Marker detection kit and detection analysis system for typing or prognosis evaluation after liver cancer thermal ablation operation and application of marker detection kit and detection analysis system

Abstract

The invention provides a marker detection kit for typing or prognosis evaluation after liver cancer thermal ablation, a detection analysis system and application of the marker detection kit and the detection analysis system. In order to solve the clinical problems that after thermal ablation treatment, hepatocellular carcinoma is prone to relapse and the mechanism is unknown, in a clinical sample and an orthotopic xenograft model derived from a patient, it is jointly confirmed that a remarkable DNA hypermethylation phenomenon exists in residual and relapsed HCC tissue after ablation, and the phenomenon is accompanied by remarkable up-regulation of DNMT1; therefore, heat stress can trigger and promote an epigenetic-metabolic cascade reaction of tumors by activating a DNA hypermethylation 5mC mechanism. On the basis, it is clear that DNMT1-mediated DNA hypermethylated 5mC is one of important mechanisms of HCC recurrence after thermal ablation, an accurate detection marker is provided for recognizing the recurrence risk, and a detection and analysis system is established to improve the layered diagnosis capacity after liver cancer thermal ablation, so that the prognosis and survival conditions of patients are improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a biomarker detection kit and detection and analysis system for postoperative classification or prognostic assessment of liver cancer by thermal ablation, and their applications. Background Technology

[0002] Thermal ablation techniques, such as microwave ablation (MWA) and radiofrequency ablation (RFA), are currently the first-line radical treatment for early-stage unresectable hepatocellular carcinoma (HCC). However, this technique has a significant clinical drawback: due to factors such as irregular tumor morphology and indistinct borders, the ablation area is often not completely covered, allowing surrounding tumor cells to survive under sublethal high temperatures (41-50°C). This results in a persistently high local recurrence rate, reaching as high as 70%. More seriously, current clinical observations and studies indicate that these residual lesions due to incomplete ablation may exhibit more malignant biological behavior, accelerating disease progression and creating a clinical paradox of "aiming for a cure but potentially promoting progression."

[0003] Regarding this issue, current technologies do not provide a deep understanding of the recurrence mechanism. Although studies have confirmed that cancer cells subjected to heat stress exhibit stronger tumorigenesis and metastasis in vivo, the core molecular mechanisms driving this sustained tumor progression remain unclear, severely hindering the prediction and assessment of recurrence. In recent years, epigenetics, particularly DNA hypermethylation, has been considered a key mechanism for cells to respond to acute environmental stress and form "memory." Literature indicates that a single extreme heat stress can induce long-term changes in genome-wide DNA hypermethylation in other biological models (References 1, 2). However, in the specific clinical context of hepatocellular carcinoma thermal ablation, whether DNA methylation mechanisms predict malignant progression of residual tumor remains an unexplored scientific gap.

[0004] Therefore, the current shortcomings lie in the insufficient understanding of biomarkers for the prognosis of HCC after thermal ablation and the lack of a systematic explanation from an epigenetic perspective. This invention application is thus proposed.

[0005] References:

[0006] 1. Zhu, S. et al. Targeting N(7)-methylguanosine tRNA modification blocks hepatocellular carcinoma metastasis after insufficient radiofrequency ablation. Mol. Ther. 31, 1596-1614 (2023). 2. Su, T. et al. Insufficient radiofrequency ablation promotes shepatocellular carcinoma metastasis through N6-methyladenosine mRNAmethylation-dependent mechanism. Hepatology. 74, 1339-1356 (2021). Summary of the Invention This invention aims to address the core technical problem in existing technologies where the lack of understanding of the mechanisms driving tumor recurrence after thermal ablation prevents early and accurate risk stratification of patients, leading to delayed clinical intervention and poor patient prognosis. This invention provides a biomarker detection kit and analysis system for post-thermal ablation classification or prognostic assessment of liver cancer, along with their applications, aiming to provide reliable biomarkers for predicting and assessing the prognosis after liver cancer thermal ablation.

