Biomarker for early diagnosis of liver cancer and application thereof

Abstract

The invention relates to the technical field of biological medicine, and discloses a biomarker for early diagnosis of liver cancer and application of the biomarker, and the biomarker is SERPINI1 and AFP. The liver cancer biomarker can be well used for early diagnosis of liver cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to biomarkers for early diagnosis of liver cancer and applications thereof. Background Art

[0002] In recent years, surgical operations, interventional embolization and chemotherapy have achieved good clinical results in the treatment of liver cancer. However, liver cancer lacks specific symptoms and signs. Many patients are already in the middle and late stages when diagnosed, and have lost the best time for treatment. Therefore, early diagnosis is crucial for its treatment and prognosis.

[0003] Good tumor markers can play a role in high-risk screening, early diagnosis and guiding treatment. Currently, the most commonly used tumor marker for screening and diagnosis of liver cancer patients is alpha-fetoprotein (AFP). When serum AFP ≥ 400 μg / L, after excluding pregnancy, active or chronic liver disease, gonadal embryonic tumors and other digestive system tumors, it is highly suggestive of liver cancer; those with mild elevations in serum AFP can be monitored regularly and compared with liver function changes, or combined with imaging examinations; but there are still reports showing that about 1 / 3 of early liver cancer patients are AFP negative. It can be seen that only by detecting the level of alpha-fetoprotein in the diagnosis of early liver cancer patients may lead to missed diagnosis.

[0004] Therefore, in clinical practice, it is necessary to supplement biomarkers for early diagnosis of liver cancer to improve the sensitivity of liver cancer diagnosis and further improve treatment effects and prognosis. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides biomarkers for early diagnosis of liver cancer and applications thereof. The liver cancer biomarkers of the present invention can be well used for early diagnosis of liver cancer.

[0006] The present invention provides biomarkers for early diagnosis of liver cancer, wherein the biomarkers are SERPINI1 and AFP.

[0007] The present invention also provides a kit for early diagnosis of liver cancer, which comprises reagents for detecting the expression of SERPINI1 and AFP.

[0008] The present invention also provides the use of the reagent for detecting the expression of SERPINI1 and AFP in preparing a tool for diagnosing early liver cancer.

[0009] Furthermore, the application comprises the following steps: S1: Collect samples of the subjects to be tested and control samples; S2: Detect and compare the expression levels of SERPINI1 and AFP in the samples of the test subjects and the control samples; When the expression levels of SERPINI1 and AFP in the sample of the test subject are higher than the expression levels of SERPINI1 and AFP in the control sample, the test subject is diagnosed as having liver cancer or being at risk of having liver cancer.

[0010] Furthermore, the control sample is derived from healthy people or healthy tissues of the test subjects.

[0011] Furthermore, the sample of the subject to be tested is one or more of serum, plasma, whole blood, pus, organ, tumor tissue, circulating tumor cells, circulating tumor DNA or urine exfoliated cells.

[0012] Furthermore, the amino acid sequence of the SERPINI1 is shown in SEQ ID No.1.

[0013] Furthermore, the nucleotide sequence of the AFP protein is edited as shown in SEQ ID No.2.

[0014] The embodiments of the present invention have the following technical effects: In the present invention, it is first discovered that the expression levels of SERPINI1 protein and AFP protein in patients with early liver cancer are significantly higher than those in healthy people. When diagnosing patients with early liver cancer, it is found that simultaneous detection of SERPINI1 protein and AFP protein can improve the sensitivity and specificity of diagnosing early liver cancer, and significantly reduce the missed diagnosis rate of liver cancer when AFP is detected alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 The present invention provides a comparison of the SERPINI1 content between liver cancer patients and healthy controls.

[0017] Figure 2 It is a ROC curve diagram provided by the present invention. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0019] In a first aspect, some embodiments of the present invention provide biomarkers for early diagnosis of liver cancer, wherein the biomarkers are SERPINI1 and AFP.

[0020] In a second aspect, some embodiments of the present invention further provide a kit for early diagnosis of liver cancer, wherein the kit comprises reagents for detecting the expression of SERPINI1 and AFP.

