Study on The Expression and Correlation of Alpha-Enolase (Eno1) in The Serum of Patients With Hepatocellular Carcinoma

Background: Serum tumor markers for the early detection of hepatocellular carcinoma is limited. This study analyzed the distribution and differences of ENO1 in the serum of healthy people, patients with chronic hepatitis B, cirrhosis and hepatocellular carcinoma. Furthermore, the distribution and correlation of ENO1 in the clinical data of patients with hepatocellular carcinoma were explored and its clinical diagnostic effect as well as combined diagnostic effect with AFP were compared. In addition, the effect of ENO1 on the survival of patients with hepatocellular carcinoma were also evaluated. The study will provide data basis for the application of ENO1 in the diagnosis of hepatocellular carcinoma, and provide new ideas for the study of molecular markers and therapeutic targets for hepatocellular carcinoma. Methods: In accordance with the inclusion and exclusion criteria, Patients diagnosed at the Infectious Diseases Department of the General Hospital of Ningxia Medical University and the healthy check-up population at the medical check-up centre from May 2012 to March 2017 were screened for inclusion in the study. T The nal study subjects were 181, 28 patients with chronic hepatitis B (CHB), 31 patients with liver cirrhosis (LC), 104 patients with liver cancer (HCC) and 18 healthy people (NC). Clinical data and sera were collected from all study subjects, tissue specimens were collected from some patients. The levels of serum ENO1 and AFP were measured by ELISA. QRT-PCR and Western Blot t were used to detect the relative expression of ENO1 mRNA and protein in liver cancer tissues and paracancerous tissues. Statistical analysis was performed by t-test, bivariate Spearman correlation test, curve regression analysis, ROC curve analysis and survival curve analysis. Results: The relative expression of ENO1 mRNA and protein was signicantly upregulated in hepatocellular carcinoma tissues relative to paraneoplastic tissues (p<0.05), ENO1 levels in the serum of HCC patients were sequentially higher than those of CHB patients, LC patients and healthy subjects, with signicant differences (p<0.05). The differences in the distribution of serum ENO1 levels in the clinical data subgroups alpha fetoprotein

Conclusion: Serum ENO1 levels were signi cantly correlated with the occurrence, progression and metastasis of hepatocellular carcinoma, staging and postoperative survival of patients. ENO1 may be a candidate diagnostic marker for early diagnosis and evaluation of hepatocellular carcinoma progression, and can be used in combination with AFP as an auxiliary diagnostic function.