[0007] The first objective of this invention is to provide the use of a reagent for detecting DNA methyltransferase 1 expression levels in the preparation of products for diagnosing recurrence of liver cancer after thermal ablation.

[0008] The second objective of this invention is to provide a diagnostic kit for liver cancer after thermal ablation.

[0009] The third objective of this invention is to provide a detection and analysis system for postoperative classification or prognostic assessment of liver cancer by thermal ablation.

[0010] A fourth objective of this invention is to provide the application of reagents for detecting the expression level of DNA methyltransferase 1 and whole-genome DNA hypermethylation 5mC in the preparation of products for assessing the risk of recurrence after thermal ablation of liver cancer.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution: This invention, for the first time, systematically demonstrates in vitro and in vivo that sublethal heat stress is a key initiating factor triggering epigenetic remodeling of liver cancer cells after ablation. It also clarifies that the upregulation of DNMT1 and the resulting genome-wide DNA hypermethylation (5mC) are the core mechanisms driving recurrence and malignant progression. Therefore, DNMT1 can be used as a biomarker for the diagnosis and classification of liver cancer recurrence after ablation. PDOX model, clinical sample validation, in vitro cell models, and ROC curves all confirm that DNMT1 levels and DNA hypermethylation (5mC) status are objective, repeatable indicators directly related to recurrence after ablation, enabling early and accurate identification of patients at high risk of recurrence. Furthermore, knocking down DNMT1 completely blocks the heat stress-induced metastatic enhancement effect. Therefore, this invention provides a biomarker, DNMT1, for predicting the prognosis after thermal ablation of liver cancer. By detecting the expression level of DNMT1 in a sample and combining it with the 5mC state of whole-genome DNA hypermethylation mediated by DNMT1, the recurrence risk of the patient after thermal ablation can be assessed. Furthermore, it can classify patients at risk of recurrence, thus solving the core technical problem in the prior art where the lack of understanding of the driving mechanism of tumor recurrence after thermal ablation prevents early and accurate stratification of recurrence risk, resulting in delayed clinical intervention and poor patient prognosis.

[0012] Therefore, the present invention provides the following applications of reagents for detecting DNA methyltransferase 1 expression levels: Application in the preparation of products for diagnosing recurrence of liver cancer after thermal ablation.

[0013] Application in the preparation of diagnostic kits for post-thermal ablation classification or prognostic assessment of liver cancer.

[0014] Preferably, the kit can distinguish between different recurrence risk groups after thermal ablation of liver cancer.

[0015] Preferably, the DNA methyltransferase 1 has a gene ID of 1786 in NCBI.

[0016] The present invention also provides the use of formulations that knock down DNA methyltransferase 1 expression in the preparation of products that block heat stress-induced enhanced growth and metastasis of liver cancer tumors.

[0017] Preferably, the preparation for knocking down DNMT1 expression is a recombinant vector, recombinant bacteria, or recombinant virus containing a knockdown DNA methyltransferase 1 expression vector.

[0018] The present invention also provides a product comprising the above-described formulation with knocked-down DNMT1 expression.

[0019] This invention provides a diagnostic kit for liver cancer after thermal ablation, containing reagents for detecting the expression level of DNA methyltransferase 1.

[0020] Preferably, the reagent is a primer for detecting the expression level of DNA methyltransferase 1, containing an upstream primer: 5′-AGAACGGTGCTCATGCTTACA-3′; and a downstream primer: 5′-CTCTACGGGCTTCACTTCTTG-3′.

[0021] This invention, by simultaneously monitoring the upstream driving factor (DNMT1 protein) and the downstream global effect (DNA hypermethylation 5mC) of the mechanism, constructs a detection and analysis system that can accurately identify patients whose tumor cells have been "armed" by heat stress and entered a high-speed progression track. This fundamentally improves the assessment of recurrence risk from "probabilistic speculation" to "mechanistic early warning," providing an irreplaceable decision-making basis for subsequent individualized intervention.

