or LOXL1-AS1 in A549 and SPC-A-1 cells.2.Interference with MALAT1 or LOXL1-AS1 can inhibit A549 and SPC-A-1 cell proliferation,cell cycle progression,clone formation,invasion,migration,epithelial-mesenchymal transition and glycolysis,and promote cell apoptosis.3.Overexpression of TRIM65 can reverse the effects of interference with MALAT1 on the biological specificity and glycolysis of A549 and SPC-A-1 cells.4.MALAT1 can target and negatively regulate the expression of miR-515-5p,and miR-515-5p can target and negatively regulate the expression of TRIM65.5.The expression of MALAT1 and miR-515-5p,miR-515-5p and TRIM65 in NSCLC tissues are negatively correlated,and the expression of MALAT1 and TRIM65 in NSCLC tissues is positively correlated.6.In NSCLC cells,MALAT1 can regulate the expression of TRIM65 through miR-515-5p.7.Nude mouse xenograft experiments confirmed that lncRNA MALAT1 is involved in regulating the progression of NSCLC through the miR-515-5p/TRIM65 axis.Conclusion: 1.MALAT1 and LOXL1-AS1 are highly expressed in NSCLC tissues and cells.Interfering with MALAT1 or LOXL1-AS1 can inhibit the growth,metastasis and glycolysis of non-small cell lung cancer cells.2.MALAT1 can be used as the ce RNA of miR-515-5p to competitively bind to miR-515-5p,thereby regulating the expression of the downstream gene TRIM65.3.MALAT1 can mediate the biological characteristics and glycolysis of NSCLC cells by regulating the miR-515-5p/TRIM65 axis,and participate in the promotion of non-small cell lung cancer.