LINC00667 Promotes Angiogenesis In Lung Adenocarcinoma Through Stabilizing VEGFA

Background and Objectives:Lung cancer is a malignant tumor with the highest incidence of male and female in the world,so it is necessary to seek accurate diagnostic biomarkers and identify therapeutic targets.These recognized biomarkers include long non-coding RNAs(lncRNAs).LncRNAs with sequences greater than 200bp are often considered to be a single transcript that does not have protein-coding capabilities,but they can regulate molecular processes at the DNA-RNA-protein level.By participating in epigenetic,transcriptional and post-transcriptional mechanisms,it plays an important role in the regulation of oncogene and tumor suppressor gene expression.The VEGF-A-VEGFR axis is critical in both physiological and pathological angiogenesis and vascular permeability.Disruption of the splicing of just one gene in the VEGF-A-VEGFR axis(VEGF-A,VEGFR-1,VEGFR-2)leads to changes in the entire signaling axis,leading to increased vegfr-2 signaling and angiogenesis abnormalities in cancer.This study we first using gene chip technology to screen out differential lncRNAs,verified by RT-PCR technique result showing that lung adenocarcinoma tissue specimen with LINC00667 high expression,and silent LINC00667 inhibiting proliferation,migration,angiogenesis of lung adenocarcinoma cancer cells.LINC00667 may be a oncogene,and be associated with VEGF-VEGFR pathway,according to the analysis of the bioinformatics software.Further study was carried to investigate mechanism of LINC00667 interacted with VEGFA to regulate pathological angiogenesis.The results of this study provide new ideas for the basic research and treatment of anti-angiogenesis mechanism of lung cancer.Methods:PART ONE Expression of LINC00667 and its clinical significance in lung adenocarcinomaMethods1.Materials including lung adenocarcinoma frozen tissue,lung cancer and pulmonary epithelial cell lines,reagent and instruments of Real-time PCR?Western-Blot and cell culture were prepared.LncRNA chips was carried out to detect differential lncRNAs.2.Real-time PCR was used to verified the reliability of lncRNAs chips results by testing expression of four lncRNAs in lung adenocarcinoma and pulmonary epithelial cell lines.3.Analyze of the correlation between LINC00667 expression and clinical characteristics.Results1.The expression level of LINCOO667 in lung adenocarcinoma was significantly increased.2.LINC00667 expression show closely relashionshipi with clinical characteristics of lung adenocarcinoma patients.PART TEO Effects of silencing LINC00667 on proliferation,migration and angiogenesis of lung adenocarcinoma cellsMethods1.Construction of silence LINC00667 recombinant lentivirus vector2.Effect of sh-LINC00667 to the proliferation of SPC-A1 and H1299 cells by Cell counting kit-8(CCK-8)and Ethynyldeoxyuridine(EdU)analysis.3.Effect of sh-LINC00667 to the migration of SPC-A1 and H1299 cells by Transwell test.4.Effect of sh-LINC00667 to the angiogenesis of SPC-A1 and H1299 cells by Tube formation assay.Results1.Sh-LINC00667 was obtained successfully.2.Proliferation and migration of SPC-A1 and H1299 cells transfected by sh-LINC00667 was restrained.3.Angiogenesis of SPC-A1 and H1299 cells transfected by sh-LINC00667 was restrained.PART THREE LINC00667 promotes angiogenesis in lung adenocarcinoma through stabilizing VEGFAMethods1.Using Microarray analysis to detect RNA bonding proteins associated with LINC00667.2.RT-PCR to test VEGFA and EIF4A3 expression.3.Effect of overexpression of VEGFA to proliferation,migration,,and angiogenesis of SPC-Aland H1299 transfected by pcDNA3.1/VEGFA overexpression vector.4.Using western-blot to Test VEGFA and EIF4A3 expression of SPC-A1and H1299 transfected by shLINC00667.5.Effect of silencing EIF4A3 on proliferation,migration,and angiogenesis of SPC-A1 and H1299 cells.Results1.Bioinformatics analysis showed that LINC00667 was related with RNA binding protein EIF4A3.2.High expression of VEGFA and EIF4A3 was detected in lung adenocarcinoma tissues.Expression of VEGFA was declined by silencing LINC00667.LINC00667 could influence the activity of VEGFA on post-translational level.3.Proliferation,migration,and angiogenesis were increased in SPC-A1 and H1299 cells transfected by pcDNA3.1/VEGFA overexpression vector.4.Silencing LINC00667 could decrease VEGFA expression,with no effect on EIF4A3 expression.5.Silencing EIF4A3 could supresse the proliferation,migration,and angiogenesis of lung adenocaricinoma.Conclusions1.High expression was detected in lung adenocarcinoma tissues,and LINC00667 was associated with lung adenocarcinoma patients clinical parameters.2.LINC00667 could promote the proliferation,migration,and angiogenesis in lung adenocarcinoma cells.3.LINC00667 could promote the angiogenesis by stabilizing VEGFA via EIF4A3.