Biomarker for liver cancer diagnosis and prognosis method thereof

Abstract

The invention discloses a biomarker for liver cancer diagnosis and a prognosis method thereof, and relates to the field of medical biological detection, and the biomarker for liver cancer diagnosis comprises a TATA binding protein related factor 3 and a sterol regulatory element binding protein 2; the expression levels of the TATA binding protein related factor 3 and the sterol regulatory element binding protein 2 are subjected to immunohistochemical detection, and when the immunohistochemical score of the TATA binding protein related factor 3 is greater than or equal to 2 +, the TATA binding protein related factor 3 is highly expressed; when the immunohistochemical score of the sterol regulatory element binding protein 2 is greater than or equal to 2 +, the sterol regulatory element binding protein 2 is highly expressed; according to the biomarker for liver cancer diagnosis and the prognosis method of the biomarker, liver cancer patients with poor prognosis can be accurately screened out by detecting the expression level and serum cholesterol concentration of the TATA binding protein related factor 3 and the sterol regulating element binding protein 2, a basis is provided for personalized treatment, and the diagnosis accuracy is remarkably improved.

Description

Technical Field

[0001] The present invention relates to medical biological detection technology, and in particular to biomarkers for liver cancer diagnosis and a prognostic method thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is a common and fatal cancer worldwide. Current surgical treatments offer a poor prognosis, primarily due to tumor metastasis and recurrence. The 5-year recurrence rate after surgical resection is >70%, and targeted drugs (such as sorafenib) only extend survival by 2-3 months. Current biomarkers for HCC diagnosis suffer from low sensitivity and insufficient specificity, making it difficult to accurately detect HCC in its early stages. Consequently, many patients are diagnosed in the advanced stages, missing the optimal treatment window. Therefore, there is an urgent need to discover new and more effective biomarkers to improve the early diagnosis of HCC.

[0003] Existing HCC prognosis assessment methods often cannot accurately predict, which makes it difficult for clinicians to formulate personalized treatment plans. The lack of effective prognostic markers makes it impossible to provide patients with accurate prognostic information, affecting the scientificity and rationality of treatment decisions. Summary of the Invention

[0004] The purpose of the present invention is to provide biomarkers for liver cancer diagnosis and a prognostic method thereof, so as to address the above-mentioned deficiencies in the prior art.

[0005] In order to achieve the above objectives, the present invention provides the following technical solution: biomarkers for liver cancer diagnosis, which are TATA binding protein-associated factor 3 and sterol regulatory element binding protein 2.

[0006] Furthermore, the expression levels of the TATA binding protein-associated factor 3 and the sterol regulatory element binding protein 2 are detected by immunohistochemistry. When the immunohistochemical score of the TATA binding protein-associated factor 3 is ≥2+, it means that the TATA binding protein-associated factor 3 is highly expressed; when the immunohistochemical score of the sterol regulatory element binding protein 2 is ≥2+, it means that the sterol regulatory element binding protein 2 is highly expressed.

[0007] A use of the biomarker for liver cancer diagnosis in the preparation of a kit for liver cancer diagnosis.

[0008] Furthermore, the kit includes specific probes or antibodies for detecting the expression levels of TATA binding protein-associated factor 3 and sterol regulatory element binding protein 2.

[0009] A prognostic method for biomarkers in diagnosing liver cancer comprises the following steps:

[0010] S1. Immunohistochemical examination of the expression levels of TATA-binding protein-associated factor 3 and sterol regulatory element binding protein 2 in tumor tissue samples removed from the patient and subjected to simple medical treatment;

[0011] S2. Detecting cholesterol concentration in serum samples removed from the patient and subjected to simple medical treatment by electrochemiluminescence;

[0012] S3. If the immunohistochemical scores of TATA-binding protein-associated factor 3 and sterol regulatory element-binding protein 2 are both ≥2+, and the serum cholesterol concentration is >5.2 mmol / L, the patient is judged to have a poor prognosis and should be treated with combined cholesterol synthesis inhibitors. If the immunohistochemical scores of TATA-binding protein-associated factor 3 and sterol regulatory element-binding protein 2 are both <2+, and the serum cholesterol concentration is <5.2 mmol / L, the patient is judged to have a good prognosis.