[0022] The present invention provides a detection and analysis system for classification or prognostic assessment after thermal ablation of liver cancer, including (1) a quantitative detection module for DNMT1 expression, (2) a quantitative detection module for DNA hypermethylation 5mC, (3) a data input module, (4) a data analysis and judgment module and (5) a result output module.

[0023] Furthermore, the quantitative detection module (1) and module (2) quantitatively detect the expression level of DNMT1 or 5mC in the sample, and transmit the measured expression level value to the data analysis and judgment module (4) through the data input module (3) for data integration and analysis, judge the result according to its judgment criteria, and output the result through the result output module (5).

[0024] Preferably, the quantitative detection module (1) and module (2) stain the paraffin sections of tumor tissue using immunohistochemistry and perform a comprehensive scoring of the percentage of positive cells.

[0025] Furthermore, data judgment and analysis are performed in the (4) data analysis and judgment module; Positive determination criteria: By comparing with known negative and positive controls, if significant DNMT1 or 5mC specific staining appears in the nuclei of tumor cells in the test sample, and the combined score of its staining intensity and the percentage of positive cells is significantly higher than that of the negative control, then it is determined that the DNMT1 or 5mC level is upregulated; specifically, it can be defined as a high-risk threshold where the IHC score of DNMT1 or 5mC is more than 1.5 times the average level of the non-ablation group, and the result is judged as positive. Negative interpretation criteria: If the IHC score of DNMT1 or 5mC is not significantly different from that of the non-ablation group or is less than 1.5 times the standard deviation of the average level of the non-ablation group, the result is judged as negative. Because high expression of DNMT1 and its primary protein function lead to enhanced 5mC hypermethylation of DNA; when DNMT1 is highly expressed but its primary protein function is inhibited, 5mC hypermethylation will not be enhanced; when DNMT1 is not highly expressed and does not perform its primary protein function, 5mC hypermethylation may be enhanced due to the action of other methyltransferases. Therefore, when DNMT1 is highly expressed but 5mC hypermethylation is not enhanced, the relapse risk can be considered medium, as protein activity may be inhibited, and when the inhibitory factor is removed, 5mC hypermethylation will be enhanced. When DNMT1 is not highly expressed but 5mC hypermethylation is enhanced, this enhancement is detached from the core factor DNMT1, and is therefore considered low relapse risk.

[0026] Output the results of the data analysis module (4) to the result output module (5) to output the classification or prognostic assessment results: If the judgment result received from module (4) is that the patient's tumor tissue has both DNMT1 and 5mC protein levels upregulated, that is, the quantitative results of modules (1) and (2) are both positive, then the patient is assessed as a high-risk group for recurrence after thermal ablation of liver cancer, and the result output is a high-risk group for recurrence after thermal ablation of liver cancer; If the DNMT1 result is positive and the 5mC result is negative, then the patient is assessed as a medium-risk group for recurrence after thermal ablation of liver cancer, and the result output is a medium-risk group for recurrence after thermal ablation of liver cancer; If the DNMT1 result is negative and the 5mC result is positive, then the patient is assessed as a low-risk group for recurrence after thermal ablation of liver cancer, and the result output is a low-risk group for recurrence after thermal ablation of liver cancer; If the quantitative results of modules (1) and (2) are both negative, then the patient is assessed as a relatively low-risk group for recurrence, and the result output is a low-risk group for recurrence after thermal ablation of liver cancer.

