The study was approved by the Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University (Grant no. 28/2021). Written informed consent was obtained from all participants before participating in the present study. All methods were performed in accordance with the relevant guidelines and regulations. Subjects were recruited from the Department of Infectious Diseases and the Physical Examination Center of the Second Affiliated Hospital of Chongqing Medical University between April 1, 2021 and October 31, 2021. This study was performed at the Second Affiliated Hospital of Chongqing Medical University. This study included discovery and validation cohorts. Each cohort included HCC patients, liver cirrhosis (LC)patients, Chronic hepatitis B(CHB) patients, and healthy subjects(NC). Healthy subjects were individuals with normal health examination results, including chest X-ray, oral examination, abdominal ultrasound, routine stool examination, routine blood examination, liver and kidney function, HBV and HCV antigen test results, and no antibodies against HIV and syphilis. Chronic hepatitis B (CHB) was defined as chronic necroinflammatory liver function caused by persistent HBV infection (positive HBsAg over 6 months with serum HBV DNA > 105 copies/ml and persistent or intermittent elevation in AST or ALT concentrations). Patients with LC were confirmed by biopsy or two imaging modalities (hepatic ultrasound with CT or MRI). HCC was diagnosed based on biopsy of the tumor or CT/MRI,and staged based on Barcelona Clinic Liver Cancer(BCLC) staging system2.We divided HCC into early HCC(BCLC 0- BCLC A) and advanced HCC(BCLC B- BCLC D).HCC patients were not treated with anticancer therapy. All patients with HCC and LC had hepatitis B virus infection. No subjects had oral disease or chronic liver disease due to other causes.
Specimens were collected between 8 a.m. and 10 a.m. and subjects were required to avoid eating, drinking and smoking for at least 2 hours before collection and to gargle with water immediately before collection12.We used the spitting method to collect whole saliva13, where the subjects spat saliva into pre-cooled 15ml centrifuge tubes. The saliva sample was centrifuged at 4000 g for 15 min at 4°C) to pellet shed cells. The supernatant was transferred to a 1.5 mL Eppendorf tube, followed by further centrifugation (12000 g, 10 min, 4°C) to completely remove the cell components. and add protease inhibitor was added to the samples (100:1) which were stored at -80°C until use.
Salivary protein extraction and labeling
Ten saliva samples in each group of the discovery cohort were pooled together to obtain one mixed sample from each group. 4 groups of mixed samples were precipitated with cold acetone, and the protein concentration of the precipitated protein was detected by Bradford assay. Salivary proteins were suspended, denatured, cysteine blocked, and digested with trypsin. Each group of samples was labeled with iTRAQ marker reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and repeated with a different label (NC group was labeled 117 and 118; CHB was labeled 119 and 121; LC was labeled 115 and 116; HCC was labeled 113 and 114).
Liquid phase separation of the samples was performed with a Shimadzu LC-20AB liquid phase system on a 5um 4.6x250mm Gemini C18 column. The elution peak was monitored at a wavelength of 214nm and one component was collected per minute. The samples were combined according to the chromatographic elution peak map to obtain 20 fractions, which were then freeze-dried.
High performance liquid chromatography
The dried peptide samples were reconstituted with mobile phase A (2% ACN, 0.1% FA), centrifuged at 20,000 g for 10 minutes, and the supernatant was taken for injection. Separation was performed on a Thermo UltiMate 3000 UHPLC (Thermo Fisher Scientific).
Mass spectrometry detection
The peptides separated by liquid chromatography were ionized by a nanoESI source and passed to a Q-Exactive HF X tandem mass spectrometer (Thermo Fisher Scientific, Inc. ) for Data Dependent Acquisition (DDA) mode detection. The ion fragmentation mode was HCD, and the fragment ions were detected via Orbitrap. ProteinPilot2.0 protein quantitative analysis software and the Uniprot database (http://www.uniprot.org/) were used for data processing. DEPs must have met the following conditions: 95% confidence interval and 5% false positive rate (FDR); protein identification requires at least 2 unique peptides; protein score selection threshold > 1.314.
Target protein determination
The top 10 up-regulated differentially expressed proteins(DEPs) were identified from the iTRAQ analysis, and were verified by the Western blot analysis of the mixed and individual saliva samples. Proteins that were identified by Western blot analysis as being significantly over-expressed in the liver cancer group were selected. Finally, proteins that were found, via immunohistochemistry(IHC) and enzyme-linked immunosorbent assay (ELISA), to be highly expressecd in liver cancer tissues and in the saliva of liver cancer patients were selected as the target proteins of this study.