[0013] Furthermore, the cholesterol synthesis inhibitor in S3 is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, and the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor includes simvastatin.

[0014] Compared with existing technologies, the biomarker for liver cancer diagnosis and its prognostic method provided by the present invention can accurately screen liver cancer patients with poor prognosis by detecting the expression levels of TATA binding protein-associated factor 3 and sterol regulatory element binding protein 2 and serum cholesterol concentration, providing a basis for personalized treatment and significantly improving diagnostic accuracy.

[0015] The kit can be used to quickly detect the expression levels of TATA-binding protein-associated factor 3 and sterol regulatory element-binding protein 2, shortening the patient enrollment screening time and improving treatment efficiency; biomarker-based efficacy prediction can avoid ineffective treatment, reduce waste of medical resources, and have significant economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments described in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0017] Figure 1 Schematic diagram of the effect of knockdown / overexpression of TAF3 gene expression on cell proliferation ability provided by an embodiment of the present invention;

[0018] Figure 2 Schematic diagram of the effect of knockdown / overexpression of TAF3 gene expression on cell invasion ability provided by an embodiment of the present invention;

[0019] Figure 3 A schematic diagram of changes in downstream signaling pathways caused by knockdown / overexpression of TAF3 based on RNA-seq data analysis provided in an embodiment of the present invention;

[0020] Figure 4 Schematic diagram of the effect of knocking down SREBP2 on the expression of genes related to cholesterol metabolism detected by qPCR provided in an embodiment of the present invention;

[0021] Figure 5 Schematic diagram of the electrochemiluminescence method for detecting intracellular cholesterol concentration after TAF3 knockdown and overexpression provided in an embodiment of the present invention;

[0022] Figure 6 A schematic diagram of analyzing the transcriptional regulation of SREBP2 by TAF3 based on the GEO database provided in an embodiment of the present invention;

[0023] Figure 7 Schematic diagram of the effect of TAF3 gene knockdown on HCC tumor growth provided by an embodiment of the present invention;

[0024] Figure 8 Schematic diagram of the correlation analysis between TAF3 expression and cholesterol content in tumor tissues provided by an embodiment of the present invention;

[0025] Figure 9 Schematic diagram of the correlation analysis between TAF3 expression in liver cancer patient tissues and tumor proliferation and EMT transformation marker E-cadherin provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0027] Example 1:

[0028] See also Figures 3 to 6 and Figure 8 , biomarkers for diagnosing liver cancer, which are TATA binding protein-associated factor 3 and sterol regulatory element binding protein 2; the expression levels of the TATA binding protein-associated factor 3 and sterol regulatory element binding protein 2 are detected by immunohistochemistry. When the immunohistochemical score of TATA binding protein-associated factor 3 is ≥2+, it means that TATA binding protein-associated factor 3 is highly expressed; when the immunohistochemical score of sterol regulatory element binding protein 2 is ≥2+, it means that sterol regulatory element binding protein 2 is highly expressed.

[0029] TATA-binding protein-associated factor 3 (TAF3) activates the transcription of sterol regulatory element binding protein 2 (SREBP2) by directly binding to the promoter region (chr7:116,543,221-116,543,579), thereby regulating cholesterol metabolism.

[0030] See also Figure 3 RNA-seq data was used to analyze the changes in downstream signaling pathways caused by knockdown / overexpression of TAF3. HepG2 liver cancer cell lines were selected and divided into four groups: a control group, which was transfected with a non-targeted negative control short hairpin RNA to provide a reference for normal cell growth; a TAF3 knockdown group, which was transfected with a TAF3-targeting shRNA to specifically reduce TAF3 expression levels in cells; a normal control group, which maintained normal gene expression and physiological status; and a TAF3 overexpression group, which was transfected with an exogenous TAF3 gene expression vector to achieve overexpression of the TAF3 gene in cells. Cells in these groups were cultured under standard conditions until the cell density reached approximately 80%-90%, at which point they were harvested for RNA extraction.

[0031] The extracted RNA samples were sent to BGI for sequencing. By comparing the differences in gene expression between the TAF3 knockdown / overexpression genome and the control group, differentially expressed genes (DEGs) were screened. Functional enrichment analysis of the screened differentially expressed genes was performed using methods such as GSEA to comprehensively and systematically understand the downstream signaling pathways regulated by TAF3.