[0021] In a third aspect, some embodiments of the present invention also provide use of the reagent for detecting the expression of SERPINI1 and AFP in the preparation of a tool for diagnosing early liver cancer.

[0022] In some embodiments, the application includes the following steps: S1: Collect samples of the subjects to be tested and control samples; S2: Detect and compare the expression levels of SERPINI1 and AFP in the samples of the test subjects and the control samples; When the expression levels of SERPINI1 and AFP in the sample of the test subject are higher than the expression levels of SERPINI1 and AFP in the control sample, the test subject is diagnosed as having liver cancer or being at risk of having liver cancer.

[0023] In some embodiments, the control sample is derived from healthy people or healthy tissue of the subject to be tested.

[0024] In some embodiments, the sample of the subject to be tested is one or more of serum, plasma, whole blood, pus, organ, tumor tissue, circulating tumor cells, circulating tumor DNA or urine exfoliated cells.

[0025] In some embodiments, the amino acid sequence of SERPINI1 is shown as SEQ ID No.1.

[0026] In some embodiments, the sequence SEQ ID No. 1 is: MAFLGLFSLLVLQSMATGATFPEEAIADLSVNMYNRLRATGEDENILFSPLSIALAMGMMELGAQGSTQKEIRHSMGYDSLKNGEEFSFLKEFSNMVTAKESQYVMKIANSLFVQNGFHVNEEFLQMMKKYFNAAVNHVDFSQNVAVANYINKWVENNTNNLVKDLVSPRDFDAATYLALINAVYFKGNWKSQFRPENTRTF SFTKDDESEVQIPMMYQQGEFYYGEFSDGSNEAGGIYQVLEIPYEGDEISMMLVLSRQEVPLATLEPLVKAQLVEEWANSVKKQKVEVYLPRFTVEQEIDLKDV LKALGITEIFIKDANLTGLSDNKEIFLSKAIHKSFLEVNEEGSEAAAVSGMIAISRMAVLYPQVIVDHPFFFLIRNRRTGTILFMGRVMHPETMNTSGHDFEEL.

[0027] In some embodiments, the nucleotide sequence of the AFP protein is edited as shown in SEQ ID No.2.

[0028]

[0029] The following is described in conjunction with specific embodiments: Embodiment 1: 1. Screening of serum markers: By screening differentially expressed genes in the two datasets GSE101728 and GSE84005 in the Gene Expression Omnibus (GEO) database, we found that SERPINI1 was upregulated in the serum of patients with liver cancer. We further verified in the GEPIA database that SERPINI1 was upregulated in the tissues of patients with liver cancer. We found in the UniProt protein library that SERPINI1 was a secreted protein.

[0030] 2. Detection of SERPINI1 protein content in the blood of patients with early liver cancer: (1) Whole blood was collected from 138 healthy controls and 153 liver cancer patients using procoagulant tubes. The blood was centrifuged at 3000 rpm for 5 minutes and the supernatant was aspirated for later use.

[0031] (2) The SERPINI1 protein level in the serum of healthy controls and HCC patients was detected using an ELISA kit for SERPINI1 (Cloud-Clone Corp.); The specific method includes the following steps: taking 100 μL of serum samples of liver cancer patients, standard products and blank controls, respectively, and adding them to a 96-well plate, incubating at 37°C for 1 hour; after the incubation, aspirating the liquid in the well, adding 100 μL of assay diluent to each well, and incubating at 37°C for 1 hour; after the incubation, aspirating the liquid in the well, washing 3 times with PBST (phosphate buffer solution), adding 100 μL of enzyme-labeled antibody to each well, and incubating at 37°C for 30 minutes; after the incubation, aspirating the liquid in the well, washing 5 times with PBST, adding 90 μL of substrate buffer to each well, and incubating at 37°C for 10-20 minutes; after the incubation, adding 50 μL of stop solution to each well. The absorbance value of each well at 450nm was detected by an enzyme reader, and the protein content of SERPINI1 was calculated.

[0032] (3) The Roche electrochemical immunoluminescence method was used to detect the AFP content in the serum of 138 healthy controls and 153 liver cancer patients.