Background
Primary liver cancer ranks sixth in tumor incidence and is the second leading cause of death due to cancer, reports show that 905,677 cases of liver cancer were diagnosed and 830,180 deaths were reported worldwide in 2020 [1] . Hepatocellular carcinoma (HCC) accounts for 90% of primary liver cancers. Hepatitis B virus (HBV) infection, hepatitis C virus (HCV) infection, liver cirrhosis, alcohol abuse, diabetes or obesity-related metabolic syndrome can all cause HCC [2][3] . According to statistics, the 5-year survival rate of Chinese liver cancer patients from 2003-2015 was only 12.5% [4] . Currently, surgery remains the main treatment for hepatocellular carcinoma, but more patients are initially diagnosed at an intermediate to advanced stage and are unable to undergo effective treatment. Therefore, early detection and treatment can effectively improve the survival rate of hepatocellular carcinoma. Serum tumor markers are one of the early diagnostic modalities for hepatocellular carcinoma and can complement the pathological diagnosis of hepatocellular carcinoma, the combination of multiple serum markers can enable early detection of hepatocellular carcinoma tumor lesions. The existing serum markers for hepatocellular carcinoma (HCC) are mainly alpha fetoprotein (AFP). However, reports show that the diagnostic sensitivity of AFP is about 39-64%, which to some extent limits the clinical utility of AFP [5] . Therefore, screening for markers or therapeutic targets for the early diagnosis of hepatocellular carcinoma is of great importance for the prevention and treatment of hepatocellular carcinoma.
Α-enolase (α-enolase, ENO1) is one of the key enzymes of the pentose phosphate pathway and plays an important role in the process of cellular energy metabolism [6][7] . The function of ENO1 is distinguished by its location in the cell. If Α-enolase is located in the cytoplasm, it acts as a rate-limiting enzyme of the glycolytic pathway, in uencing the metabolism of tumor cells and thus regulating tumourigenesis and development [8] . If Α-enolase is located on the cell surface, it acts as a brinogen receptor and promotes tumor cell invasion by activating brinolytic enzymes and accelerating the degradation of ECM [9] . If Αenolase is located in the nucleus, its encoded protein can in uence tumourigenesis and development through transcriptional repression [10] . Studies have shown that ENO1 is aberrantly highly expressed in a variety of tumor cells, and that its high expression promotes proliferation, invasion and suppresses apoptosis [11] . In many tumors, ENO1 or anti-ENO1 antibodies have the potential to function as tumor markers. For example, Pan et al. identi ed ENO1 as a lung cancer-related protein marker for clinical application through semi-quantitative proteomic analysis [12] . ENO1 is highly expressed in ovarian cancer and can be used as a diagnostic marker and therapeutic target for ovarian cancer [13] . ENO1 has been associated with breast cancer, and a study by Mittal L et al. demonstrated that the expression of ENO1 was signi cantly reduced in triple-negative breast cancer treated with electrochemical therapy, accompanied by activation of related signaling pathways and ultimately apoptosis, suggesting that ENO1 may be a possible target for anticancer therapy in breast cancer [14] . A recent study, through bioinformatic analysis of the distribution and expression of ENO1 in hepatocellular carcinoma expression pro le microarrays in public databases such as TCGA, found that ENO1 expression was signi cantly upregulated in hepatocellular carcinoma tissues and cells relative to normal liver tissues or hepatocellular carcinoma paracancerous tissues, and its expression was signi cantly correlated with the survival time of patients [15][16] . Unfortunately, there are fewer reports on whether the detection rate of HCC can be improved by detecting the concentration of ENO1 in blood, and extensive in-depth studies are needed.
Therefore, studying the distribution and correlation of serum ENO1 in clinical indicators of patients with hepatocellular carcinoma is of great signi cance for the early diagnosis and prevention of HCC.
This experiment intends to collect clinical data and sera from healthy people, patients with chronic hepatitis B, cirrhosis and hepatocellular carcinoma. The expression levels of ENO1 and AFP in their sera will be measured., to analyze the distribution and differences of ENO1 in the sera of healthy population, chronic hepatitis B, cirrhosis and hepatocellular carcinoma patients, to explore its distribution and correlation in the clinical data of hepatocellular carcinoma patients, compare its clinical diagnostic effect with AFP and combined diagnostic effect, and evaluate the effect of ENO1 on the survival of hepatocellular carcinoma patients. The study will provide theoretical and data basis for the application of ENO1 in the diagnosis and healing evaluation of hepatocellular carcinoma, and provide new ideas for the research of molecular markers and therapeutic targets of hepatocellular carcinoma.

Study subjects and grouping
The study subjects were from May 2012 to March 2017 at the Department of Infectious Diseases, General Hospital of Ningxia Medical University. Based on the inclusion and exclusion criteria, 181 cases were nally identi ed as eligible for enrollment in the study, 28 patients with chronic hepatitis B (CHB), 31 patients with liver cirrhosis (LC), 104 patients with liver cancer (HCC) and 18 cases in the healthy population (NC). All subjects signed an informed consent form prior to the survey sampling. The study protocol and all amendments were approved by the institutional review board or ethics committee of participating site and were conducted per the principles expressed in the Declaration of Helsinki. All patients provided written informed consent to participate.  [17] . Liver function status was graded according to the Child Class score and the MELD (Model for end-stage liver disease, MELD) [18] . Patients with hepatocellular carcinoma were staged according to the American Joint Committee on Cancer 6th edition TNM staging criteria [19]

