Application of sNINJ1 as a serum marker in the diagnosis, prognosis, efficacy evaluation, and treatment of liver cancer

Abstract

The present invention belongs to the field of tumor detection technology, and specifically discloses the use of sNINJ1 as a serum marker in the diagnosis, prognosis, efficacy evaluation, and treatment of liver cancer. The present invention found that sNINJ1 was significantly highly expressed in the serum of liver cancer patients. Based on this, the use of sNINJ1 as a serum marker in the preparation of a kit for liver cancer diagnosis, prognosis, and efficacy evaluation was proposed. The present invention provides a new biomarker with high sensitivity and specificity for the diagnosis, prognosis, and efficacy evaluation of liver cancer, which is more suitable for clinical promotion and application. At the same time, the present invention also proposes that a new liver cancer targeted drug can be developed with sNINJ1 and its related pathways as targets, providing new ideas for the treatment of liver cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor detection, and in particular to the application of sNINJ1 as a serum marker in the diagnosis, prognosis, efficacy evaluation and treatment of liver cancer. Background Art

[0002] Currently, commonly used clinical methods for diagnosing liver cancer include pathological examination, imaging, and serological marker testing. Pathological examination is the gold standard for diagnosing liver cancer, but it is invasive and therefore not widely applicable. Abdominal ultrasound, due to its flexibility, noninvasiveness, and ease of use, is recommended by many domestic and international guidelines as a screening method for liver cancer. However, ultrasound examinations are often subject to subjective factors such as the thickness of the patient's subcutaneous fat and the operator's experience, technique, and meticulousness in diagnosing early-stage liver cancer. For patients who test positive for ultrasound combined with alpha-fetoprotein (AFP) screening, dynamic contrast-enhanced CT and multimodal MRI can be used to confirm the diagnosis. However, both CT and MRI have disadvantages such as lengthy examinations and high costs. CT also carries the risk of contrast agent nephrotoxicity and radiation damage. Another technique discloses a method for diagnosing liver cancer, which discovered that the NINJ1 protein is highly expressed in liver cancer and is associated with its development and metastasis. Based on this finding, the NINJ1 gene has been proposed for diagnosis and treatment of liver cancer, as well as NINJ1 agonists. However, this technique detects a gene sequence as its target, and detects the full-length NINJ1 gene. Since protein levels are regulated at multiple levels, including gene transcription, post-transcription, translation, and post-translation, high gene expression in tissues does not necessarily lead to an increase in protein content. Therefore, diagnosing liver cancer by detecting the NINJ1 gene in liver tissue lacks certain accuracy. At the same time, this technology detects the presence of target substances in liver tissue. Since components in the blood are affected by cell release and stability, there is no necessary connection between the levels in tissue contents and the levels in the blood. In addition, compared with blood samples, liver tissue sampling is more difficult and the detection cost is also higher.

[0003] Compared to the aforementioned technical methods, serum tumor marker testing is a more commonly used method for tumor diagnosis. It has considerable clinical value in diagnosing malignant tumors, observing treatment efficacy, assessing disease progression, and monitoring recurrence. It offers advantages such as simplicity and non-invasiveness, quantitative and objective results, repeatability for dynamic monitoring, and relatively low cost. Currently, serological markers such as alpha-fetoprotein (AFP), AFP variants, abnormal prothrombin, and ferritin are primarily used in the diagnosis and treatment evaluation of liver cancer.

[0004] Although serological diagnosis of liver cancer has become relatively mature, the biomarkers currently used in clinical practice still have certain limitations, with certain false positive and false negative rates, which cannot meet the needs of precise diagnosis and treatment. For example, AFP, a classic marker for diagnosing liver cancer, is also susceptible to active liver disease, pregnancy, and gonadal embryonal tumors and digestive tract tumors, which has certain limitations for early diagnosis of liver cancer. Based on this, the development of a new serum marker with high sensitivity, good specificity, suitable for early diagnosis of liver cancer, and more suitable for clinical promotion and application will further help improve the diagnosis and treatment of liver cancer in my country. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of the present invention is to provide the application of sNINJ1 as a serum marker in the diagnosis, prognosis, efficacy evaluation and treatment of liver cancer, providing a new and more reliable technical means for the diagnosis, prognosis and efficacy evaluation of liver cancer.

[0006] To achieve the above-mentioned and other related purposes, the present invention provides the use of sNINJ1 as a serum marker in the preparation of a kit for liver cancer diagnosis, prognosis and efficacy evaluation.

[0007] Furthermore, the expression level of sNINJ1 was positively correlated with the severity of HCC.

[0008] Furthermore, the expression level of sNINJ1 protein was positively correlated with the severity of HCC.

[0009] Furthermore, the kit uses serum / blood as a test sample.