[0032] Figure 3 A shows the changes in gene expression between TAF3 knockdown and control groups; Figure 3 B shows the changes in gene expression between TAF3 overexpression and the normal control group. In the case of TAF3 knockdown, 303 genes were downregulated, 397 genes were upregulated, and 5313 genes showed no significant changes. Some genes were significantly upregulated or downregulated after TAF3 knockdown. In the case of TAF3 overexpression, 6 genes were downregulated, 162 genes were upregulated, and 2624 genes showed no significant changes. Compared with TAF3 knockdown, TAF3 overexpression led to more gene upregulation.

[0033] Figure 3 C compared the TAF3 gene expression knockdown group with the control group and analyzed the enrichment of the HALLMARK_CHOLESTEROL_HOMEOSTAS IS (cholesterol homeostasis) gene set; Figure 3D compares the TAF3 overexpression group with the normal control group and similarly analyzes the enrichment of the HALLMARK_CHOLESTEROL_HOMEOSTAS IS gene set. In the comparison between the TAF3 knockdown group and the control group, the enrichment score curve for the cholesterol homeostasis gene set peaks in the middle of the dataset, indicating that these genes may be upregulated or downregulated after TAF3 knockdown. In the comparison between the TAF3 overexpression group and the normal control group, the enrichment score curve for the cholesterol homeostasis gene set also shows a similar trend, demonstrating the impact of TAF3 overexpression on genes related to cholesterol homeostasis.

[0034] Knockdown of TAF3 led to changes in the expression of numerous genes, with a significant impact on a gene set involved in cholesterol homeostasis. Overexpression of TAF3 also caused changes in gene expression, with a particularly large number of upregulated genes, also affecting a gene set involved in cholesterol homeostasis. These results suggest that changes in TAF3 expression can significantly influence the expression of downstream genes, particularly in pathways related to cholesterol metabolism.

[0035] See also Figure 6 Based on the GEO database, we analyzed the transcriptional regulation of SREBP2 by TAF3. There was an obvious signal peak near the SREBF2 gene region, which indicated that TAF3 was significantly enriched in this region. This enrichment meant that TAF3 had a higher binding activity in the promoter region of SREBP2.

[0036] In liver cells, SREBP2 is involved in the synthesis and metabolism of cholesterol, and the regulatory role of TAF3 may affect the regulation of cholesterol metabolism. From a disease perspective, the SREBP2 gene is closely related to cholesterol metabolism, and its dysregulated expression may be associated with diseases such as hypercholesterolemia. Abnormal regulation of its transcription by TAF3 can further affect cholesterol metabolism, which is a key feature of hepatocellular carcinoma (HCC).

[0037] See also Figure 8 , the expression of SREBP2 and the correlation between TAF3 and cholesterol content in liver cancer tissues were analyzed. The experimental groups were divided into TAF3 low expression group and TAF3 high expression group, and the expression of SREBP2 gene was detected by real-time fluorescence quantitative PCR (qPCR) ( Figure 8 A), cholesterol content in tumor tissue was determined using a cholesterol determination kit ( Figure 8 B).

[0038] See also Figure 8A shows the SREBP2 gene expression levels in liver cancer tissues with high and low TAF3 expression levels. As can be seen from the figure, the SREBP2 gene expression level in the TAF3 high expression group was significantly higher than that in the TAF3 low expression group; see Figure 8 Figure B shows liver cholesterol levels in tumor tissues with high and low TAF3 expression levels. The cholesterol level in the TAF3 high-expression group was significantly higher than that in the TAF3 low-expression group. Combining the results of these two figures, we can speculate that high TAF3 expression in liver cancer tumor tissues regulates SREBP2 expression, thereby affecting cholesterol metabolism and accumulation.

