Application of ubald2 as a marker in hepatocellular carcinoma diagnostic product

Abstract

The application belongs to the technical field of biomarker diagnostic products, and relates to application of UBALD2 as a marker in a hepatocellular carcinoma diagnostic product, and application of a substance for detecting UBALD2 expression level in an in-vitro detection reagent, a detection kit and / or an in-vitro prognosis evaluation product. The application proves that UBALD2 is highly expressed in hepatocellular carcinoma tissues from two aspects of protein level and mRNA level, and indicates that UBALD2 can be used as one of molecular indexes reflecting the occurrence and development state of hepatocellular carcinoma. The application proposes that high expression of UBALD2 is related to poor overall survival of patients, and is related to a later T stage, and indicates that the substance for detecting UBALD2 can be used for prognosis risk evaluation, prognosis stratification and clinical auxiliary judgment products of hepatocellular carcinoma patients, and provides a reference basis for individualized management of patients.

Description

Technical Field

[0001] This invention belongs to the technical field of biomarker diagnostic products, and relates to the application of UBALD2 as a prognostic assessment / risk stratification marker for hepatocellular carcinoma, as well as the application of substances used to detect UBALD2 expression levels in in vitro detection reagents, detection kits and / or in vitro prognostic assessment products. Background Technology

[0002] Hepatocellular carcinoma is one of the most common malignant tumors, characterized by its complex development and significant intratumoral and intertumoral heterogeneity. Even after surgical resection, ablation, or systemic therapy, there are still significant differences in recurrence, metastasis, and overall survival among patients. Clinically, there is an urgent need for more refined in vitro risk stratification and prognostic assessment products to guide follow-up management and treatment strategy development.

[0003] Current prognostic assessments for hepatocellular carcinoma (HCC) primarily rely on clinicopathological indicators such as TNM staging, BCLC staging, and pathological grading. However, significant differences in outcomes among patients with the same stage / grade are common in clinical practice, suggesting that relying solely on traditional staging and grading methods is insufficient to fully reflect tumor biological behavior and individualized prognostic risk.

[0004] Serological markers and some histological biomarkers can be used for auxiliary assessment of hepatocellular carcinoma, but they still suffer from insufficient sensitivity and specificity, limited applicability to different subtypes, and unstable ability to stratify recurrence / metastasis risk. Furthermore, molecular subtyping or prognostic models based on multi-gene expression profiles still face obstacles in standardization, cost, consistency of detection platforms, and compatibility with conventional FFPE samples. Therefore, there is an urgent need to provide a molecular biomarker and its in vitro detection product and assessment method that can be stably detected in patient-derived tissue samples for prognostic risk stratification to meet the clinical management needs of hepatocellular carcinoma and related tumors. Summary of the Invention

[0005] This invention addresses the problems existing in the traditional hepatocellular carcinoma prognostic assessment process by proposing the application of substances for detecting UBALD2 expression levels in in vitro detection reagents, detection kits, and / or in vitro prognostic assessment products.

[0006] To achieve the above objectives, the present invention is implemented using the following technical solution: This invention proposes the application of substances for detecting the expression levels of the UBALD2 gene and / or UBALD2 protein in liver cancer diagnostic reagents or kits.

[0007] This invention also proposes the application of substances that detect the expression levels of the UBALD2 gene and / or UBALD2 protein in reagents or kits for detecting the proliferation, migration and invasion abilities, and apoptosis levels of liver cancer cells.

[0008] This invention provides a liver cancer diagnostic kit, comprising primer pairs for detecting UBALD2 and internal control primer pairs, the sequences of which are as follows: UBALD2 positive: 5'-CCACCACCACCAGATGATGT-3'; UBALD2 reverse: 5'-CTGAAGTTTGCAGGTGGGGA-3'; GAPDH positive: 5'-CCAGGGCTGCTTTTAACTCT-3'; GAPDH reverse: 5'-GGACTCCACGACGTACTCA-3'.

[0009] In hepatocellular carcinoma (HCC) tissues, the immunohistochemical staining level of UVALD2 was significantly higher than that of adjacent normal tissues. UVALD2 expression levels can stratify the overall survival of HCC patients, with the high-expression group showing significantly worse overall survival. High UVALD2 expression is correlated with later T stages (T3 / T4), suggesting its association with tumor progression. Furthermore, in vitro functional experiments showed that intervention in UVALD2 expression can significantly affect the proliferation, migration, and apoptosis levels of HCC cells. In vivo tumorigenesis experiments showed that intervention in UVALD2 expression can significantly affect the growth rate and endpoint tumor volume / weight of xenografts in nude mice. Therefore, UVALD2 has application value as a prognostic stratification marker and a potential intervention target.

