New serum biomarker identification and analysis by mass spectrometry in cervical precancerous lesion and acute cervicitis in South China

Abstract

Background: According to the statistics of WHO/IARC, cervical cancer (CC) has become the fourth malignant cancer of female worldwide and it is one of the main causes of death of women in developing countries. Purpose: Potential plasma and metabolic biomarkers for CC precancerous lesions and cervicitis were indicated by LC-MS techniques, and their underlying mechanisms and functions were analyzed. Methods: Plasma samples were selected from healthy people (control), low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions (HSIL), CC, and post-treatment patients. All polypeptide types and sequences were detected by LC-MS/MS and the results were normalized by using Pareto-scaling. Potential metabolic biomarkers were screened by applying MetaboAnalyst 4.0 software and XCMS software, and analysis of variance and enrichment analysis were performed. Metabolic pathway analysis and functional enrichment analysis were used to further investigate the significance and pathological mechanisms of potential biomarkers. Results: Compared with healthy people, 9 differentially expressed metabolites were screened, 4 of which were up-regulated and 5 were down-regulated. LSIL group screened 7 differentially expressed metabolites, 5 of which were up-regulated and 2 were down-regulated; CC group screened 12 differentially expressed metabolites were screened, of which 9 were up-regulated and 3 were down-regulated. Eight differentially expressed metabolites were screened in the IF group, of which 5 showed up-regulation and 3 showed down-regulation. In functional enrichment analysis, differential metabolism was found to be associated with addition and coagulation cascades. Among all potential biomarkers, 2-amino-3-methyl-1-butanol, L-carnitine, Asn Asn Gln Arg, Ala Cys Ser Trp, Soladulcidine, Ala Ile Gln Arg, 2-amino-3 -Methyl-1-butanol, L-carnitine, Asn Asn Gln Arg, Ala Cys Ser Trp, Soladulcidine, Ala Ile Gln Arg can be used as predictors of precancerous lesions at different stages of CC. Among all biomarkers, 6α-fluoro-11β1,17-dihydroxypren-4-ene-3,20-dione has higher expression in the CC and HSIL groups and lower expression in the treatment group. Conclusion: By applying molecular markers to assess the progression of the disease, the accuracy and specificity of the diagnosis can be improved, which has certain prospects in clinical applications.

Keywords: LC-MS; acute cervicitis; cervical precancerous lesion; metabonomics; serum biomarkers.

Figure 1

PCA analysis and OPLS-DA analysis of serum metabolites in each group of patients. (A) PCA analysis of CON, IF, LSIL, HSIL and CC groups. (B) PLS-DA analysis of CON, IF, LSIL, HSIL and CC groups.

Figure 2

Volcano Plot of serum metabolites in each group of patients. (A) Volcanic map of serum metabolites in LSIL patients compared to healthy individuals. (B) Volcanic map of serum metabolites in HSIL patients compared to healthy individuals. (C) Volcanic map of serum metabolites in patients with CC compared to healthy subjects. (D) Volcanic map of serum metabolites in patients with cervical IF compared to healthy subjects. Red dots were significantly up-regulated metabolites (FC >2, p<0.05), and blue dots were significantly down-regulated metabolites (FC <0.5, p<0.05). Abbreviations: CON, control; IF, inflammation; ISIL, low grade squamous intraepithelial lesion; HSIL, high grade squamous intraepithelial lesion; CC, cervical cancer; FC, fold change analysis.

Figure 3

Heat map cluster analysis of potential serum metabolic biomarkers in patients with cervical cancer, precancerous lesions and cervicitis. The abscissa is the sample grouping and the ordinate is the M/z of the metabolite. Abbreviations: CON, control; IF, inflammation; ISIL, low grade squamous intraepithelial lesion; HSIL, high grade squamous intraepithelial lesion; CC, cervical cancer.

Figure 4

Analysis of serum metabolic biomarkers in patients from each group. (A) 2-Amino-3-methyl-1-butanol was continuously up-regulated in the LSIL, HSIL group compared with CON. (B) L-Carnitine is continuously up-regulated in the LSIL and HSIL groups compared with CON. (C) Asn Asn Gln Arg is continuously up-regulated in the LSIL and HSIL groups compared with CON. (D) Ala Cys Ser Trp is continuously up-regulated in the LSIL, HSIL group compared with CON. (E) Soladulcidine was continuously down-regulated in the LSIL and HSIL groups compared with CON. (F) Ala Ile Gln Arg is continuously down-regulated in the LSIL and HSIL groups compared with CON. (G) Prosopinine was continuously up-regulated in the CON, IF, LSIL, HSIL and CC groups and began to be lowered in the TREAT group. (H) 6alpha-Fluoro-11beta, 17-dihydroxypregn-4-ene-3, 20-dione was higher in the cervical cancer and precancerous lesions than in the CON group and significantly decreased in the TREAT group. *p<0.05, **p<0.01, ***p<0.001 vs Con group. Abbreviations: CON, control; IF, inflammation; ISIL, low grade squamous intraepithelial lesion; HSIL, high grade squamous intraepithelial lesion; CC, cervical cancer.

Figure 5

Differential metabolic material enrichment analysis results. (A) Enrichment analysis of differential metabolites in healthy people with LSIL. The results showed that the differential metabolites were mainly enriched in Phenylacetate Metabolism, Thiamine Metabolism, Phosphatidylethanolamine Biosynthesis and Phosphatidylcholine Biosynthesis. (B) Enrichment analysis of differential metabolites in healthy people with HSIL. The results showed that the differential metabolites were mainly enriched in Phosphatidylethanolamine Biosynthesis, Phosphatidylcholine Biosynthesis, Beta Oxidation of Very Long-Chain Fatty Acids, Betaine Metabolism and Carnitine Synthesis. (C) Enrichment analysis of differential metabolites in healthy people with cervical inflammation showed that the differential metabolites were mainly enriched in Phosphatidylethanolamine Biosynthesis, Phosphatidylcholine Biosynthesis, Pantothenate and CoA Biosynthesis, Phenylacetate Metabolism and Thiamine Metabolism. Abbreviations: CON, control; IF, inflammation; ISIL, low grade squamous intraepithelial lesion; HSIL, high grade squamous intraepithelial lesion; CC, cervical cancer.