[0033] (4) Statistical analysis of SERPINI1 detection results Comparison of SERPINI1 levels between patients with liver cancer and healthy controls ( Figure 1 ), the correlation analysis between SERPINI1 and clinical related indicators was performed, and the ROC curve of SERPINI1 and AFP was drawn ( Figure 2 ).

[0034] exist Figure 1 It was found that the expression level of SERPINI1 in the serum of liver cancer patients was significantly higher than that in healthy controls, confirming that the expression level of SERPINI1 would increase in liver cancer patients.

[0035] The ROC curve was used to analyze the independent diagnosis and combined diagnosis test results of SERPINI1 protein and AFP protein. Figure 2 As shown, SERPINI1 and AFP have good diagnostic effects on liver cancer, but the combined diagnostic effect is the best.

[0036] From this result, it can be seen that the detection of SERPINI1 protein or AFP protein content alone has a certain diagnostic effect on liver cancer, but the diagnostic specificity and sensitivity are still insufficient, and the combination of the two can achieve excellent specificity and sensitivity. Therefore, SERPINI1 and AFP can be used to diagnose liver cancer together. The specific results are shown in Table 1.

[0037] Table 1 Significance of detection of SERPINI1 protein and AFP protein in patients with liver cancer

[0038] <0.01 compared with SERPINI1+AFP group In Table 1, it can be seen that when the SERPINI1 protein and AFP protein content are detected separately, it is found that the sensitivity, specificity and AUC value of the detection are significantly lower than the combined use of SERPINI1 protein and AFP protein to diagnose liver cancer. And in the present invention, when SERPINI1 protein and AFP protein are used in combination to diagnose liver cancer, the specificity and AUC value are significantly higher than 0.9. When the specificity and AUC value of the detection are, the detection method has a higher accuracy. It should be known to those skilled in the art that although there are many biomarkers for cancer, it is not that any biological indicator can be combined for detection, and the specificity and AUC value of the detection can be higher than 0.9. Therefore, in the present invention, it is found that the selected SERPINI1 protein and AFP protein can be used to jointly diagnose liver cancer.

[0039] 3. Correlation analysis between serum SERPINI1 protein levels and clinical parameters of liver cancer patients: Serum SERPINI1 levels were correlated with case information of liver cancer patients. The specific correlation is shown in Table 2.

[0040] Table 2 Correlation between serum SERPINI1 levels and clinical parameters in 153 patients with liver cancer

[0041] Statistical analysis of the data in Table 2 revealed that the expression of SERPINI1 protein was significantly correlated with tumor size, differentiation grade, vascular invasion, lymph node metastasis, and tumor stage in patients with liver cancer. This also further verified that the expression of SERPINI1 would increase in patients with liver cancer.

[0042] In summary, the SERPINI1 protein in the present invention can be used as a biomarker for liver cancer, and can be used in combination with the AFP protein to improve the sensitivity and specificity of detection.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the technical solutions of the embodiments of the present invention.

Claims

1. A biomarker for early diagnosis of liver cancer, characterized in that: The biomarkers are SERPINI1 and AFP.

2. A kit for early diagnosis of liver cancer, characterized in that: The kit comprises reagents for detecting the expression of SERPINI1 and AFP.

3. Use of reagents for detecting the expression of SERPINI1 and AFP in the preparation of tools for the diagnosis of early liver cancer.

4. The use according to claim 3, characterized in that: The following steps are involved: S1: Collect samples of the subjects to be tested and control samples; S2: Detect and compare the expression levels of SERPINI1 and AFP in the samples of the test subjects and the control samples; When the expression levels of SERPINI1 and AFP in the sample of the test subject are higher than the expression levels of SERPINI1 and AFP in the control sample, the test subject is diagnosed as having liver cancer or being at risk of having liver cancer.

5. The use according to claim 4, characterized in that: The control sample is derived from healthy people or healthy tissues of the test subject.

6. The use according to claim 4, characterized in that: The sample of the subject to be tested is one or more of serum, plasma, whole blood, pus, organ, tumor tissue, circulating tumor cells, circulating tumor DNA or urine exfoliated cells.

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