ELISA Assay
The study population of 163+8 people ate regularly for 3 days prior to blood sampling, fasting for 8 hours on the day before the blood draw, 5 ml of fasting venous blood was drawn on the following morning, within 2 h of the blood being drawn, the blood was taken to the laboratory, the serum is separated by centrifugation at 1000 rpm for 5 min at room temperature. Using the ENO1 (alpha-enolase, alpha-enolaSe) ELISA kit (abcam, UK) and AFP (alpha-fetoprotein) ELISA kits (Invitrogen, USA), serum ENO1 levels were measured by a multifunctional chemiluminescence instrument (Promega, USA) for each group of samples. The remaining blood specimens were centrifuged to obtain plasma and stored at -80°C . ELISA assay steps.
(1) Remove the ELISA plate and allow to stand for 20 minutes at room temperature before equilibrating the plate components.
(2) Dilute the standards in a 2-fold gradient using sample diluent and add 50 uL of diluted sample to each well using sample diluent. Take 10 uL of serum sample and mix with 40 uL of diluent and add to the assay wells.
(3) Seal the 96-well ELISA culture plate with air-seal plate lm, shake and mix, centrifuge the plate transiently and incubate for 60 min in a 37°C thermostatic incubator for the reaction.
(4) Remove the plate, discard the liquid from the plate, pat the plate dry on stream lter paper, add 100 uL of wash buffer per well and leave for 1 min at room temperature, discard the wash buffer and drain the plate again, repeat 5 times.
(5) Add 50 uL each of substrate A and substrate B to the wells and incubate for 15 min at 37°C in an incubator protected from light, add 50 uL of termination solution to terminate the reaction, read the OD value of the wells at 450 nm using a uorescent enzyme marker, set up 3 replicate wells for each sample.

RNA preparation and reverse transcription
Lysis and extraction of total RNA from tissues using TRIzol ® Reagent (Invitrogen USA) verify RNA quality by electrophoresis of 0.3 μg RNA(1.3% agarose gel electrophoresis, 1× TAE buffer, 120 V, 20 min). The RNA was reverse transcribed into cDNA using the PrimeScript® RT reagent kit(Thermo USA), the reverse transcribed RNA template was 500ng, The reverse transcription conditions are: 5 minutes at 25°C, 42°C for 60 minutes and 70°C for 5 minutes, the remaining RNA and cDNA were stored at -80°C.

Relative expression of ENO1 gene mRNA in hepatocellular carcinoma tissues by QRT-PCR
The cDNA was obtained from each group, the relative expression of ENO1 gene was measured by QRT-PCR using TransStart Tip Green qPCR SuperMix (Quan-Style Gold Biology, Beijing, China) on a LightCycler 480II quanti cation platform (Roche, USA). β-actin was used as the internal reference gene and the details of primer sequences used in the experiment were showed in Table1  . Grinding of tissue specimens to powder using liquid nitrogen in a mortar add 400µL of lysate, grind again to a powder, After the lysate powder has thawed to a liquid, the liquid is collected, centrifuge in a tube for 10 minutes under the condition of 4°C and 12,000 rpm, The supernatant is the protein extract. The concentration of the protein extract was determined using a BCA protein quanti cation kit (Nanjing KGI, China). 20 µg of protein specimens were taken, the proteins were separated in 10% SDS-PAGE electrophoresis. The proteins were transferred onto PVDF membranes (Millipore Corp, USA) using a semi-dry transfer machine (Biorad Trans-Blot SD, BIO RAD, USA). A Sealing uid was made by dissolving 50 g of skimmed milk powder in 100 ml of 1 x TBST, The incubator was closed for 3h, then the sealing uid was discarded Wash 3 times with 1×TBST and add ENO1 primary antibody (monoclonal antibody, 1:1000 dilution, abcam, UK) and β-actin primary antibody (monoclonal antibody, 1:1000 dilution, abcam, UK) to the incubation cassette. The incubation cassette was placed on a plate shaker at 50 rpm 4°C at room temperature and incubated overnight, 1 x TBST washed 5 times for 5 min each After washing, IgG secondary antibody (1:5000 dilution, Proteintech, USA) was added to the incubation cassette Incubation cassettes were placed on a plate shaker at 50 rpm for 2h and 1× TBST washed 5 times. 1:1 mixture of liquid A and liquid B of the ECL(WesternBright ECL advansta USA)was prepared as the assay solution, The bands were observed after exposure of the PVDF membrane using a multicolor uorescent gel formation system(ChemiDoc MP Imaging System Bio-Rad, USA). The protein bands were quanti ed using Image J software.