[0027] This invention discovers that sublethal heat stress rapidly triggers the direct binding of the molecular chaperone HSP90α to DNMT1 and upregulates DNMT1. This discovery reveals the rapid response mechanism of epigenetic regulators under heat stress. Therefore, detecting DNMT1 protein levels directly captures one of the upstream key events driving recurrence triggered by the therapeutic event of "thermal ablation," making prediction mechanism-specific and early. The upregulation of DNMT1 is the cause, while the genomically generated hypermethylated DNA 5mC produced by it is the consequence leading to malignant phenotypes. This invention not only detects the gene (DNMT1) but also its functional output (DNA hypermethylated 5mC), forming a complete functional verification loop. This mechanism-based dual-indicator approach greatly improves the accuracy and reliability of prediction, effectively avoiding false positives or false negatives caused by individual differences or technical errors.

[0028] Therefore, this invention assesses the risk of recurrence in patients with hepatocellular carcinoma (HCC) after thermal ablation by detecting the expression level of DNA methyltransferase 1 (DNMT1) and the level of DNA hypermethylation 5mC throughout the genome in tumor tissues.

[0029] Therefore, this invention provides the application of a reagent for detecting the expression level of DNA methyltransferase 1 (DNMT1) in the preparation of products for assessing the risk of recurrence after thermal ablation of liver cancer.

[0030] This invention also provides the application of reagents for detecting the expression level of DNA methyltransferase 1 and the level of 5mC hypermethylation of genome-wide DNA in the preparation of products for assessing the risk of recurrence after thermal ablation of liver cancer.

[0031] In addition, the present invention also provides the application of DNA methyltransferase 1 as a drug target in screening drugs for preventing recurrence of liver cancer after thermal ablation; preferably, it provides the application of DNMT1 inhibitors in the preparation of drugs for preventing recurrence of liver cancer after thermal ablation.

[0032] The present invention has the following beneficial effects: This invention establishes for the first time the complete causal molecular chain of "heat stress → upregulation of DNMT1 protein stability → genome-wide DNA hypermethylation 5mC" as the core biological basis for predicting recurrence after thermal ablation of liver cancer. Based on this, a multi-dimensional joint prediction model system based on DNMT1 protein level and DNA hypermethylation 5mC status is created. By detecting the expression level of DNA methyltransferase 1 (DNMT1) and the genome-wide DNA hypermethylation 5mC level in tumor tissues of hepatocellular carcinoma (HCC) patients, it can be used for subtyping or prognostic assessment after liver cancer thermal ablation. This solves the core technical problem in existing technologies where the lack of understanding of the driving mechanism of tumor recurrence after thermal ablation prevents early and accurate risk stratification of patients, leading to delayed clinical intervention and poor patient prognosis.

[0033] Meanwhile, the present invention has the following advantages: Existing technologies have only observed a high recurrence rate and increased malignancy after thermal ablation, but the underlying persistent driving mechanism remains unclear. This invention reveals for the first time a complete causal chain of "sublethal heat stress → DNMT1-specific upregulation → genome-wide DNA hypermethylation 5mC," establishing DNMT1 and DNA hypermethylation 5mC as biomarkers based on a profound understanding of the mechanisms, rather than a simple correlation finding. Furthermore, it confirms that DNMT1 levels and DNA hypermethylation 5mC status are objective, repeatable indicators directly related to recurrence after ablation, enabling early and accurate identification of patients at high risk of recurrence.

[0034] This invention provides crucial decision-making support for personalized treatment by accurately identifying high-risk relapse populations. This enables closer follow-up or more aggressive adjuvant therapy (e.g., epigenetic therapy targeting DNMT1) for high-risk patients, thus shifting from a "one-size-fits-all" approach to "personalized precision prevention and treatment," ultimately improving long-term patient survival.

[0035] This invention has broad application prospects and can guide new drug development. It not only provides predictive tools but also identifies intervention targets. DNMT1, as a key effector molecule in this cascade reaction, provides a clear and effective target for developing innovative drugs to prevent recurrence of liver cancer after thermal ablation (such as DNMT1 inhibitors), possessing significant clinical translational value and market potential. Attached Figure Description

[0036] Figure 1 The image shows the results of Western blot validation of tumor cells with knocked-down DNMT1.