[0039] See also Figure 4 , qPCR detection of the effect of knocking down SREBP2 on the expression of cholesterol metabolism-related genes: The qPCR reaction system contains cDNA template, upstream and downstream primers, SYBR Green fluorescent dye, dNTPs, Taq DNA polymerase and buffer. The reaction was carried out on a real-time fluorescence quantitative PCR instrument. The reaction conditions were 95℃ pre-denaturation for 5 minutes, followed by 40 cycles, each cycle including 95℃ denaturation for 15 seconds, 55-65℃ annealing for 30 seconds, and 72℃ extension for 30 seconds. According to the fluorescence signal intensity, 2 -ΔΔCt The data were processed by the TAF3 knockdown method. The expression levels of the target genes were normalized with the internal reference genes. The relative expression folds of the target genes in the TAF3 knockdown group were calculated with the knockdown control group as a reference, thereby analyzing the expression changes of cholesterol metabolism-related genes in cells of different treatment groups.

[0040] See also Figure 4 Knocking down the SREBP2 gene significantly affected the expression of genes involved in cholesterol metabolism. Expression of different genes varied, and there were certain differences between the two cell lines. SREBP2 is an important member of the sterol regulatory element binding protein family, primarily involved in cholesterol synthesis and uptake. Knocking down SREBP2 affects cholesterol metabolism by regulating the transcription of genes involved in cholesterol metabolism.

[0041] See also Figure 5 Electrochemiluminescence (ECL) was used to measure intracellular cholesterol concentration in HepG2 and MHCC97H cells. A specific ECL reagent reacts with intracellular cholesterol, generating a light signal whose intensity is proportional to the cholesterol concentration. The intracellular cholesterol concentration was calculated by measuring the light signal intensity.

[0042] See also Figure 5A, In HepG2 cell lines, the intracellular cholesterol concentration was significantly decreased in the TAF3 knockdown group compared with the control group, indicating that downregulation of TAF3 inhibited cholesterol synthesis or promoted cholesterol metabolism; the intracellular cholesterol concentration was significantly increased in the TAF3 overexpression group compared with the control group, indicating that upregulation of TAF3 may promote cholesterol synthesis or accumulation.

[0043] See also Figure 5 B, In the MHCC97H cell line, the intracellular cholesterol concentration was significantly decreased in the TAF3 knockdown group compared with the control group, further supporting the regulatory role of TAF3 in cholesterol metabolism; the intracellular cholesterol concentration was significantly increased in the TAF3 overexpression group compared with the control group, which is consistent with the results of the HepG2 cell line.

[0044] TAF3 expression levels are closely correlated with intracellular cholesterol concentrations. In both cell lines, TAF3 knockdown resulted in a significant decrease in cholesterol concentrations, while TAF3 overexpression significantly increased cholesterol concentrations. This suggests that TAF3 influences intracellular cholesterol levels by regulating the expression of genes involved in cholesterol metabolism.

[0045] In summary, TAF3 and SREBP2 can be used as biomarkers for the diagnosis of liver cancer.

[0046] Example 2:

[0047] This embodiment provides a technical solution based on the first embodiment: the use of biomarkers for liver cancer diagnosis in the preparation of a kit for liver cancer diagnosis, the kit comprising specific probes or antibodies for detecting the expression levels of TATA-binding protein-associated factor 3 and sterol regulatory element binding protein 2.

[0048] Example 3:

[0049] See also Figure 1 and Figure 2 This embodiment provides a technical solution based on the first embodiment: the effect of knocking down / overexpressing TAF3 gene expression on the proliferation and invasion ability of liver cancer cells.

[0050] 1. Effects of knockdown / overexpression of TAF3 gene expression on the proliferation ability of liver cancer cells:

[0051] Liver cancer cell lines HepG2 and MHCC97H were selected, and the cells were divided into four groups: the knockdown control group, which was transfected with non-targeted negative control short hairpin RNA (shRNA) to provide reference data under normal cell growth conditions; the TAF3 knockdown group, which was transfected with shRNA targeting the TAF3 gene to specifically reduce the expression level of the TAF3 gene in cells; the overexpression control group, which maintained normal gene expression and physiological state of the cells, served as the control group; the TAF3 overexpression group, which used gene transfection technology to introduce exogenous TAF3 gene expression vectors to achieve overexpression of the TAF3 gene in cells, in order to explore the effect of high TAF3 expression on cell proliferation.