[0010] The present invention found that sNINJ1 is significantly overexpressed in the serum of liver cancer patients. By detecting its serum content, it can provide a new and more reliable basis for the diagnosis, prognosis and efficacy evaluation of liver cancer. The polypeptide sequence of sNINJ1 is:

[0011] MDSGTEEYELNGGLPPGTPGSPDASPARWGWRHGPINVNHYASKKSAAESMLDIAL.

[0012] Furthermore, the kit measures the expression level of sNINJ1 by ELISA method.

[0013] Furthermore, the kit also includes a mass spectrometry identification reagent, and / or an antibody or an antigen-binding fragment thereof; the mass spectrometry identification reagent, antibody or an antigen-binding fragment thereof is associated with sNINJ detection, and / or with liver cancer diagnosis, prognosis and efficacy evaluation.

[0014] The present invention also provides the use of sNINJ1 as a serum marker in combination with other liver cancer biomarkers in the preparation of a kit for liver cancer diagnosis, prognosis and efficacy evaluation.

[0015] Furthermore, the other liver cancer biomarkers are selected from at least one of AFP, alpha-fetoprotein isoforms, abnormal prothrombin, and ferritin.

[0016] The present invention also provides a kit for liver cancer diagnosis, prognosis and efficacy evaluation, and the kit includes a reagent for detecting the serum marker sNINJ1.

[0017] Furthermore, the kit includes a reagent for detecting the serum marker sNINJ1 protein.

[0018] Furthermore, the kit also includes reagents for detecting other liver cancer biomarkers.

[0019] The present invention also provides the use of sNINJ1 in the preparation and / or screening of liver cancer targeted drugs, wherein the liver cancer targeted drugs use sNINJ1 and its related pathways as targets.

[0020] Furthermore, the liver cancer targeted drug targets the sNINJ1 protein and its related pathways.

[0021] The present invention also provides a liver cancer targeted drug, which targets sNINJ1 and its related pathways.

[0022] As described above, the use of sNINJ1 as a serum marker in the diagnosis, prognosis, efficacy evaluation, and treatment of liver cancer has the following beneficial effects:

[0023] This invention provides a new serum marker, sNINJ1, for the diagnosis and treatment of liver cancer. It can serve as a target molecule for liver cancer diagnostic reagents and be used in early diagnosis, efficacy observation, and prognosis assessment of liver cancer patients. Compared with existing technologies, it is more suitable for clinical promotion and application and is expected to improve the diagnosis and treatment of liver cancer in my country. Furthermore, this invention proposes the development of a new liver cancer-targeted drug targeting sNINJ1 and its related pathways, providing new insights into liver cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Shown is a schematic diagram of the expression difference of sNINJ1 content in the serum of subjects with different diseases and normal controls in the examples of this application;

[0025] Figure 2 Shown is a schematic diagram of the correlation analysis between sNINJ1 and AFP, abnormal prothrombin (PIVKAII), LDH, 5'NT and VEGF in the serum of liver cancer patients in the examples of this application;

[0026] Figure 3 Shown is a schematic diagram of the difference in sNINJ1 expression in the serum of liver cancer patients before and after treatment in the embodiment of the present application;

[0027] Figure 4 Shown is a schematic diagram of the difference in sNINJ1 expression in the serum of patients with large tumor volume and patients with small tumor volume in the examples of this application;

[0028] Figure 5 Shown is a schematic diagram of the difference in sNINJ1 expression in the serum of patients with liver cancer that has metastasized and patients with liver cancer that has not metastasized in the examples of this application;

[0029] Figure 6 Shown is a schematic diagram of the difference in sNINJ1 expression in the serum of liver cancer patients with different CNLC stages in the examples of this application;

[0030] Figure 7 Shown is the ROC curve of serum sNINJ1 and AFP for single diagnosis and combined diagnosis of liver cancer patients in the embodiment of the present application;

[0031] Figure 8 Shown is a schematic diagram of the expression of NINJ1 in liver cancer and normal tissues analyzed by GEPIA database in the examples of this application.

[0032] Figure 9 Shown is a schematic diagram of the difference in overall survival rate of liver cancer in the NINJ1 high expression group and low expression group analyzed by the GEPIA database in the examples of this application. DETAILED DESCRIPTION

[0033] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0034] To overcome the deficiencies of the above-mentioned prior art, the present invention provides a serum marker, soluble nerve injury-induced protein 1 (sNINJ1), which is more conducive to the diagnosis, prognosis and efficacy evaluation of liver cancer.