[0037] Figure 2 To verify in vivo experiments the effect of DNMT1 knockdown on tumor growth and metastasis in the insufficiently ablated group (Figure A: tumor size phenotype; B: tumor volume; C: tumor section staining results; D: number of metastatic tumors).

[0038] Figure 3 To verify the effect of DNMT1 knockdown on tumor growth and metastasis in the insufficiently ablated group in the secondary transplantation experiment (Figure A: tumor size phenotype; B: tumor volume; C: tumor section staining results; D: number of metastatic tumors).

[0039] Figure 4 The effect of knocking down DNMT1 on tumor growth and metastasis in the insufficiently ablated group was shown in Figure A (staining results; B shows the number of metastatic nodules per lung).

[0040] Figure 5 Figure A shows the results of DNMT1 and DNA hypermethylation assays in the ablation-deficient group (Figure A shows the H&E staining results; Figure B shows the protein expression levels of 5mC and DNMT1; Figure C shows the immunohistochemical (IHC) staining results).

[0041] Figure 6 The results show the expression of DNA genome 5mC and DNMT1 protein in recurrent HCC tissue after thermal ablation (Figure A shows the IHC staining results; Figure B shows the IHC quantification results).

[0042] Figure 7 To analyze the expression of DNA methylation markers (Figure A shows the results of spot blot analysis; Figure B shows the results of protein blot analysis).

[0043] Figure 8This is a graph showing the change in 5mC level after knocking down DNMT1.

[0044] Figure 9 ROC curve for evaluating DNMAT1 as a predictor of relapse risk. Detailed Implementation

[0045] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0046] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0047] In the examples, DNMT1 is a methyltransferase that mainly interacts with methylated 5mC. Since methylation is a state and there is more than one type, the entire text is described using DNA hypermethylated 5mC.

[0048] Example 1: The role of DNMT1 in mediating the growth and metastasis of hepatocellular carcinoma after ablation Based on the molecular mechanism discovered in previous biological studies, sublethal heat stress (41-50°C) during thermal ablation enhances the binding of HSP90α to DNMT1 and promotes USP7-mediated DNMT1 deubiquitination, leading to increased DNMT1 protein stability and accumulation in the cell nucleus. This was verified by knocking out DNA methyltransferase 1 (DNMT1, DNA methyltransferase 1 [Homosapiens (human)], gene ID: 1786 in NCBI).

[0049] 1. Effects of DNMT1 knockdown on tumor cells: The highly metastatic human hepatocellular carcinoma cell line HCCLM3 (purchased from ATCC) was used. A stably knocked-down DNMT1 cell line and a control cell line (shNC) were constructed via lentiviral transfection. After tumor formation by subcutaneous seeding of cells in nude mice, the tumors were surgically transplanted into the livers of another batch of nude mice to establish an orthotopic xenograft model. The knockdown sequence used was: shDNMT1#1: 5′-GGAAGAAGAGTTACTATAAT-3′; shDNMT1#2: 5′-GCTTCAGTGTGTACTGTAAT-3′.

[0050] Western blot verification yielded shDNMT1#1 and shDNMT1#2, as follows: Figure 1 As shown.

[0051] Experimental grouping and treatment: Tumor-bearing mice were randomly divided into four groups (n=6): (1) shNC sham-operated group; (2) shNC incomplete ablation group; (3) shDNMT1#1 incomplete ablation group; (4) shDNMT1#2 incomplete ablation group. Ablation treatment was performed using a clinical microwave ablation system, monitored by an infrared thermal imager, with the highest temperature at the tumor center controlled at 60-65°C to simulate the situation of incomplete clinical ablation and the formation of a peripheral sublethal zone (41-50°C). The sham-operated group only underwent laparotomy to expose the tumor and did not receive hyperthermia.