[0052] The CCK8 method was used to quantitatively detect cell proliferation. Before the experiment began, each group of cells in the logarithmic growth phase was digested with trypsin and made into a single cell suspension. 5000-10000 cells per well were inoculated in a 96-well cell culture plate, and 100 μL of cell suspension was added to each well to ensure that the cells were evenly distributed in the culture plate. The culture plate was placed in a cell culture incubator at 37°C and 5% CO2 to allow the cells to grow adherently. The assay was performed at three time points: 24h, 48h, and 72h after inoculation. During the assay, 20 μL of CCK8 reagent was added to each well, the culture plate was gently shaken to allow the reagent to fully contact the cells, and then the cells were incubated in the incubator for another 4h. The tetrazolium salt in the CCK8 reagent is reduced by the dehydrogenase in the living cells to a highly water-soluble formazan product, which has strong absorbance at a specific wavelength (450nm). The absorbance (OD value) of each well was measured at a wavelength of 450 nm using a microplate reader. The OD value is proportional to the number of living cells. By comparing the OD values ​​of each group of cells at different time points, the cell proliferation activity can be indirectly reflected.

[0053] See also Figure 1 In MHCC97H cells, as the culture time prolonged (0h, 24h, 48h, 72h), the relative OD450 value of the TAF3 knockdown group was significantly lower than that of the knockdown control group, indicating that knockdown of the TAF3 gene could inhibit the proliferation of MHCC97H liver cancer cells, and this inhibitory effect gradually appeared over time, indicating that the TAF3 gene played an important role in maintaining the normal proliferation of MHCC97H cells; the relative OD450 value of the OE group was higher than that of the OENC group, indicating that overexpression of the TAF3 gene could enhance the proliferation ability of MHCC97H cells.

[0054] In HepG2 cells, after knocking down the TAF3 gene, the relative OD450 value of HepG2 cells was lower than that of the knockdown control group, indicating that knocking down the TAF3 gene inhibited the proliferation of HepG2 cells; the relative OD450 value of the TAF3 overexpression group was higher than that of the overexpression control group, indicating that overexpression of the TAF3 gene can promote the proliferation of HepG2 cells.

[0055] The overall results show that the TAF3 gene plays an important positive regulatory role in the proliferation of liver cancer cells (MHCC97H and HepG2). Knocking down the TAF3 gene inhibits cell proliferation, while overexpressing the TAF3 gene promotes cell proliferation.

[0056] 2. Effects of knockdown / overexpression of TAF3 gene expression on the invasion ability of liver cancer cells:

[0057] HepG2 and MHCC97H cell lines were selected. Before the experiment, the cells were cultured to the logarithmic growth phase, digested with trypsin, and single-cell suspensions were prepared. The cell concentration was adjusted to 1×10 cells per ml. 5 -5×10 5 cells. Transwell l chambers were used for cell invasion experiments. The pore size of the polycarbonate membrane of the chamber is usually 8μm, which can effectively simulate the extracellular matrix barrier in vivo. Before the experiment, Matrigel matrix gel was diluted with serum-free culture medium at a ratio of 1:10. Then 50μL of the diluted Matrigel was evenly spread on the bottom of the upper chamber of the Transwell chamber and incubated in a 37°C incubator for 3 hours to allow Matrigel to form a gel-like extracellular matrix layer, simulating the basement membrane environment in vivo. After incubation, 200μL of cell suspension (approximately 5000 cells) was added to the upper chamber, and 600μL of culture medium containing chemokines (2% fetal bovine serum) was added to the lower chamber. Chemotactic factors can attract cells to migrate under the membrane. The Transwell l chamber was placed in a cell culture incubator at 37°C and 5% CO2 for 72 hours. After the culture is completed, the Transwell chamber is removed, and the cells that have not passed through the membrane in the upper chamber are gently wiped with a cotton swab. The chamber is then placed in a 4% paraformaldehyde solution for fixation for 30 minutes. After fixation, it is washed twice with PBS buffer. Next, the cells that have passed through the membrane are stained with 0.1% crystal violet solution for 10 minutes. After staining, they are washed again with PBS buffer to remove excess staining solution. Under a microscope, three fields of view are randomly selected and the stained transmembrane cells are counted. The more transmembrane cells there are, the stronger the cell's invasive ability. By comparing the differences in the number of transmembrane cells between different groups, the effect of knocking down or overexpressing the TAF3 gene on cell invasion ability was evaluated.

