Abstract:

Objective To investigate the value of collagen oligopeptides（COP）in the tumor microenvironment in the diagnosis of lung adenocarcinoma. Methods A total of 47 patients with lung adenocarcinoma from Shanghai Chest Hospital of Shanghai Jiao Tong University School of Medicine from March 23，2011 to March 22，2019 were enrolled，and cancer tissues and paired adjacent tissues were collected. The metabolite profiles of cancer tissues and adjacent tissues were analyzed using broad-targeted metabolomics technology to determine the metabolic differences of oligopeptides. An animal model of lung adenocarcinoma was established in C57BL/6J mice（in situ lung tumor formation），and the metabolic differences of oligopeptides between lung cancer tissues and normal lung tissues in mice were analyzed，and the differences in total collagen and cathepsin（CTS）L expression were compared. A549 cells were transfected with negative control small interfering RNA（siRNA）（NC-siRNA），CTSL-siRNA1，CTSL-siRNA2 and CTSL-siRNA3，respectively，and the metabolic changes of oligopeptides in the cell culture supernatant were determined. Pearson correlation analysis was used to evaluate the correlation between amino acid oligopeptides and collagen，and receiver operating characteristic（ROC）curve was used to evaluate the efficacy of single and combined determination of amino acid oligopeptides in the diagnosis of lung adenocarcinoma. Results A total of 256 differential metabolites from cancer and adjacent tissues were identified. Among the amino acids and their metabolites，25 oligopeptides were abnormally enriched，with the top 3 most enriched oligopeptides being L-glycyl-isoleucine（Ile），L-aspartyl （Asp）-L-phenylalanine（Phe），L-leucylglycine（Leu）-glycine（Gly）. The diagnostic areas under curves（AUC）of all Gly oligopeptides in cancer tissues and adjacent tissues of lung adenocarcinoma patients were 0.932，0.947，0.873，0.849 and 0.901 for Gly-Ile，Leu-Gly，Gly-tryptophan（Trp），Gly-tyrosine（Tyr） and Gly-Gly-Phe，respectively. The combined determination of these 5 differentially expressed Gly oligopeptides based on 3 machine learning algorithms [random forest（RF），support vector machine（SVM）and logistic regression（LR）] had AUC of 1.000，0.944 and 0.944，respectively，for the diagnosis of lung adenocarcinoma. Compared with normal mouse lung tissues，the levels of L-threonyl（Thr）-glutamic acid（Glu），collagen and CTSL in mouse lung cancer tissues were increased（P<0.05）. The level of Thr-Glu was positively correlated with collagen expression（r=0.824，P<0.05）. The analysis based on the Cancer Genome Atlas Program（TCGA）database showed that COL1A1，COL1A2，COL2A1，COL3A1 and CTSL were highly expressed in lung adenocarcinoma tissues compared with normal adjacent tissues（P<0.05）. Compared with normal human bronchial epithelial cells BEAS-2B，the CTSL mRNA and protein were highly expressed in human lung cancer cell lines H460，A549 and H1437. Compared with the NC-siRNA group，the expression of CTSL mRNA and protein in the CTSL-siRNA1 group，CTSL-siRNA2 group and CTSL-siRNA3 group was decreased（P<0.001）. The level of COP（Gly-Glu）in the cell culture supernatant of CTSL-siRNA3 group was decreased（P<0.01）. Conclusions The metabolism of COP in the tumor microenvironment of lung adenocarcinoma patients is elevated，which may serve as a biological indicator for the diagnosis of lung adenocarcinoma.

Key words: Collagen oligopeptide, Lung adenocarcinoma, Collagen, Cathepsin L