[0052] Mice were euthanized 4 weeks after treatment, and the liver tumor was completely dissected. The tumor volume was measured and calculated using calipers (formula: V = major axis × minor axis). 2 / 2). Lung tissue was removed, fixed in 4% paraformaldehyde, embedded in paraffin, stained with hematoxylin and eosin, and lung metastatic nodules were counted under a microscope. The percentage of metastatic lesion area to total lung area was calculated using ImageJ software.

[0053] The results are as follows Figure 2 As shown in Figures AB, the tumor volume in the insufficient shNC ablation group was reduced by 30.6% compared to the shNC sham surgery group, indicating that thermal ablation has a direct killing effect. However, the two DNMT1 knockdown groups (shDNMT1#1 and #2) achieved tumor volume inhibition rates of 85.3% and 82.5% respectively after receiving the same ablation, and their tumor suppression effect was significantly better than that of the insufficient shNC ablation group. P <0.001).

[0054] Furthermore, it was shown that all shNC-treated mice developed lung metastases, such as... Figure 2 As shown in CD, compared with the sham-operated group, the number of lung metastatic nodules in mice in the shNC ablation insufficiency group was significantly increased ( P <0.01). Conversely, in the shDNMT1 knockdown ablation group, only a few mice showed sporadic metastases, and the metastatic burden (number of nodules) was significantly suppressed compared to the shNC insufficiency ablation group. P <0.001).

[0055] 2. Secondary transplantation experiment to verify the cancer-promoting effect of DNMT1: To eliminate the interference of the direct killing effect of thermal ablation on the results, the surviving tumor tissue after the initial ablation was re-transplanted into a new batch of mice. The results are as follows: Figure 3 As shown, tumors from the shNC ablation group grew faster after secondary transplantation, with a volume 1.8 times that of tumors from sham surgery; while tumors from the shDNMT1 knockdown ablation group showed significantly inhibited growth, with a volume only 15.2%-21.7% of the control. Lung metastasis results were consistent with the trend in the primary transplantation experiment.

[0056] Further experimental lung metastasis model was constructed by injecting HCCLM3 cells pretreated with sublethal heat stress (47°C water bath for 15 minutes) via tail vein. Results are as follows... Figure 4 As shown, heat-stressed shNC cells formed far more metastatic foci in the lungs than untreated cells; and knockdown of DNMT1 completely blocked the heat-stress-induced metastatic enhancement effect.

[0057] The above results, validated by an in vivo model, show that upregulation of DNMT1 is a key driver of sublethal heat stress promoting residual tumor growth and metastasis after thermal ablation of liver cancer, and that inhibiting DNMT1 can effectively reverse the cancer-promoting risk brought about by thermal ablation.

[0058] Example 2: Methylation and its biomarker analysis after HCC thermal ablation Following existing research methods (Chen, Y., Bei, J., Liu, M., Huang, J., Xie, L., Huang, W., Cai, M., Guo, Y., Lin, L., & Zhu, K. (2021). Sublethal heat stress-induced O-GlcNAcylation coordinates the Warburg effect to promote hepatocellular carcinoma recurrence and metastasis after thermal ablation. Cancer letters, 518, 23–34. https: / / doi.org / 10.1016 / j.canlet.2021.06.001.), two independent orthotopic xenograft (PDOX) mouse models derived from hepatocellular carcinoma (HCC) patients were constructed. Incomplete thermal ablation was performed on model animals using a clinical microwave ablation (MWA) system. Real-time monitoring with an infrared thermometer ensured that the maximum temperature within the tumor was controlled at 60-65°C, guaranteeing the formation of a central lethal zone (>50°C) and a peripheral sublethal zone (41-50°C). A sham-operated group of mice served as a control (without thermal ablation). Twenty-four hours post-ablation, residual tumor tissue was collected for H&E staining and immunohistochemical (IHC) analysis, and reduced characterization bisulfite sequencing (RRBS) was used for DNA methylome analysis.

