| Objective:To investigate the glucose metabolism characteristics of oral squamous cell carcinoma(OSCC)-associatedfibroblasts(CAFs)and related regulatory molecules.Methods:(1)The primary CAFs were obtained by tissue culture method,and 0.25%trypsin digestion was passaged,and the cells were purified by mechanical scraping and enzymatic digestion.The cells were preliminarily identified by morphological observation and protein expression detection.(2)CCK8,Flow Cytometry and Seahorse Extracellular Flux(XF)Analyzers were used to detect the growth,proliferation,apoptosis and glucose metabolism of CAFs and NFs.(3)Label-free quantitative proteomics was used to study the differential proteins in the mitochondria of CAFs/NFs,and Gene Ontology KEGG was used to analyze the differential proteins and screen the key proteins of CAFs metabolism regulation.(4)Key protein was overexpressed by transfecting CAFs with lentivirus CAFs and their metabolic regulation function was verified.Results:(1)CAFs were successfully isolated,cultured and identified.Under the light microscope,there were differences in the morphology between CAFs and NFs.Cellular immunohistochemistry showed that both of CAFs and NFs expressed Vimentin positively and CK negatively.However,CAFs significantly higher expressed protein α-SMA than NFs.(2)CCK8 and Flow Cytometry results showed that the proliferation activity of CAFs was significantly enhancedas compared to NFs,while the apoptosis rate was significantly decreased,with statistically significant differences(P<0.0001).(3)The results of the Seahorse XF Analyzersshowed that CAFs had stronger mitochondrial activity,mainly through oxidative phosphorylation(OXPHOS)metabolism,which could produce more ATPas compared to NFs.Moreover,CAFs highly expressed peroxisome proliferator-activated receptor y coactivator-1α(PGC-1α),transcription factor A mitochondrial(TFAM)as compared to NFs.(4)CAFs and NFs were successfully isolated,purified and identified,and their ultrastructure were observed under electron microscopy.Label-free quantitative proteomics assay showed that 183 differentially expressed proteins were identified in mitochondria of CAFs and NFs,95 were up-regulated and 88 were down-regulated.Gene Ontology and KEGG database showed that 26 proteins were tightly related to OXPHOS,in which the expression level of ATP synthase(ATP50)was significantly increased with a fold change of 3.84(FC=3.84),while the expression level of tumor necrosis factor receptor-related protein 1(TRAP 1)was significantly decreased with FC=2.00.(5)When CAFs were transfected with lentivirus and TRAP1 were overexpressed,the OXPHOS was significantly lower than that of the vector group and control group.Conclusions:(1)The proliferation activity of CAFs was stronger,but the apoptosis of CAFs was lower as compared to NFs.(2)CAFs have strong mitochondrial function and are mainly metabolized by OXPHOS,which can promote tumor growth and progression.(3)Our resultsindicated that TRAP1 is one of the important regulatory molecules of CAFs glucose metabolism.Further study on the regulatory mechanism of TRAP 1 has important theoretical significance and application value for elucidating the role of oral CAFs in glucose metabolism and discovering new therapeutic targets of OSCC. |