Statistical analysis
Data were collated and analyzed by using SPSS 23.0 (IBM, USA) software, All measurements are presented as mean ± standard deviation (x ± s), Comparative analysis of the signi cance of continuous measures and subgroup data using the T-test and one-way ANOVA methods; Analysis of the correlation between serum ENO1 levels and AFP levels using the bivariate Spearman correlation test; Analysis of the correlation between ENO1 levels and hepatitis B virus using curve regression; Analysis of the sensitivity and speci city of serum ENO1 in the diagnosis of hepatocellular carcinoma using the ROC curve (receiver operating characteristic curve); The survival curve was used to evaluate the relationship between serum ENO1 and survival of patients with hepatocellular carcinoma. All signi cant results were considered statistically signi cant at p<0.05 and highly signi cant at p<0.01.

Distribution of ENO1 in different liver diseases
The expression levels of serum ENO1 were measured by ELISA and the clinically relevant test results were collected from the investigated subjects, as shown in Table 2. The results showed that the expression level of ENO1 in the serum of the healthy population was low, approximately 19.39±10.81 mg/L. Serum ENO1 expression levels were signi cantly higher in the CHB group (80.70±90.79), LC group (37.51±27.94) and HCC group (154.06±166.74) than in the NC group of the healthy population (p<0.01), among them, the expression level of ENO1 was signi cantly higher in the HCC group than in the LC and CHB groups (p<0.01). The results suggest that the development of liver diseases such as chronic hepatitis B, cirrhosis and hepatocellular carcinoma will lead to a signi cant increase in serum ENO1 expression levels, with the highest differential upregulation in hepatocellular carcinoma, and ENO1 has the potential to become a serum marker for early hepatocellular carcinoma. The expression of ENO1 gene mRNA and protein levels in hepatocellular carcinoma and paraneoplastic tissues were detected by qRT-PCR and Western blot methods. The results showed that the expression level of serum ENO1 was signi cantly higher in the serum of patients with liver cancer than that of patients with non-hepatocellular carcinoma and the healthy population (p=0.001) (Figure 1-A). The mRNA expression of ENO1 gene in paraneoplastic tissues was normalized to control base 1 and β-actin was used as an internal reference gene The mRNA expression level of ENO1 was signi cantly upregulated in hepatocellular carcinoma tissues relative to the paraneoplastic tissues (p=0.001), with a difference of approximately 3.5-4.5-fold (Figure 1-B). Randomly selected cancer/paraneoplastic tissues from two patient, protein expression showed consistent expression results with ENO1 mRNA, i.e. the relative expression of ENO1 protein was signi cantly higher in liver cancer tissues than in paraneoplastic tissues (p = 0.003) (Figure 1-C). The correlation results further suggested that the up-regulation of ENO1 expression might be associated with the development of hepatocellular carcinoma. The results showed that there was no statistically signi cant difference in the distribution of serum ENO1 levels in AFP status and TNM stage (II vs III, III vs IV) in the clinical data groups (p>0.05), There were statistically signi cant differences (p<0.05) in the distribution of gender, tumour size, whether the tumour was metastatic or not, TNM stage (I vs II) and Edmondson's classi cation in the clinical data group, the difference in the distribution of clinical data grouped by tumour recurrence was highly signi cant (p<0.01). The results further suggest that serum ENO1 can be used as a marker for the diagnosis of earlystage hepatocellular carcinoma and the diagnosis of recurrence of hepatocellular carcinoma. In addition, the high expression of ENO1 in hepatocellular carcinoma may be associated with the process of hepatocellular carcinoma development, metastasis and recurrence, and the related mechanism is unknown. (Table 3) Legend AFP: alpha-fetoprotein; ENO1: α-enolasel; TNM: Tumor Node Metastasis The cut-off value for a positive AFP is 7ng/L.

Analysis of the sensitivity of serum ENO1 in hepatocellular carcinoma and its correlation with AFP
To explore the correlation between serum ENO1 levels and AFP levels by bivariate Spearman correlation analysis, the sensitivity and speci city of ENO1, AFP and their synergy in the diagnosis of liver cancer were analyzed by ROC curves (Figure 3) The results showed that serum ENO1 levels were signi cantly correlated with the survival of patients with hepatocellular carcinoma, Within 40 months, patients with high ENO1 levels survived signi cantly less than those with low ENO1 levels (p < 0.05) ( Figure 5). The correlation results suggest that ENO1 levels can provide a guide and basis for patient survival after surgery.