[0010] Compared with the prior art, the advantages and positive effects of the present invention are as follows: 1. This invention confirms that UBALD2 is highly expressed in hepatocellular carcinoma tissues at both the protein and mRNA levels, indicating that UBALD2 can serve as one of the molecular indicators reflecting the development and progression of hepatocellular carcinoma.

[0011] 2. This invention proposes that high expression of UBALD2 is associated with poor overall patient survival and shows a correlation trend with later T stage, suggesting that substances that detect UBALD2 can be used for prognostic risk assessment, prognostic stratification, and clinical auxiliary judgment products for hepatocellular carcinoma patients, providing a reference for individualized patient management. Attached Figure Description

[0012] Figure 1 The image shows the detection results of elevated UBALD2 expression levels in liver cancer tissues. In the image, A shows the immunohistochemical staining scores of UBALD2 in liver cancer tissues and paired adjacent normal tissues; B shows the results of differential mRNA expression of UBALD2 between liver cancer tissues and normal liver tissues based on the analysis of the TCGA and GTEx databases.

[0013] Figure 2The graph shows the results of the analysis of the correlation between UBALD2 expression and prognosis in patients with liver cancer. In the graph, A is the overall survival curve of the UBALD2 high expression group and the low expression group in the included patient cohort; B is the overall survival curve of the UBALD2 high expression group and the low expression group based on samples from public databases.

[0014] Figure 3 The figures show the results of the construction and validation of the UBALD2 overexpression cell model. In the figure, A is the result of qRT-PCR detection of UBALD2 mRNA expression level in HCCLM3 cells and SNU449 cells; B is the result of Western blot detection of UBALD2 protein expression level in HCCLM3 cells and SNU449 cells.

[0015] Figure 4 The figure shows the experimental results of the effect of UBALD2 overexpression on the clonogenic ability of liver cancer cells.

[0016] Figure 5 The figure shows the experimental results of the effect of UBALD2 overexpression on the migration ability of liver cancer cells.

[0017] Figure 6 The figure shows the experimental results of the effect of UBALD2 overexpression on the apoptosis level of liver cancer cells.

[0018] Figure 7 The figure shows the experimental results of the effect of UBALD2 overexpression on the tumorigenicity of liver cancer cells in vivo. Detailed Implementation

[0019] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described below with reference to specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0020] Numerous specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways than those described herein, and therefore the invention is not limited to the specific embodiments disclosed in the following specification. Example 1

[0021] I. Analysis of the Expression Characteristics and Clinical Significance of UBALD2 in Hepatocellular Carcinoma To assess the expression level of UVALD2 in hepatocellular carcinoma (HCC) and its clinical significance, immunohistochemical staining was performed using a HCC tissue microarray (HLivH180Su30) purchased from Shanghai ChipSuper Biotechnology Co., Ltd. Samples with tissue loss after staining were excluded, and 54 paired HCC and adjacent normal tissue samples were ultimately included for analysis. Patient clinicopathological data, including gender, age, T stage, and history of cirrhosis, were collected and analyzed. Furthermore, to further validate the expression characteristics of UVALD2 in HCC and its correlation with patient prognosis, a combined analysis was performed on 371 HCC samples from the TCGA database and 160 normal liver tissue samples from the GTEx database.

[0022] 1. Immunohistochemical detection (1) Reagents and materials: UBALD2 antibody: Thermo Fisher Scientific, PA5-54676.

[0023] Antibody diluent: Subcoin, BMU103.

[0024] Universal histochemistry kit (containing endogenous peroxidase inhibitor and enzyme-labeled goat anti-mouse / rabbit IgG polymer): Zhongshan Jinqiao, PV-6000.

[0025] Sodium citrate antigen repair solution: Bosideng, AR0024.

[0026] DAB colorimetric reagent kit: Zhongshan Jinqiao, ZLI-9017.

[0027] Sappanin: Biyuntian, C0107.

[0028] Hydrochloric acid ethanol rapid differentiation solution: Beyotime, C0163.