The concentration of extracted proteins was determined via an Enhanced BCA Protein Assay kit (Beyotime Institute of Biotechnology, Haimen, China).The proteins of the 4 groups of mixed saliva samples in the discovery cohort were extracted and diluted with 5X loading buffer (Beyotime, China), then heated at 100℃ for 5 minutes to fully denature the proteins. Protein samples were subjected to 10% SDS-PAGE electrophoresis and transferred to a PVDF membrane (Millipore Corporation, Bedford, MA, USA).Blocked with 5% BSA and TBST for 2 hours at room temperature. The membrane was then incubated with primary antibodies (all 1:1000; anti-AFP, ab169552, Abcam, UK; anti-ORM1, ab134042, Abcam, UK; anti-MMP9, ab246539, Abcam, UK; anti-Haptoglobin, ab256454, Abcam, UK; anti-Arginase, ab124917, Abcam, UK; anti-FCG3B, A7894, ABclonal, China;anti-COTL1, A4550, ABclonal, China; anti-GAPDH, 5174S, Cell Signaling Technology, America) at 4℃ for 12 hours, and washed thrice with 1x TBST. The membrane was incubated with HRP-IgG antibodies(1:5000;Santa Cruz Biotechnology, USA) at room temperature for 1 hour, washed again with 1x TBST. ECL reagents were prepared at a ratio of 1:1, and the bands were analysed using a ChemiDoc MP imaging system (Bio-Rad Laboratories).
The expression of AFP, ORM1 and HP in HCC was detected via immunohistochemistry using an HCC tissue array (OD-CT-DgLiv03-003, Outdo Biotech,China), which included 31 HCC tissues and 31 matched normal adjacent tissues.The tissue chips were rehydrated in xylene and an ethanol gradient, washed with double distilled water and soaked in 3% H2O2 for 10 min to quench endogenous peroxidase activity. The tissue arrays were blocked with BSA for 30 min, and incubated with AFP, ORM1 and HP primary antibodies overnight at 4℃. AFP, ORM1 and HP expression was measured using a DAKO EnVision + System, HRP (DakoCytomation, Glostrup, Denmark) under 200x magnification. Nuclear staining intensity was classified as negative, weak, medium and strong, represented by the numbers 0,1,2,3 respectively. The number of positive cells was divided into five grades: 0, 1, 2, 3 and 4, indicating that the number of positive cells accounted for 0%, 1%-25%, 26%-50%, 51%-75% and 76%-100% of the total number of cells, respectively. Staining index (SI) was defined as the product of nuclear staining intensity score and positive cell number score (scope: 0–12).
Elisa and experimental indicators were detected
The 200 saliva samples from the validation cohort were tested for AFP (ELH-AFP-1, Raybiotech, Peachtree Corners, GA, USA) and ORM1 (243675, Abcam, UK) using ELISA kits. Serum AFP levels were determined by LIAISON automatic chemiluminescence analyzer (DiaSorin S.p.A., Italy). The measurement protocol was performed according to the instructions of the respective ELISA kits. Saliva and blood tests were performed independently by different researchers in two different laboratories. As the specimens were numbered randomly, the researchers did not know the basic information of the patients.
All experiments were performed at least in triplicate. Statistical analyses were performed using SPSS 20.0 (IBM, Armonk, NY, USA). The quantitative variables of the normal distribution were represented as mean ± standard deviation (SD). The Student's-t test was used for comparison between the two groups, and analysis of variance was used for comparison between multiple groups. The quantitative variables of the non-normal distribution were represented by median (range), and the Mann-Whitney U test was used between two groups. Comparisons between multiple groups were analyzed using the Kruskal-Wallis H Test. For classification variables, we used the Chi-square test for differences between groups and Spearson correlation analysis to determine the correlation between two continuous variables (Saliva AFP and serum AFP). Receiver operating characteristic (ROC) curve analysis was used to evaluate the diagnostic performance of the target proteins. For the two markers combined diagnostic model we used the binary logistic regression model. P values less than 0.05 were considered to be statistically significant, and all significance tests were two-sided tests. Based on the area under the ROC curve, a type 1 error (α) of 5% and statistical power (1-β) of 90% (two-sided), we used Power Analysis and Sample Size (PASS) software to calculate the minimum sample size required.