Discussion
The incidence of hepatocellular carcinoma (HCC) is extremely high in our country and is a serious health risk to patients. Early diagnosis of hepatocellular carcinoma has a tremendous contribution to improving the treatment outcome of patients with hepatocellular carcinoma. At present, the most commonly used serum marker in hepatocellular carcinoma clinics is AFP, which is a histone protein secreted through primitive embryonic tissue, and its ability to be speci cally expressed in the serum of hepatocellular carcinoma patients, thus playing a role in establishing the diagnosis or differential diagnosis. However, some studies have found that there are limitations to the application of AFP in the diagnosis of hepatocellular carcinoma, because serum AFP also abnormally upregulated in patients with cirrhosis, viral hepatitis, reproductive tumors, or metabolic syndrome [20] . In addition, there are false negatives for AFP in the diagnosis of hepatocellular carcinoma, and all of these factors limit the practicality of AFP in the clinical adjunct diagnosis of hepatocellular carcinoma to some extent [21] . Fortunately, other hepatocellular carcinoma diagnostic markers can work synergistically with AFP to improve diagnostic e ciency. For example, abnormal prothrombin (Des-gamma-carboxyprothrombin, DCP) is undetectable in the serum of healthy populations, but is abnormally highly expressed in the serum of patients with hepatocellular carcinoma, which has a sensitivity of 56% for the diagnosis of hepatocellular carcinoma alone, shows greater speci city in the characterization of hepatocellular carcinoma invasion, and is a serum marker for differentiating benign/malignant hepatocellular carcinoma [22] . Golgi Protein 73 (GP73), also upregulated in expression during hepatocarcinogenesis and lowly expressed in hepatocytes of healthy population, has a sensitivity of about 76% for diagnosis alone, and GP73 is able to synergize with AFP to improve the sensitivity and speci city of hepatocellular carcinoma diagnosis [23] . The sensitivity of interleukin IL-6 alone in diagnosing early-stage hepatocellular carcinoma was only 46%, but its serum levels showed a signi cant positive correlation with AFP levels and could complement the AFP diagnosis to some extent [24] . However, the tissue speci city of IL-6 is extremely poor, and its expression can be upregulated in a variety of diseases and multiple organs, thus limiting its diagnostic value, but the idea of nding positive AFP-related markers deserves further in-depth study. Therefore, exploring new HCC serological markers can effectively improve the detection rate and detection sensitivity of early hepatocellular carcinoma.
Α-enolase (α-enolase, EN01) is one of the key enzymes of the glycolytic pathway, which acts as a glycolytic rate-limiting enzyme in the cytoplasm to regulate the metabolism of tumor cells and thus affect their proliferation. It functions as a brinogen receptor on the cell surface, accelerating the degradation of the extracellular matrix and thus affecting tumor cell invasion and migration, and in the nucleus, it acts as a transcriptional repressor through encoded proteins and thus affects tumorigenesis and progression. Therefore, ENO1 is closely related to tumorigenesis and development and is a potential excellent target for studying tumor-related mechanisms. Reports have con rmed that ENO1 is aberrantly expressed in tumor tissues of non-small cell lung cancer [25] , liver cancer [20] , colorectal cancer [26] , and endometrial cancer [26] , and could be a potential diagnostic marker for these tumors. Through preliminary studies and bioinformatic analysis of ENO1-related data in existing databases, we found that the relative expression of both mRNA and protein of ENO1 was signi cantly up-regulated in hepatocellular carcinoma tissues, which, together with the literature, further con rmed its ability to serve as a potential diagnostic marker for hepatocellular carcinoma [27] .
In this study, 28 serum samples of chronic hepatitis B (CHB), 31 serum samples of cirrhosis (LC), 56 serum samples of hepatocellular carcinoma (HCC) and 26 serum samples of healthy population were collected. The levels of ENO1 in the serum of different populations were detected, and the results showed that the expression of ENO1 in HCC was sequentially higher than that in the CHB group, the LC group, and the healthy population. This suggests that serum ENO1 levels are signi cantly associated with liver disease and its speci c high expression in the HCC group indicates its potential as a marker for hepatocellular carcinoma diagnosis. The related ndings are consistent with the existing reports [15][16] . In addition, the expression of ENO1 in the serum of HCC patients was signi cantly higher than its expression in CHB patients and LC patients, indicating that it possesses tissue speci city along with the speci city of HCC disease, avoiding the interference of other liver