[0029] Neutral resin: Beyotime, C0173.

[0030] Histology pen: White Shark Biology, BC003.

[0031] PBS buffer: White Shark Biotechnology, BL601A.

[0032] In the embodiments, unless otherwise specified, the concentration of a substance is a mass fraction.

[0033] (2) The specific steps are as follows: The tissue chip was baked in a 65℃ constant temperature oven for 2 h; then it was soaked in xylene I (250 mL) and xylene II (250 mL) for 30 min each for dewaxing; after dewaxing, it was sequentially hydrated in anhydrous ethanol solution I (250 mL), anhydrous ethanol solution II (250 mL), 95% ethanol solution (250 mL, V / V, the same below), 85% ethanol solution (250 mL) and 75% ethanol solution (250 mL), and then soaked in PBS buffer (250 mL) 3 times, 3 min each time. Then the tissue chip was placed in sodium citrate antigen retrieval solution (300 mL), and antigen retrieval was performed by microwave heating. After heating on high for 8 min, the temperature was turned to medium-low for 25 min. After naturally cooling to room temperature, it was soaked in PBS buffer (250 mL) 3 times, 3 min each time. The tissue region was then delineated using a histochemical pen, and 200 μL of endogenous peroxidase inhibitor was added. The mixture was incubated at room temperature in the dark for 15 min. After incubation, the tissue was immersed three times in PBS buffer, 250 mL of fresh buffer each time, for 3 min each time. UBALD2 antibody was diluted 1:50 (v / v) with antibody dilution buffer, and 200 μL was added to the tissue region. The tissue was then incubated in a humidified chamber at 4°C for 12 h. The next day, after returning to room temperature, 200 μL of enzyme-labeled goat anti-mouse / rabbit IgG polymer was added, and the mixture was incubated at room temperature for 30 min. DAB working solution (200 μL) was then prepared according to the DAB kit instructions for color development, with a development time of 2 min. After color development, 200 μL of hematoxylin was added to counterstain the cell nuclei for 5 min, followed by differentiation with 250 mL of hydrochloric acid-ethanol rapid differentiation buffer for 5 s. The cells were then rinsed under running water for 10 min to regain their blue color. Finally, the slides were mounted with neutral resin. After the mounting medium has cured, it is scanned and analyzed using a digital slide scanner (3DHISTECH CaseViewer), and the staining results are independently scored by two pathologists.

[0034] 2. Immunohistochemical scoring criteria The UBALD2 immunohistochemical staining results were evaluated using a semi-quantitative scoring method. The score consisted of two parts: a positive cell percentage score and a staining intensity score. Positive cell percentage ≤10% was scored as 1 point, 11%–40% as 2 points, 41%–70% as 3 points, and >70% as 4 points. Staining intensity was as follows: no positive staining was scored as 0 points, pale yellow as 1 point, brownish-yellow as 2 points, and brownish-red as 3 points. The final score was the product of the positive cell percentage score and the staining intensity score. Based on the scoring results, patients were divided into a UBALD2 high-expression group and a low-expression group, with a score ≥6 defined as high expression.

[0035] 3. UBALD2 is highly expressed in hepatocellular carcinoma tissues. Immunohistochemical staining results showed that, compared with paired adjacent normal tissues, UBALD2 protein expression was significantly increased in liver cancer tissues, and the immunohistochemical score was significantly increased. Figure 1 A, P <0.05 indicates that UBALD2 is highly expressed in hepatocellular carcinoma (HCC) tissues. To further validate these results at the transcriptional level, 371 HCC samples from the TCGA database and 160 normal liver tissue samples from the GTEx database were analyzed using the GEPIA platform. The results showed that the mRNA expression level of UBALD2 in HCC tissues was significantly higher than that in normal liver tissues (<0.05), indicating that UBALD2 was highly expressed in HCC tissues. Figure 1 B, p<0.01). The above results demonstrate, from both the protein and mRNA levels, that UBALD2 is abnormally highly expressed in liver cancer.