[0059] The results are as follows Figure 5 As shown in Figure A, H&E staining confirmed the presence of residual live tumor cells in the adjacent necrotic area within the ablation group tumor tissue.

[0060] Immunohistochemical (IHC) staining results as follows Figure 5 As shown in BC, the protein expression levels of DNA methylation markers 5-methylcytosine (5mC) and DNA methyltransferase 1 (DNMT1) were significantly upregulated in the residual tumor after ablation, by at least 1.5-fold. However, the expression of other methyltransferases, DNMT3A and DNMT3B, did not show significant changes.

[0061] Example 3: Retrospective Analysis of Clinical Samples Clinical samples were collected from 41 patients with hepatocellular carcinoma (HCC), including 19 patients who underwent reoperation after thermal ablation (post-ablation group) and 22 patients who underwent primary tumor resection without ablation (non-ablation group). There were no significant differences in baseline characteristics between the two groups. IHC staining and quantitative analysis were performed on the samples.

[0062] IHC quantification results are as follows Figure 6 As shown in Figures AB, the results indicated that the levels of 5mC and DNMT1 proteins in recurrent tumor tissue after ablation were significantly higher than those in non-ablated primary tumor tissue. No significant differences were found in the expression of other methyltransferases, DNMT3A and DNMT3B.

[0063] Example 4: Validation of an in vitro cell model Human HCC cell lines Huh7 and HCCLM3 (purchased from ATCC) were used. Cells were heated in a 47°C water bath for 15 minutes to simulate sublethal heat stress, while control cells were maintained at 37°C. Cells were collected at different time points after treatment, and the expression of DNA methylation markers 5mC and DNMT1, as well as other methyltransferases DNMT3A and DNMT3B, was analyzed by dot blot and Western blot analysis.

[0064] Dot imprint analysis, such as Figure 7 As shown in Figure A, the genomic DNA 5mC level in Huh7 and HCCLM3 cells significantly increased after sublethal heat treatment.

[0065] Protein blots, such as Figure 7 As shown in Figure B, after sublethal heat treatment, the expression of DNMT1 protein was upregulated in both cell types, while the expression of DNMT3A remained unchanged, and the expression of DNMT3B decreased slightly.

[0066] Example 5: Effects of DNMT1 knockdown on HCC cell lines To confirm the function of DNMT1, a stable DNMT1 knockdown HCC cell line was constructed, following the same method as in Example 1. The results were obtained through dot blot analysis. Figure 8As shown, knocking down DNMT1 effectively prevents the increase in 5mC levels induced by sublethal heat stress, proving that DNMT1 is a key executor of heat stress-triggered DNA hypermethylation, indicating that sublethal heat stress induces DNA hypermethylation in residual HCC cells after ablation by upregulating DNMT1.

[0067] In summary, through retrospective analysis of PDOX animal models, clinical samples, and in vitro cell experiments, we have jointly demonstrated that sublethal heat stress can specifically upregulate DNMT1 protein expression and induce genome-wide DNA hypermethylation. This provides a solid experimental basis for using DNMT1 and DNA hypermethylation status as biomarkers for predicting recurrence after thermal ablation of liver cancer.

[0068] Example 6: Validation of biomarkers for post-thermal ablation classification or prognostic assessment of liver cancer We selected samples from 51 patients with hepatocellular carcinoma who remained relapse-free for 5 years after ablation and 43 patients with hepatocellular carcinoma who relapsed 5 years after ablation. Receiver operating characteristic (ROC) curves were used to evaluate the ability of the diagnostic biomarker DNMT1 gene to distinguish between the two groups (high recurrence vs. low recurrence). Biomarker values ​​were sorted from highest to lowest (higher values ​​indicate a higher likelihood of recurrence). Each possible value (or a series of thresholds) was used as a "cutoff point," with values ​​above this point classified as "high recurrence" and values ​​below as "low recurrence." At each cutoff point, the corresponding sensitivity (ordinate) and 1-specificity (radical) were calculated to evaluate the discriminative power of the 5-year recurrence prediction model. The ROC curve shows 1-specificity (false positive rate) on the x-axis and sensitivity (true positive rate) on the y-axis.