[0035] Nerve injury-induced protein 1 (NINJ1) is a novel adhesion molecule that is ubiquitously expressed in the damaged peripheral nervous system and is present in various tissues throughout the body, including embryonic and adult epithelial tissues such as the liver, kidney, thymus, lymphocytes, leukocytes, and the surface of vascular endothelial cells. It plays a crucial role in tissue development and neural repair. The NINJ1 protein has two hydrophobic transmembrane domains and an extracellular NH2-terminal (ENT) domain. This extracellular NH2-terminal region can be cleaved by matrix metalloproteinase 9 (MMP9) and released into the bloodstream, forming soluble NINJ1 (sNINJ1). sNINJ1 mediates cell-cell adhesion in vitro through homologous binding.

[0036] The present invention found through analysis that the sNINJ1 content in serum samples of non-liver cancer patients is low, but the sNINJ1 content in the serum of liver cancer patients is significantly increased. Therefore, detecting the sNINJ1 content level in serum samples can be used for the diagnosis, prognosis and efficacy evaluation of liver cancer.

[0037] Furthermore, the present invention utilizes the characteristic of high expression of sNINJ1 in the serum of liver cancer patients. Subsequently, based on the specific expression level of sNINJ1, the relationship between sNINJ1 and the patient's liver cancer stage, metastasis, and prognosis survival was explored. By combining specific clinical data, follow-up, and mathematical model establishment, a liver cancer prognosis model based on sNINJ1 was constructed, providing a new direction for the treatment and prognosis evaluation of liver cancer patients.

[0038] It should be noted that the target substance detected by the present invention is sNINJ1 protein, and its polypeptide sequence is:

[0039] MDSGTEEYELNGGLPPGTPGSPDASPARWGWRHGPINVNHYASKKSAAESMLDIAL.

[0040] While prior art has shown that the NINJ1 gene is highly expressed in liver cancer tissue, it's important to note that sNINJ1 is a soluble form of NINJ1, a truncated form produced by intracellular enzymatic cleavage that can be released into the blood, whereas NINJ1 itself is not released into the blood. Therefore, the detection of sNINJ1 protein in blood is fundamentally different from the detection of NINJ1 gene in tissue.

[0041] To better illustrate the purpose, technical solutions, and advantages of the present invention, the present invention will be further described below with reference to the accompanying drawings and specific embodiments. The embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making creative efforts are within the scope of protection of the present invention.

[0042] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0043] Example 1

[0044] 1. Detection method

[0045] In this example, the sNINJ1 content in the serum of liver cancer patients and normal healthy subjects was detected by ELISA.

[0046] 1. The clinical samples in this example are peripheral blood (102 newly diagnosed primary liver cancer patients, 41 newly diagnosed lung cancer patients, 40 newly diagnosed colorectal cancer patients, 33 systemic lupus erythematosus patients, 28 rheumatoid arthritis patients, and 67 healthy subjects). Relevant samples should be collected clinically. The procedure for collecting peripheral blood samples is as follows:

[0047] 2.5-3.5 mL of fasting venous blood from the research subjects was placed in a coagulant tube, allowed to stand vertically at room temperature for 25-35 minutes, and then centrifuged at 2500-3500 rpm for 5 minutes at room temperature. The upper layer of liquid was aspirated into a labeled EP tube and stored at -80°C for later use.

[0048] 2. ELISA test steps

[0049] After equilibration at room temperature for 10 minutes, remove the required strips from the ELISA kit, seal the remaining strips with a ziplock bag and return them to 4°C; add 100ul of sample or standard of different concentrations to the corresponding wells, add 100ul of universal diluent to the blank wells, cover with a sealing film and incubate at 37°C for 1 hour; remove the ELISA plate, discard the liquid, do not wash, directly add 100ul of biotinylated antibody working solution to each well, cover with a sealing film and incubate at 37°C for 1 hour; discard the liquid, and add 300ul of biotinylated antibody working solution to each well. 1x washing solution, let it stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat the washing process 3 times; add 100ul of enzyme conjugate working solution to each well, cover with sealing film and incubate at 37℃ for 30 minutes; discard the liquid and wash according to the washing method in step 4, and wash the plate 5 times; add 90ul of substrate (TMB) to each well, cover with sealing film, and incubate at 37℃ in the dark for 15 minutes; remove the ELISA plate, directly add 50ul of stop solution to each well, immediately measure the OD value of each well at a wavelength of 450nm, and calculate the concentration of sNINJ1 in each group of serum according to the standard curve.

[0050] 3. Statistical analysis of serum sNINJ1 detection results

[0051] The serum sNINJ1 levels in patients with different diseases were compared with those in healthy subjects. The correlation between sNINJ1 and clinical related indicators was analyzed, and the ROC curve of sNINJ1 was drawn. The serum sNINJ1 levels in patients with liver cancer of different tumor areas were compared and analyzed.