[0036] 4. High expression of UBALD2 is associated with poor prognosis in patients with liver cancer. After confirming the high expression of UFALD2 in liver cancer tissues, its relationship with patient prognosis was further analyzed. Kaplan-Meier survival analysis showed that in the patient cohort included in this example, the overall survival rate of patients in the high UFALD2 expression group was lower than that of the low expression group, and the Log-rank test indicated a statistically significant difference. Figure 2 A, P <0.05). Further survival analysis using samples from public databases also showed that patients in the UBALD2 high-expression group had poorer overall survival (…). Figure 2 (B) Among them, the median overall survival of patients in the UFALD2 high expression group was 45.08 months, while that in the low expression group was 70.01 months, with a hazard ratio (HR) of 1.63 and a 95% confidence interval of 1.15–2.31. These results suggest that high UFALD2 expression is significantly associated with poor prognosis in patients with hepatocellular carcinoma.

[0037] 5. Relationship between UBALD2 expression and clinicopathological parameters Patients were further divided into high-expression and low-expression groups based on UBALD2 expression levels, and the relationship between UBALD2 expression and clinicopathological features was analyzed. The results are shown in Table 1. UBALD2 expression was related to patient gender (…). P =0.252), age ( P =0.562) and history of cirrhosis ( P =0.460) showed no significant correlation; however, it was significantly correlated with T stage (Table 1, P =0.013). Among them, the proportion of patients with high UBALD2 expression in T3 / T4 stage patients was significantly higher than that in patients with T1 / T2 stage patients, suggesting that high expression of UBALD2 is closely related to local tumor progression.

[0038] Table 1. Relationship between UBALD2 expression and clinicopathological features

[0039] In summary, combined analysis using tissue microarray immunohistochemical staining and public databases showed that UBALD2 was abnormally highly expressed in hepatocellular carcinoma (HCC) tissues, and this high expression was closely associated with poor overall survival outcomes in patients. Furthermore, UBALD2 expression levels were significantly correlated with T stage, suggesting its potential involvement in HCC progression. Therefore, UBALD2 holds promise as a candidate biomarker for HCC diagnosis, prognostic assessment, and stratified management.

[0040] II. Cell Culture HCCLM3 cell line was purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd., and SNU449 cell line was a gift from the National Key Laboratory of Biotherapy, Sichuan University. HCCLM3 cells were cultured in DMEM high-glucose medium (Gibco, catalog number: C11995500BT). SNU449 cells were cultured in PRMI-1640 medium (Gibco, catalog number: C11875500BT). Both media were supplemented with 10% fetal bovine serum (EcoSy, catalog number: FSP500). The cell culture conditions were 37°C and 5% CO2 (v / v).

[0041] III. Construction and Validation of UBALD2 Overexpression Cell Model 3.1 Viral Transfection The expression vector GL181 (Shanghai Heyuan Biotechnology Co., Ltd.) was used to construct the UBALD2 overexpression system, and the lentivirus LV-UBALD2 was also constructed by Shanghai Heyuan Biotechnology Co., Ltd. Following the product instructions, Polybrene was used to assist in lentiviral infection to improve transfection efficiency. After infection, the overexpression efficiency of UBALD2 was verified using real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot, respectively.

[0042] 3.2 RNA extraction and real-time quantitative PCR detection UBALD2 mRNA expression Total RNA was extracted from cells using a FOREGENE Total RNA Extraction Kit (Foji, catalog number: RE-03113) according to the manufacturer's instructions, and RNA concentration was measured using a Thermo Fisher microplate reader (USA). The extracted RNA was then reverse transcribed using a PrimeScript RT kit (Takara, catalog number: RR036A) according to Takara's manufacturer's instructions to obtain cDNA. Using the cDNA as a template, quantitative real-time PCR (qRT-PCR) was performed using a Roche PCR instrument with a TB Green Premix Ex Taq II kit (Takara, catalog number: RR037A) according to Takara's manufacturer's instructions to detect the RNA. UBALD2 mRNA expression levels. GAPDH As an internal reference gene, the relative expression level of the target gene was analyzed using the 2^-ΔΔCt method. The primer sequences used were: UBALD2 positive: 5'-CCACCACCACCAGATGATGT-3'; UBALD2 reverse: 5'-CTGAAGTTTGCAGGTGGGGA-3'; GAPDH positive: 5'-CCAGGGCTGCTTTTAACTCT-3'; GAPDH reverse: 5'-GGACTCCACGACGTACTCA-3'.

[0043] The test results showed that, Figure 3 As shown in A, UBALD2 The mRNA expression levels in virus-transfected HCCLM3 and SNU449 cells were significantly increased, indicating that a stable UBALD2 overexpression cell model was successfully established.