[0058] See also Figure 2In MHCC97H cells, the number of invasive cells in the TAF3 knockdown group was significantly less than that in the control group, indicating that knocking down the TAF3 gene can inhibit the invasion of MHCC97H liver cancer cells. The number of invasive cells in the TAF3 overexpression group was greater than that in the control group, indicating that overexpression of the TAF3 gene can enhance the invasive ability of MHCC97H cells. Microscopic images also intuitively show the invasion of cells in each group. Fewer cells in the TAF3 knockdown group invaded into the lower layer, while more cells were seen in the TAF3 overexpression group.

[0059] In HepG2 cells, the number of invasive cells in the TAF3 knockdown group was less than that in the knockdown control group, indicating that the knockdown of the TAF3 gene inhibited the invasion of HepG2 cells. The number of invasive cells in the overexpression group of the TAF3 gene was more than that in the overexpression control group, indicating that the overexpression of the TAF3 gene can promote the invasion of HepG2 cells. Microscope images also support this conclusion. The number of invasive cells in the TAF3 knockdown group was less, and the number in the TAF3 overexpression group was more. Overall, the TAF3 gene plays an important role in promoting the invasion of liver cancer cells (MHCC97H and HepG2).

[0060] Example 4:

[0061] This embodiment provides a technical solution based on the first embodiment: a prognostic method for biomarkers of liver cancer diagnosis, comprising the following steps:

[0062] S1. Detect the expression levels of TATA-binding protein-associated factor 3 and sterol regulatory element binding protein 2 in tumor tissue samples that have been removed from the patient and undergone simple medical treatment by immunohistochemistry; the simple medical treatment in this step includes but is not limited to the use of neutral buffered formaldehyde solution (such as 4% paraformaldehyde) to fix the tissue samples to maintain the morphological structure and antigenicity of the tissue and prevent cell autolysis and corruption.

[0063] S2. Detecting the cholesterol concentration in a serum sample that has been removed from the patient and subjected to simple medical treatment by electrochemiluminescence. The simple medical treatment in this step includes but is not limited to placing the collected blood sample in a centrifuge tube and centrifuging it at a centrifugal force of 1000-2000g for 10-15 minutes to separate the serum and ensure that the serum is fully separated from components such as blood cells.

[0064] S3. If the immunohistochemical scores for TATA-binding protein-associated factor 3 and sterol regulatory element binding protein 2 are both ≥2+ and the serum cholesterol concentration is >5.2 mmol / L, the patient is considered to have a poor prognosis and should be treated with a cholesterol synthesis inhibitor. If the immunohistochemical scores for TATA-binding protein-associated factor 3 and sterol regulatory element binding protein 2 are both <2+ and the serum cholesterol concentration is <5.2 mmol / L, the patient is considered to have a good prognosis. Cholesterol synthesis inhibitors are 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, which include simvastatin.

[0065] Embodiment 5:

[0066] See also Figure 7 This embodiment provides a technical solution based on the first embodiment: the effect of TAF3 gene knockdown on HCC tumor growth (tumor volume and weight) in an HCC mouse model (subcutaneous transplant tumor model).

[0067] Six-week-old BALB / c nude mice were selected, and the barrier system was stable for 2 weeks. Under sterile conditions, HepG2 knockdown control group and TAF3 knockdown group cells in the logarithmic growth phase were digested with trypsin to prepare single-cell suspensions, and the cell concentration was adjusted to 2×10 cells per ml. 7 The cell suspension was inoculated subcutaneously in the right axilla of mice, with 100 μL per mouse. After inoculation, the inoculation site was gently massaged to ensure even cell distribution. The mice were divided into a knockdown control group and a TAF3 knockdown group. Throughout the experiment, the health of the mice was observed in real time as tumors grew to a certain size, and tumor growth was measured every two days.

[0068] See also Figure 7 A. Changes in tumor volume. The tumor volume of the knockdown control group increased significantly over time, indicating that the tumors in the knockdown control group mice were growing normally; the tumor volume of the TAF3 knockdown group grew significantly slower, and the final volume was significantly smaller than that of the knockdown control group; on the 30th day, the tumor volume of the TAF3 knockdown group was approximately 30% of that of the knockdown control group. The difference between the two groups was highly statistically significant, indicating that TAF3 gene knockdown significantly inhibited tumor growth.

