The Correlation Between Differentially Expressed Lipid Molecules In Colorectal Cancer And Bisphenol A Exposure Based On UPLC-MS Lipidomics

Objective Lipidomics analysis based on ultra performance liquid chromatography-mass spectrometer（UPLC-MS）to identify differentially expressed lipid molecules in colorectal cancer（CRC）tissues and adjacent tissues,and to analyze the differentially expressed lipid molecules correlation with bisphenol A（BPA）exposure and clinical characteristics,and explore the impact of environmental BPA exposure on lipid metabolism changes in the occurrence and development of CRC from the perspective of lipid metabolomics,and provide new biomarkers for the prevention and control of environmental BPA exposure induced CRC.Methods Recruit patients who underwent CRC tumor resection in the Department of Gastrointestinal Surgery of the First Affiliated Hospital of Anhui Medical University from July 2016 to October 2019,and collected CRC patients morning urine,clinical basic data,cancer and adjacent tissues.The control group was healthy population who underwent physical examination in the health examination center of the First Affiliated Hospital of Anhui Medical University during the same period.Morning urine and physical examination data of the control group were collected.Lipid molecules in CRC cancer tissues and adjacent tissues were detected by UPLC-MS and differentially expressed lipid molecules were identified by multivariate statistical analysis combined with the univariate statistical analysis T-test and variance analysis.Liquid chromatography-mass spectrometer（LC-MS）was used to detect urine BPA concentration and urine creatinine（Cr）was used for calibration（BPAcr）to analyze the relationship between differentially expressed lipid molecules and the clinical characteristics of CRC,as well as relationship between BPAcr,differential lipid molecules expression and clinical characteristics of CRC.Result1. Analysis of lipid metabolism profile of CRC cancer tissues and adjacent tissuesAccording to the first principal component linear score graph,Hotelling T²and DMod X control graph of the PCA model established from the training cohort,it can be concluded that there were no outliers in this experiment.Due to the largely individual differences in clinical samples,unsupervised PCA does not separate cancer tissues from adjacent tissues.The supervised PLS-DA model R²Y in positive ion mode was 0.974,Q²was 0.959,R²Y in negative ion mode was 0.846,and Q²was 0.544,and permutation test Q²was-0.289,-0.569,respectively,and OPLS-DA model R²Y in positive ion mode was 0.974,Q²was 0.703,R²Y in negative ion mode was 0.846,Q²was 0.583,and permutation test Q²was-0.455,-0.709,respectively.It shows that the PLS-DA and OPLS-DA models can distinguish cancer tissues and adjacent tissues and the model has not been over-fitted.The differential expression of lipid molecules was determined by VIP>1 in OPLS-DA model and the results of univariate analysis,then 29 differentially expressed lipid molecules identified by the training cohort and the validation cohort were selected as candidate lipid molecules.Metabolic topology and pathway analysis showed that the glycerophospholipid metabolism pathway was the most disturbed,the two positions where PE and PC were located were mapped.2. The correlation analysis of serum tumor markers and differentially expressed lipid moleculesThe Nonparametric test was used to compare the peak areas of 29 candidates differentially expressed lipid molecules identified by the CEA,CA19-9 positive and negative groups.The results showed that the expression of TG（16:0/18:2/18:2）,TG（18:0/16:0/22:4）,TG（18:1/18:2/18:2）,TG（20:1/18:1/18:1）,TG（18:1/18:1/18:1）in the CEA and CA19-9 positive group decreased,while the expression of PC（32:1e）,PC（36:5）in the CEA and CA19-9 positive group was up-regulated,the differences were statistically significant（P<0.05）.3. The correlation analysis of BPA exposure and the occurrence and development of CRCThe univariate analysis showed that there was no significant difference in gender,age,BMI,smoking,drinking,BPAcr between the case group and the control group,and the multivariate Logistic regression analysis showed that BPAcr was a risk factor for CRC.The risk of exposure to high concentration BPAcr in vivo was 1.826 times that of low concentration exposure（P<0.05）.The Chi-square test results showed that the pathological infiltration depth of BPAcr high-concentration group tumors in serous membrane layer（T₃）,regional lymph node metastasis>4（N₂）,distant metastasis（M₁）and moderate differentiation ratio（70.5%,34.1%,36.4%,84.1%,respectively）,higher than the low concentration BPAcr group（41.9%,14.0%,11.6%,60.5%,respectively）,the differences were statistically significant（P<0.05）.4. The correlation analysis of urine BPAcr and serum tumor markers in CRC patientsThe Spearman rank correlation analysis showed that urine BPAcr concentration was significantly correlated with serum CEA,CA125 and CA19-9,and the correlation coefficients were 0.324,0.295,0.316,and the correlations were statistically significant（P<0.05）,and the correlation between BPAcr and AFP was not statistically significant（P=0.838）.The Whitney U test showed that the concentration of CEA and CA19-9,serum tumor markers was higher in the high concentration of urine BPAcr than in the low concentration of BPAcr,with statistically significant differences（P<0.05）.5. The correlation analysis of urine BPAcr and differentially expressed lipid molecules in CRC patientsThe Nonparametric tests were performed on 29 differentially expressed lipid molecules identified in the high and low concentrations of urine BPAcr.The results showed that in CRC cancer tissues,the expression of TG（16:0/18:2/18:2）,TG（18:0/16:0/22:4）,TG（18:1/18:2/18:2）,TG（20:1/18:1/18:1）,TG（18:1/18:1/18:1）levels in the high BPAcr concentration was decreased,while the expression of PC（32:1e）,PC（36:5）levels in the high BPAcr concentration were up-regulated.6. The mesomeric effect of BPA exposure to lipid metabolic changes in CRC patients was studiedThe mesomeric effect analysis showed that BPA exposure resulted in serum tumor markers abnormal was a partial mesomeric effect.In the total effect between BPA exposure and serum tumor markers abnormal,the proportion of lipid metabolism changes accounted for 0.138,and the mesomeric effect explained the variance variation of the dependent variable was 22%.Conclusion1.There were indeed lipids change in the progression of CRC.2.The glycerolphospholipid metabolism pathway was disturbed significantly during the progress of CRC.3.The abnormal metabolism of TG（16:0/18:2/18:2）,TG（18:0/16:0/22:4）,TG（18:1/18:2/18:2）,TG（20:1/18:1/18:1）,TG（18:1/18:1/18:1）,PC（32:1e）,PC（36:5）lipid molecules may play an important role in the occurrence and development of CRC.4.BPA exposure was a risk factor for the development of CRC and may contribute to its further development.5.The occurrence and development of CRC induced by BPA exposure may be related to TG（16:0/18:2/18:2）,TG（18:0/16:0/22:4）,TG（18:1/18:2/18:2）,TG（20:1/18:1/18:1）,TG（18:1/18:1/18:1）,PC（32:1e）,PC（36:5）abnormal metabolism,these 7 lipid molecules may be potential biomarkers for CRC diagnosis and treatment.