[0044] 3.3 Western blot detection of UBALD2 protein expression Cells in the logarithmic growth phase, cultured in 10cm culture dishes, were washed twice with 2mL of pre-chilled PBS buffer at 4°C each time. After washing, 200 μL of RIPA lysis buffer (Beyotime, catalog number: P0013B) was added to the culture dish for lysis. This RIPA lysis buffer contained a 1:100 volume ratio of protease inhibitor and phosphatase inhibitor mixture (Beyotime, catalog number: P1045), and the mixture was incubated at 4°C and 40 rpm on a shaker for 30 min. The lysis buffer was then collected into 1.5 mL EP tubes using a cell scraper and incubated at 4°C on a shaker for another 30 min. After lysis, the cells were centrifuged at 12,000 rpm for 15 min, and 200 μL of the supernatant was transferred to a new EP tube. Protein concentration was determined using a BCA protein assay kit (Beyotime, catalog number: P0010) according to the manufacturer's instructions. Next, 50 μL of 5× SDS-PAGE protein loading buffer (Beyotime, catalog number: P0015L) was added to the sample, and the sample was heated at 98℃ for 5 min to obtain the protein sample for subsequent detection.

[0045] 40 μg of total protein was loaded onto a 12.5% ​​SDS-polyacrylamide gel (Yamei, catalog number: PG113) for electrophoresis separation. The stacking gel voltage was set to 80 V, and the separating gel voltage was set to 120 V. After electrophoresis, the separated proteins were transferred to a polyvinylidene fluoride membrane (PVDF membrane, Merck Millipore, catalog number: ISEQ00010) and blocked with 50 mL of 5% skim milk at room temperature for 1.5 h. After blocking, the PVDF membrane was incubated with 5 mL of UBALD2 primary antibody (NOVUS, 1:1000) and 5 mL of β-actin primary antibody (Cell Signaling Technology, 1:5000) at 4 °C for 15 h. After the primary antibody incubation, 5 mL of HRP-labeled goat anti-rabbit IgG secondary antibody (Baode Biotechnology, China) was added and incubated at room temperature for 1 h. Finally, signal detection was performed using a chemiluminescence analyzer (Senxi Science & Technology, China) to analyze the expression level of UBALD2 protein.

[0046] Test results as follows Figure 3 As shown in Figure B, the protein expression level of UBALD2 was significantly increased in both virus-transfected HCCLM3 and SNU449 cells, further demonstrating that a stable UBALD2 overexpression cell model has been successfully constructed.

[0047] IV. Cloning Experiment Transfected cells in good condition and in the logarithmic growth phase from section 3.3 were seeded at a density of 500 cells per well in six-well plates and cultured for 12 days, with the culture medium changed every 3 days during the culture period. Culture was terminated once visible cell clones formed without contact between cells. After discarding the culture medium, 1 mL of 4% paraformaldehyde (White Shark, BL539A) was added to each well for fixation for 30 min. After fixation, the cells were washed twice with 1 mL of PBS buffer, and then 1 mL of 1% crystal violet staining solution (Solepro, catalog number: G1062) was added, and staining was performed at room temperature in the dark for 30 min.

[0048] The test results showed that, Figure 4 As shown, compared with cells transfected with the control virus solution (LV-NC), the number of colonies formed in the UBALD2 overexpression group (LV-UBALD2) HCCLM3 cells and SNU449 cells was significantly increased, and the difference was verified to be statistically significant. P The result (<0.05) suggests that UBALD2 overexpression can enhance the clonogenic ability of liver cancer cells. These results indicate that UBALD2 can promote the proliferative potential of liver cancer cells.

[0049] V. Cell Migration Experiment Take 1 × 10⁶ transfected cells from well 3.3 that are in good condition and in the logarithmic growth phase, and distribute them at a ratio of 1 × 10⁶ cells per well. 5 Cells were seeded at a density of 100 μL in the upper chamber of a Transwell chamber (Corning Costar, catalog number: 3422) with 200 μL of serum-free medium (DMEM and 1640); 500 μL of medium containing 10% fetal bovine serum was added to the lower chamber. After 48 h of culture, the Transwell chambers were removed and fixed with 500 μL of 4% paraformaldehyde (White Shark, BL539A) for 30 min, and the unmigrated cells in the upper chamber were gently wiped away with a cotton swab. Subsequently, the cells were stained with 500 μL of 1% crystal violet staining solution (Solepro, catalog number: G1062) at room temperature in the dark for 30 min. Finally, the cells were observed and photographed under a microscope.

