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Fibrinogen Like Protein 1 As a Potential Biomarker for Hepatitis B Virus Related Hepatocellular Carcinoma

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Fibrinogen Like Protein 1 As a Potential Biomarker for Hepatitis B Virus Related Hepatocellular Carcinoma Fibrinogen Like Protein 1 as a Potential Biomarker for Hepatitis B Virus Related Hepatocellular Carcinoma Cai Xin Renmin Hospital of Wuhan University Tang Dongling Renmin Hospital of Wuhan University Chen Juanjuan Renmin Hospital of Wuhan University Li Huan Renmin Hospital of Wuhan University Hu Yuanhui Renmin Hospital of Wuhan University Zhang Pingan ( [email protected] ) Renmin Hospital of Wuhan University Research Article Keywords: FGL1, AFP, HBV, HCC, Biomarker, Diagnosis Posted Date: August 19th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-741807/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/13 Abstract Background There is an urgent need for new serum biomarkers for early screening of HBV-related hepatocellular carcinoma (HCC). Fibrinogen like protein 1 (FGL1) may develop the potential diagnostic value of alpha fetoprotein (AFP) in HBV-related HCC. Methods The TCGA database was used to screen out genes related to liver cancer and perform differential expression analysis. Enzyme-linked immunosorbent assay and chemiluminescence immunoassay were used to detect concentrations of FGL1 and AFP. Using immunouorescence semi-quantitative method to detect the mean uorescence intensity of FGL1. Result FGL1 is lower in tumor tissues than in normal tissues. The serum levels of FGL1 and AFP in patients with HBV-related HCC are signicantly higher than others for each group. Compared with other groups, the area under the receiver operating curve (AUC) of FGL1 is higher than that of AFP when compared with the normal group, and the AUC of other groups is lower than that of AFP. The combination of the two can increase the AUC to 0.862 (95%CI, 0.786 ~ 0.918) in distinguishing benign liver disease from HBV-related HCC. The specicity of FGL1 and AFP in the diagnosis of HBV-related HCC is 98.39% and 70.97%, respectively. The specicity of the combination was 93.55%. In distinguishing the A and B stages in the BCLC staging, the combination of the two increased the AUC from 0.584 to 0.647. When distinguishing benign liver disease from HBV-related HCC, the AUC of FGL1 reached 0.849, with a specicity of 100%. Conclusion FGL1 can be used as a non-invasive biomarker for HCC. When combined with AFP, the diagnostic eciency and specicity were improved. 1. Background Globally, primary hepatocellular carcinoma (HCC) is the sixth most common cancer and the third leading cause of cancer death, with approximately 906,000 new cases and 830,000 deaths [1]. Hepatitis B virus (HBV) accounts for half of the global viral hepatitis deaths, half of all liver cancer deaths and one third of all liver cirrhosis deaths [2]. In China, HBV infection accounts for 63% of the total deaths from cirrhosis and other chronic liver diseases, and 53% of the total deaths from liver cancer. China is the country with the largest number of hepatitis B and C virus infections in the world [3]. Although China vigorously popularizes hepatitis B vaccine injection, it is estimated that there are still 77 to 97 million chronic HBV infections, of which more than 20 to 30 million are in active liver disease [4], and about 7 million develop cirrhosis or liver cells Cancer [5]. Although the diagnosis and treatment of HCC have made great progress in recent years, it still cannot change the fact that most of the conrmed cases of HBV-related HCC are in the middle and late stages, and the metastasis and recurrence of HCC have led to no signicant improvement in the ve-year survival rate of patients. So far, Alpha fetoprotein (AFP) has been widely used in the laboratory for early screening of HBV-related HCC, but its diagnostic value still needs to be improved. Some HCC patients express low level AFP and cause AFP negative. The commonly used positive cut-off value is AFP≥ 20 ng / ml [6]. Therefore, there is an urgent need for new serum biomarkers for early screening of HBV-related HCC. Advances in bioinformatics and the development of high-throughput sequencing have surfaced some tumor markers. For example, it found that GBP5 gene is highly correlated with immune inltration [7]. DPP9 plays an important role in the pathogenesis of human HCC [8]. The appearance of these tumor markers is helpful for the diagnosis of tumors. In this study, immune-related biomarkers were screened in order to nd markers related to HCC. FGL1 contains a brinogen related domain in its C-terminal portion, which is similar to tenascins, broleukin, angiopoietins, brinogen β and γ chains[9]. The difference is that FGL1 lack of brinogen’s three functional domains, such as the platelet binding-site, the cross- linking region and the thrombin-sensitive site. It is regarded as an acute phase reactant which is secreted by liver [10]. 2. Materials And Methods 2.1 Oncolnc Database Analysis OncoLnc (http://www.oncolnc.org/) contains survival data from 8,647 patients and 21 cancer studies conducted by the Cancer Genome Atlas (TCGA), as well as mRNA and miRNA RNA-SEQ expression from TCGA, and from the lncRNA expression of MiTranscriptome beta. Use OncoLnc for hepatocellular carcinoma-related gene screening. 