diseases with the diagnostic results of ENO1, a property that makes ENO1 more potential and valuable than IL-6 in hepatocellular carcinoma. autoantigen to stimulate the body's immune system and induce local in ammation [28] . The expression of ENO1 can be detected in the serum of patients with various autoimmune diseases, such as autoimmune hepatitis, systemic lupus erythematosus, and rheumatoid arthritis [29] . Therefore, it is reasonable to suspect that ENO1 plays a role in the host immune process and lesions caused by hepatitis B virus infection of the host, and the related mechanisms need to be studied in depth. A study by TAKASHIMA [30] con rmed that high expression of ENO1 was closely associated with tumor size and tumor recurrence in hepatocellular carcinoma, and our experiments veri ed this conclusion from ENO1 serum levels. In addition, previous studies have shown that high expression of ENO1 promotes tumor cell proliferation and invasion, accelerates tumor metastasis, and inhibits tumor cell apoptosis [7][8][9][10][11] . This suggests that the ndings of ENO1 at the cellular level are consistent with those at the serum level. The related ndings further suggest that serum ENO1 can be used as a marker for early hepatocellular carcinoma diagnosis and hepatocellular carcinoma recurrence diagnosis.
On the basis that serum ENO1 can be used as a clinical diagnostic marker for hepatocellular carcinoma. We explored the correlation between serum ENO1 levels and AFP levels by bivariate Spearman correlation analysis, and analyzed the sensitivity and speci city of ENO1, AFP and their synergy in the diagnosis of hepatocellular carcinoma by ROC curves. The results showed that serum ENO1 levels showed a signi cant positive correlation with AFP levels; ROC curve analysis showed that the diagnostic sensitivity of ENO1 in this group of HCC patients was 67.3% (speci city was 83.1%). In comparison, the sensitivity and speci city of AFP were 71.2% and 72.9%, respectively. If ENO1 and AFP were used for co-detection, the sensitivity was increased to 74%. The correlation results suggest that ENO1 can be used not only as a diagnostic test for hepatocellular carcinoma in isolation, but also in combination with AFP. Its advantage was higher than molecular markers of hepatocellular carcinoma such as abnormal prothrombin (DCP) and interleukin IL-6, and slightly weaker than phosphatidylinositol proteoglycan 3 (GPC3) and Golgi glycoprotein 73 (GP73). and basis for postoperative survival of patients. This is consistent with the ndings of related reports [31] .
However, we did not nd other relevant reports that studied ENO1 from the serum marker level, so it was not possible to make comparisons and corrections to the available data. However, the relevant data have been able to clarify that ENO1 is a new serological marker for the diagnosis of hepatocellular carcinoma. Further data cohorts and patient sub-groups need to be expanded for more in-depth studies and analyses.
Notably, it can be speculated from our data that ENO1 levels are signi cantly associated with migration, invasion, and progression of hepatocellular carcinoma. The mechanism of ENO1 in hepatocarcinogenesis and progression also possesses research value. Interference with ENO1 was reported to be effective in inhibiting cell proliferation, migration and invasion of human hepatoma cells HepG2 at the cellular level [16] . Some reports found that ENO1 may regulate biological functions such as proliferation and migration of hepatocellular carcinoma cells through Notch signaling pathway. There are also reports showing that ENO1 in tumor cells can be able to be activated by glycolytic enzymes, and thus expression is upregulated to function with Warburg effect, enhancing glucose metabolism in tumor cells, and thus accelerating tumor cell proliferation [31] . However, the speci c mechanism of these speculations has not been elucidated, and further in-depth studies are needed.
In summary, we concluded that ENO1 plays an important role in the development, metastasis, and healing of hepatocellular carcinoma. ENO1 is a new diagnostic serum marker for hepatocellular carcinoma, which plays an important auxiliary diagnostic function in the con rmation, staging and grading, evaluation of metastasis, and healing and recurrence stages of hepatocellular carcinoma. The evaluation of its sensitivity and speci city requires statistical analysis of a larger cohort sample, and the associated mechanisms deserve in-depth study, which is the direction of our further research.

Conclusion
Serum ENO1 levels were signi cantly correlated with the occurrence, progression, metastasis, staging and grading of hepatocellular carcinoma, and postoperative survival of patients. ENO1 may serve as a candidate diagnostic marker for early hepatocellular carcinoma diagnosis and evaluation of hepatocellular carcinoma progression, and is able to perform an auxiliary diagnostic function in combination with AFP.