[0050] Test results as follows Figure 5 (Magnification 100X) Compared with cells transfected with control virus solution (LV-NC), the number of transmembrane cells in the UBALD2 overexpression group (LV-UBALD2) HCCLM3 cells and SNU449 cells was significantly increased, and the difference was verified to be statistically significant. P The value of UBALD2 < 0.05 indicates that overexpression of UBALD2 can enhance the migration ability of liver cancer cells, suggesting that UBALD2 plays an important role in promoting the malignant biological behavior of liver cancer cells.

[0051] VI. Apoptosis Experiment Take transfected cells from section 3.3 that are in good condition and in the logarithmic growth phase, and divide them into groups of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 cells / well in six-well plates. After 48 h of culture, cells were collected and analyzed using an apoptosis detection kit (BD Pharmingen, catalog number: 559763) according to the manufacturer's instructions. The cells were then analyzed by flow cytometry (BD Pharmingen, USA) to assess the level of apoptosis.

[0052] The test results showed that, Figure 6 As shown, compared with cells transfected with the control virus solution (LV-NC), the proportion of apoptotic cells in both HCCLM3 cells and SNU449 cells in the UBALD2 overexpression group (LV-UBALD2) was significantly reduced, and the difference was verified to be statistically significant. P The result (<0.05) indicates that UBALD2 overexpression can inhibit apoptosis in liver cancer cells. This result suggests that the role of UBALD2 in promoting the growth and migration of liver cancer cells is at least partly related to its reduction of apoptosis levels.

[0053] VII. Subcutaneous tumor transplantation experiment in nude mice Take transfected cells from section 3.3 that are in good condition and in the logarithmic growth phase, cultured in 10cm dishes, digest them with 2mL of 0.25% trypsin (Gibco, catalog number: 25200-072), count the cells, and resuspend them in an appropriate amount of culture medium to adjust the cell density to 3×10⁻⁶. 7 Cells / mL. After mixing the cell suspension, 100 μL was injected subcutaneously into the groin of 4-week-old male nude mice (Guangdong Yaokang Biotechnology Co., Ltd.). Tumor size was measured and recorded every 2 days after inoculation. Mice were sacrificed on day 16 post-inoculation, and tumor tissue was dissected and photographed.

[0054] The results are as follows Figure 7 As shown, compared with cells transfected with the control virus solution (LV-NC), the tumors formed by the UBALD2 overexpression group (LV-UBALD2) had significantly increased volume and significantly faster growth rate, with statistically significant differences. P <0.05). The above results indicate that UBALD2 overexpression can significantly promote the tumorigenicity of liver cancer cells in vivo, further demonstrating that UBALD2 has the effect of promoting liver cancer progression.

[0055] VIII. Statistical Analysis Data are expressed as mean ± standard deviation, and all experiments were repeated at least three times. Statistical analysis of differences between groups was performed using t-tests, including differences between cell lines, changes in expression after transfection, and differences in cell function experiments. Statistical analysis was performed using SPSS 26.0 or GraphPad Prism 9.0 software, and a p-value < 0.05 was considered statistically significant.

[0056] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments for application in other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. Application of substances that detect the expression levels of the UBALD2 gene and / or UBALD2 protein in liver cancer diagnostic reagents or kits.

2. Application of substances that detect the expression levels of UBALD2 gene and / or UBALD2 protein in reagents or kits for detecting the proliferation, migration, invasion, and apoptosis levels of liver cancer cells.

3. Application of substances that detect the expression levels of the UBALD2 gene and / or UBALD2 protein in overall survival stratification diagnostic products for hepatocellular carcinoma patients.

4. A liver cancer diagnostic kit, characterized in that, It contains primer pairs for detecting UBALD2 and internal control primer pairs, with the following sequences: UBALD2 positive: 5'-CCACCACCACCAGATGATGT-3'; UBALD2 reverse: 5'-CTGAAGTTTGCAGGTGGGGA-3'; GAPDH positive: 5'-CCAGGGCTGCTTTTAACTCT-3'; GAPDH reverse: 5'-GGACTCCACGACGTACTCA-3'.

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