[0069] See also Figure 7 B. Comparison of tumor weights: The tumor weight in the knockdown control group was higher, with an average weight of approximately 0.65 g, while the tumor weight in the TAF3 knockdown group was significantly reduced, with an average weight of approximately 0.25 g. The difference between the two groups was highly statistically significant, further confirming the inhibitory effect of TAF3 gene knockdown on tumor growth.

[0070] TAF3 gene knockdown significantly inhibited tumor growth in the HCC mouse model, with both tumor volume and weight significantly reduced. This suggests that TAF3 plays an important role in promoting HCC tumor growth. By knocking down the TAF3 gene, tumor growth can be significantly slowed and the final tumor volume and weight can be reduced.

[0071] Example 6:

[0072] See also Figure 9 This embodiment provides a technical solution based on the first embodiment: correlation analysis between TAF3 expression in liver cancer patient tissues and tumor proliferation and EMT transformation marker E-cadherin.

[0073] Liver cancer tissue samples and corresponding non-tumor liver tissue samples as controls were fixed overnight in 4% paraformaldehyde solution. The fixed tissues were dehydrated, transparentized, and wax-impregnated before being embedded in paraffin to create tissue wax blocks. The tissue wax blocks were cut into 4μm-thick sections using a rotary microtome and mounted on anti-shedding glass slides for subsequent staining and other procedures. Liver cancer tissue samples were divided into low and high TAF3 expression groups based on TAF3 expression levels.

[0074] Immunohistochemical staining: Place the slide with the tissue section in an oven and bake. Then soak it in xylene several times for a few minutes each time until the section is completely dewaxed and becomes water-based. Then, hydrate the slide by placing it in ethanol solutions of varying concentrations (from high to low) and finally rinse with distilled water.

[0075] Place the sections in a container of citrate repair solution and heat-repair them in a pressure cooker or microwave. This exposes antigens whose conformation has been altered by factors such as fixation, facilitating antibody binding. Allow the heated repair solution to cool naturally to room temperature, then rinse the sections with distilled water.

[0076] Specific primary antibodies targeting Ki 67 and E-CAD are added, respectively. The incubated slides are placed in a humidified chamber and incubated overnight at approximately 4°C to allow the primary antibodies to fully bind to the corresponding antigens in the tissue sections. The next day, the slides are removed and washed thoroughly with a buffer solution such as phosphate-buffered saline (PBS) to remove any unbound primary antibody. The corresponding secondary antibody is added and incubated at room temperature for approximately half an hour to allow the secondary antibody to bind to the primary antibody. The slides are washed again with PBS, followed by the addition of a chromogenic substrate (such as DAB). The slides are observed under a microscope and the color development time is controlled. Once the desired brownish-yellow precipitate appears on the slides, the color development reaction is terminated by rinsing with distilled water, thereby demonstrating the localization of the antigen-antibody reaction. The stained sections are observed under a microscope, and images of Ki 67 and E-CAD staining are captured. Representative fields of view are photographed for subsequent analysis and H-score evaluation.

[0077] H-score quantitative analysis: A sufficient number of visual field images were taken under a microscope for tissue sections stained with Ki 67 and E-CAD to ensure that the imaged visual field represented the staining of the entire section. H-score quantitative calculation was performed using ImageJ image analysis software. The H-score value for each image was calculated according to the staining intensity grading standard (0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining) and the proportion of positive cells at the corresponding intensity. The H-score formula is as follows:

[0078] H-score=∑(Pi×i),

[0079] Pi represents the proportion of positive cells with staining intensity i, and i represents the staining intensity level.

[0080] After calculating the H-score values ​​of Ki67 and E-CAD for multiple sample images of the same group, data collation and statistical analysis were performed to compare the differences in H-score values ​​between different groups.

[0081] Ki67 is a nuclear antigen associated with cell proliferation. It is expressed in the G1, S, G2, and M phases of the cell cycle, but not in the resting phase (G0 phase) cells. Therefore, Ki67 is a commonly used marker for evaluating cell proliferation activity. E-CAD (E-Cadherin) is a calcium-dependent transmembrane glycoprotein in epithelial cells. It belongs to the cadherin family and is one of the hallmark molecules of EMT.