[0052] 4. Bioinformatics analysis of the database

[0053] The GEPIA database was used to analyze the expression of NINJ1 in liver cancer tissue samples and normal tissue samples, and the relationship between NINJ1 high expression group, low expression group and overall survival rate of liver cancer was analyzed.

[0054] 2. Experimental Results and Analysis

[0055] The experimental results obtained by the above detection and data analysis methods are as follows Figures 1 to 9 Based on Figures 1 to 9 , we can conclude that:

[0056] 1. Figure 1 The diagram shows the expression difference of sNINJ1 in serum of patients with different diseases and normal controls. Figure 1 It can be seen that the expression level of serum sNINJ1 in patients with liver cancer is significantly higher than that in healthy controls; serum sNINJ1 is highly expressed in patients with liver cancer (HCC), and its expression level is higher than that in patients with lung cancer (LCA), colorectal cancer (CRCA), systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA).

[0057] 2. Figure 2 The figure shows the correlation analysis between sNINJ1 and AFP, abnormal prothrombin (PIVKAII), LDH, 5'NT and VEGF in the serum of patients with liver cancer. Figure 2 It can be seen that the serum sNINJ1 level in patients with liver cancer is significantly correlated with alpha-fetoprotein, abnormal prothrombin (PIVKAII), LDH, 5'NT and VEGF.

[0058] 3. Figure 3 Schematic diagram showing the difference in sNINJ1 expression in serum of HCC patients before and after treatment. Figure 3 It can be seen that the serum sNINJ1 level in patients with liver cancer after TACE (left figure) and partial hepatectomy was significantly lower than that before surgery.

[0059] 4. Figure 4 Schematic diagram showing the difference in sNINJ1 expression in the serum of patients with large tumor volume and patients with small tumor volume. Figure 4 It can be seen that the serum sNINJ1 level in liver cancer patients with large tumor area is significantly higher than that in liver cancer patients with small tumor area.

[0060] 5. Figure 5 Schematic diagram showing the difference in sNINJ1 expression in the serum of patients with metastatic liver cancer and those without metastasis. Figure 5 It can be seen that the serum sNINJ1 level in patients with metastatic liver cancer is significantly higher than that in patients with non-metastatic liver cancer.

[0061] 6. Figure 6 Schematic diagram showing the difference in sNINJ1 expression in the serum of HCC patients with different CNLC stages. Figure 6 It can be seen that the serum sNINJ1 level of liver cancer patients with CNLC stage III and IV was significantly higher than that of liver cancer patients with CNLC stage I and II.

[0062] 7. Figure 7 Shown is the ROC curve of serum sNINJ1 and AFP for the diagnosis of liver cancer patients alone and in combination. Figure 7 It can be seen that serum sNINJ1 has a strong diagnostic efficacy for liver cancer, and its combination with AFP can further enhance the diagnosis of liver cancer.

[0063] 8. Figure 8 Shown is a schematic diagram of NINJ1 expression in liver cancer and normal tissues analyzed by GEPIA database. Figure 9 The figure shows the difference in overall survival rate between the NINJ1 high expression group and the low expression group of liver cancer analyzed by GEPIA database. Figure 8 and Figure 9 It can be seen that the higher the serum sNINJ1 expression level, the lower the overall survival rate of liver cancer patients.

[0064] Therefore, serum sNINJ1 is a new biomarker with high sensitivity and specificity for diagnosing liver cancer and assessing its prognosis and therapeutic efficacy. Furthermore, combined detection of serum sNINJ1 with existing biomarkers can further improve the sensitivity and specificity of liver cancer diagnosis and assess the therapeutic efficacy and clinical prognosis of liver cancer patients. Furthermore, targeting sNINJ1 and related pathways may also provide new insights into the development of targeted liver cancer drugs.

[0065] In summary, the present invention provides a new serum marker for liver cancer, sNINJ1, which can achieve a simpler, more accurate and rapid diagnosis of liver cancer, as well as the prognosis and treatment effect evaluation of liver cancer patients. Its promotion and application in clinical practice is expected to improve the diagnosis and treatment level of liver cancer in my country.

[0066] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. Use of a reagent for detecting serum markers in the preparation of a kit for liver cancer diagnosis, prognosis, and efficacy evaluation, characterized in that: The serum markers include sNINJ1; The kit uses serum / blood as the test sample; The expression level of sNINJ1 protein is positively correlated with the severity of liver cancer; The liver cancer diagnosis is used to differentiate liver cancer from lung cancer, colorectal cancer, systemic lupus erythematosus, and rheumatoid arthritis.

2. The use according to claim 1, characterized in that: The kit measures the expression level of sNINJ1 by ELISA method.

3. The use according to claim 1, characterized in that: The serum markers further include at least one of AFP, alpha-fetoprotein isoforms, abnormal prothrombin, and ferritin.

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