Effects Of GLUT1 And VEGFR2 Interaction On The Proliferation,Apoptosis,and Migration Of Lung Adenocarcinoma Cells

Objective:Lung cancer is a malignant tumor with the highest morbidity and mortality in the world.One of the main subtypes of lung cancer is lung adenocarcinoma(LUAD).It has been shown that glucose transport type 1(GLUT1)and vascular endothelial growth factor 2(VEGFR2)play a key role in the progression of LUAD.However,whether there is an interaction between the two molecules is still unclear.Therefore,this study aimed to investigate the effects of GLUT1 and VEGFR2 on the proliferation,apoptosis,and migration of LUAD cells,as well as the interaction of GLUT1 and VEGFR2.Methods:1.A meta-analysis was performed to evaluate the GLUT1 protein expression level of non-small cell lung cancer(NSCLC).Bioinformatics analysis was used to detect the GLUT1 m RNA expression level and survival differences in samples from TCGA.Meanwhile,functional and network analysis was conducted to detect significant signaling pathways and key genes related to GLUT1 with the Gene Expression Omnibus(GEO)dataset.2.Immunohistochemistry and in situ hybridization were used to detect the protein and m RNA expression levels of GLUT1and VEGFR2 in LUAD samples,respectively.Then Person coefficient was used to analyze the correlation between GLUT1 and VEGFR2.3.It was divided into two parts in vitro experiment of A549 cell:GLUT1/VEGFR2 inhibition and GLUT1overexpression.CCK-8 was used to detect cell viability.Using CFSE(Carboxyfluorescein succinimidyl ester)tracer dye and PI(Propidium iodide)staining to detect the cell proliferation ability.Then,Annexin V-APC/PI staining was performed to calculate the cell apoptosis.A wound-healing assay was used to evaluate the migration of A549 cells.The invasion was assessed by a transwell invasion experiment.The expression of GLUT1 and VEGFR2 protein and RNA levels were detected through immunofluorescence assay and quantitative real-time PCR(q RT-PCR),respectively.4.A549 cells were injected subcutaneously into nude mice.After the formation of palpable subcutaneous tumors,the mice received the inhibition of GLUT1 and VEGFR2,as well as the tumor volume,which was measured for 7days.Results:1.Results by Meta-analysis showed that GLUT1 was overexpressed in NSCLC tissues compared to para-carcinoma tissues(RD=0.56).Besides,patients with high GLUT1 expression levels had a poor clinical prognosis(P<0.05).The results of the GEO dataset analysis showed that GLUT1 might contribute to the p53signaling pathway,cytochrome P450,and other biological pathways in patients with lung adenocarcinoma.Moreover,The GLUT1 expression was correlated with KDR(VEGFR2).2.Compared to para-carcinoma tissues,GLUT1 and VEGFR2 were expressed higher in lung adenocarcinoma tissues(P<0.05),and the expressions of the two molecules were correlated(r~2=0.5746,P<0.05).3.Results showed that the cell viability,proliferation,early apoptosis,migration,and invasion were suppressed when GLUT1 was inhibited(P<0.05).However,GLUT1 overexpression promoted the abilities of cell proliferation,migration,and early apoptosis(P<0.05).Besides,the inhibition of VEGFR2 weakened the cell viability,proliferation,migration,and invasion while increasing the early apoptotic cells(P<0.05).Furthermore,inhibiting the VEGFR2 when GLUT1 was overexpressed,the cell proliferation and migration were suppressed while the cell early apoptosis ability was promoted(P<0.05).The expression of VEGFR2 protein was decreased by inhibiting GLUT1 while increased when GLUT1 was highly expressed.When the VEGFR2 was suppressed,the expression of GLUT1 protein,which was originally overexpressed,was reduced.The expression of VEGFR2 RNA increased when GLUT1 was inhibited while the expression level of VEGFR2 RNA decreased when GLUT1 was overexpressed.After inhibiting the VEGFR2,the expression of GLUT1 RNA increased.4.Tumor volume growth rate of mice treated with GLUT1 or VEGFR2 inhibitors was significantly slower than that of the control group(P<0.05).Conclusion:1.Tumor cells in which GLUT1 was overexpressed could inhibit tumor cell proliferation and promote tumor cell apoptosis by inhibiting VEGFR2 expression.2.The expression of GLUT1 and VEGFR2 molecules might have a feedback effect.