[0082] See also Figure 9A. Immunohistochemical staining images. Ki67 staining revealed lighter nuclear staining in the TAF3 low expression group (Low TAF3), indicating lower proliferation activity; darker nuclear staining in the TAF3 high expression group (High TAF3), indicating higher proliferation activity. E-CAD staining revealed clear cell-to-cell boundaries and higher E-CAD expression in the TAF3 low expression group, indicating stronger cell adhesion. In the TAF3 high expression group, cell-to-cell boundaries were blurred and E-CAD expression was decreased, suggesting weakened cell-to-cell adhesion and a possible EMT process.

[0083] See also Figure 9 B. Quantitative analysis of H-score: Ki67 H-score: The H-score value of the TAF3 low expression group was lower, indicating low proliferation activity; the H-score value of the TAF3 high expression group was significantly increased, indicating that the TAF3 expression level was positively correlated with the proliferation activity of tumor cells.

[0084] E-CAD H-score: The H-score value of the TAF3 low expression group was higher, indicating high E-CAD expression level and strong cell adhesion; the H-score value of the TAF3 high expression group was significantly reduced, indicating that TAF3 expression level was negatively correlated with E-CAD expression, suggesting that TAF3 may promote the EMT process.

[0085] High expression of TAF3 in liver cancer tissues is closely associated with enhanced tumor cell proliferation and the EMT process. Tumor tissues with high TAF3 expression exhibit higher proliferation activity (increased Ki 67H-score) and lower intercellular adhesion (decreased E-CAD H-score). This suggests that TAF3 may play an important role in the occurrence and progression of liver cancer by promoting tumor cell proliferation and the EMT process, and may serve as a potential therapeutic target and prognostic marker.

[0086] The above description is merely illustrative of certain exemplary embodiments of the present invention. It goes without saying that those skilled in the art will be able to modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and description are illustrative in nature and should not be construed as limiting the scope of protection of the claims.

Claims

1. A biomarker for liver cancer diagnosis, characterized in that: They are TATA binding protein-associated factor 3 and sterol regulatory element binding protein 2.

2. The biomarker for liver cancer diagnosis according to claim 1, characterized in that The expression levels of the TATA binding protein-associated factor 3 and sterol regulatory element binding protein 2 are detected by immunohistochemistry. When the immunohistochemical score of TATA binding protein-associated factor 3 is ≥2+, it means that TATA binding protein-associated factor 3 is highly expressed; when the immunohistochemical score of sterol regulatory element binding protein 2 is ≥2+, it means that sterol regulatory element binding protein 2 is highly expressed.

3. Use of the biomarker for liver cancer diagnosis according to claim 2 in the preparation of a kit for liver cancer diagnosis.

4. Use of the biomarker for liver cancer diagnosis according to claim 3 in the preparation of a kit for liver cancer diagnosis, characterized in that: The kit comprises specific probes or antibodies for detecting the expression levels of TATA binding protein-associated factor 3 and sterol regulatory element binding protein 2.

5. A prognostic method for liver cancer diagnosis using biomarkers, characterized in that: The following steps are involved: S1. Immunohistochemical examination of the expression levels of TATA-binding protein-associated factor 3 and sterol regulatory element binding protein 2 in tumor tissue samples removed from the patient and subjected to simple medical treatment; S2. Detecting cholesterol concentration in serum samples removed from the patient and subjected to simple medical treatment by electrochemiluminescence; S3. If the immunohistochemical scores of TATA-binding protein-associated factor 3 and sterol regulatory element-binding protein 2 are both ≥2+, and the serum cholesterol concentration is >5.2 mmol / L, the patient is judged to have a poor prognosis and should be treated with combined cholesterol synthesis inhibitors. If the immunohistochemical scores of TATA-binding protein-associated factor 3 and sterol regulatory element-binding protein 2 are both <2+, and the serum cholesterol concentration is <5.2 mmol / L, the patient is judged to have a good prognosis.

6. The prognostic method for liver cancer diagnosis based on biomarkers according to claim 5, characterized in that: S3 The cholesterol synthesis inhibitor is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, and the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor includes simvastatin.

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