2.2 GEPIA Database Analysis Page 2/13 GEPIA (Gene Expression Proling Interactive Analysis) (http://www.oncolnc.org/) has always been a valuable and highly cited resource for gene expression analysis based on tumor and normal samples from TCGA and GTEx databases. Contains RNA sequencing data based on 9,736 tumor samples and 8,587 normal samples from TCGA and GTEx databases. We use Plotly to organize and plot GEPIA data. 2.3 Research Object Selection The proportion of males in HCC patients in China is higher than that of female patients, so this part of study only included male patients. On the other hand, it also controlled for the interfering factors brought by gender variables. All patients were recruited in the Department of Oncology, Department of Infectious Diseases and Physical Examination Center, Renmin Hospital of Wuhan University, Hubei Province, China from April 2021 to June 2021. According to the guidelines of the Chinese Society of Hepatology, the Chinese Society of Infectious Diseases and the Chinese Medical Association[11], all 153 patients and normal controls were divided into HBV-related HCC group (55 cases), HBV-infected liver cirrhosis (LC) group (31 cases), and chronic hepatitis B (CHB) group (31 cases) and the control group of healthy people (HC) (36 cases). The pathological tissues of liver cancer patients in the Department of Oncology, Renmin Hospital of Wuhan University, Hubei Province, China from April 2021 to June 2021 were collected. According to relevant guidelines, they were divided into HCC group (4 cases) and normal population control group (3 cases). All HCC patients were conrmed by liver pathological examination, X-ray examination or MRI examination. Exclusion criteria for included cases: (1) Exclude malignant tumors other than primary liver cancer. (2) Exclude other hepatitis virus infections other than HBV. (3) Exclude diseases such as severe diabetes, hyperthyroidism, and cardiovascular disease. (4) Exclude pregnant patients. All normal control populations tested negative for HBV, HCV, syphilis and HIV as well as all biochemical tests were normal. This study has been reviewed and approved by the Medical Ethics Review Committee of Renmin Hospital, Wuhan University. According to the requirements of the Medical Ethics Review Committee of Renmin Hospital, Wuhan University, all the included persons agreed and signed the informed consent form. 2.4 Sample Collection Venous blood was collected from patients who had been fasted for more than 8 hours in the morning, centrifuged at 3500/rpm for 15 minutes at room temperature, and transferred the serum from the collection tube with separating gel to the cryotube, and stored it at -80°C for further use. Blood was collected using an anticoagulant tube, immediately reversed and mixed, centrifuged after several minutes, and plasma was collected. We use 10% paraformaldehyde to x the fresh tissue, use gradient concentration of alcohol to dehydrate the tissue block, and then immerse it in xylene. After the tissue block is transparent, it is immersed in paran for embedding and sectioning, and stored at room temperature. 2.5 Laboratory Analysis Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP),γ-glutamyl transpeptidase (γ-GT), albumin (ALB), total bilirubin (TBIL), hypersensitive C-reactive protein (hs-CRP) and direct bilirubin (DBIL) was analyzed by enzymatic method using the fully automatic biochemical analyzer ADVIA 2400 (Siemens, Germany). SysmexCA-7000 (Kobe, Japan) was used to detect brinogen (FIB) and d-dimer. We use ABI ViiA7 real-time uorescent quantitative polymerase chain reaction system (ABI, America) to detect HBV DNA. AFP was detected using Siemens ADVIA Centaur XP (Siemens, Germany). We use a commercial enzyme-linked immunosorbent assay (ELISA) kit (product number JL47875) produced by Jianglai Biological Company to detect FGL1. The kit uses a double-antibody one-step sandwich method for enzyme-linked immunosorbent assay. The detection range is 46.875pg/ml~1500pg/ml, and the specimens were diluted 100 times for testing, and the auxiliary hole is set to ensure the detection accuracy. 2.6 Immunouorescence Quantication Purchase the FGL1 antibody (Cat. No.: 67391-1-Ig) produced by Proteintech Group, and entrust Siwega Biotechnology (Wuhan) to perform immunouorescence quantitative experiments. 2.7 Data Analysis The experimental data was analyzed using SPSS version 20.0 (IBM Corp, NY) and MedCal 15.2.2 (Ostend, Belgium), GraphPad Prism 6.0 (GraphPad Software, Inc, La Jolla, CA) was used for image rendering, and Image J 1.53J (National Institutes of Health, Bethesda) for immunouorescence quantitative analysis. The measurement data uses the single-sample Kolmogorov-Smirnov method to test whether the data of each group conforms to normality, the normal distribution data is represented by ±s, the comparison between multiple groups is performed by analysis of variance, and the further pairwise comparison is performed by LSD-t test.
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