[0069] The results are as follows Figure 9 As shown, the AUC is 0.78, indicating good discriminative ability within the range of 0.7 ≤ AUC < 0.9. This suggests that the model possesses stable predictive ability throughout the observation period.

[0070] Example 7: Application of biomarker detection and analysis for post-thermal ablation classification or prognostic assessment of liver cancer. Based on the above research showing that DNMT1 is a diagnostic marker for recurrence after thermal ablation of liver cancer, and combined with the DNA hypermethylation 5mC status, it can be used for subtyping or prognostic assessment of liver cancer after thermal ablation to diagnose and differentiate different recurrence risk groups after thermal ablation, and accurately predict the risk of recurrence. This embodiment provides a detection and analysis system for subtyping or prognostic assessment of liver cancer after thermal ablation, including (1) a quantitative detection module for DNMT1 expression, (2) a quantitative detection module for DNA hypermethylation 5mC, (3) a data input module, (4) a data analysis and judgment module, and (5) a result output module.

[0071] The quantitative detection module quantitatively detects the expression levels of DNMT1 and 5mC in the sample and transmits the measured expression levels to the data analysis module through the data input module. The data input module then transmits the data to the data analysis and judgment module for data integration and analysis. The module judges the results according to its judgment criteria and finally outputs the results through the result output module.

[0072] The specific application steps of this system are as follows: 1. Sample Acquisition Sample source: The samples to be tested are tumor tissue samples obtained from patients with hepatocellular carcinoma through surgical resection or biopsy. The patients include, but are not limited to, those who are scheduled to receive thermal ablation therapy, those who are currently receiving thermal ablation therapy, or those who have completed thermal ablation therapy.

[0073] The biomarker used was DNA methyltransferase 1 (DNMT1), and the protein expression level at 5mC during DNA hypermethylation was also detected.

[0074] 2. Detection of biomarkers The DNMT1 expression level was detected and quantified using the (1) DNMT1 expression quantitative detection module. The specific detection method can be immunohistochemistry (IHC) to stain paraffin sections of tumor tissue.

[0075] The expression of protein with high methylation of DNA 5mC was detected by using the (2) DNA high methylation 5mC quantitative detection module. The specific detection method can be to use immunohistochemistry (IHC) to stain paraffin sections of tumor tissue.

[0076] 3. Data input and processing Using the (3) data input module, the DNMT1 and 5mC expression results from modules (1) and (2) are input into the (4) data analysis and judgment module.

[0077] 4. Data analysis and judgment The data analysis module (4) is used to perform judgment and analysis on the data input from module (3): Positive Criteria: By comparing the sample with known negative controls (such as primary HCC tissue from the non-ablation group) and positive controls, if significant DNMT1 or 5mC-specific staining is observed in the nuclei of tumor cells in the test sample, and the combined score of staining intensity and percentage of positive cells (H-Score) is significantly higher than that of the negative controls, then the DNMT1 or 5mC level is considered upregulated. Specifically, a high-risk threshold is defined as an IHC score for DNMT1 and 5mC that is 1.5 times or more above the average level of the non-ablation group, indicating a positive result.

[0078] Negative determination criteria: If the IHC score of DNMT1 or 5mC is not significantly different from that of the non-ablation group or is less than 1.5 times the standard deviation of the average level of the non-ablation group, the result is judged as negative.

[0079] Because high expression of DNMT1 and its primary protein function lead to enhanced 5mC hypermethylation of DNA; when DNMT1 is highly expressed but does not perform its primary protein function due to inhibited protein activity, 5mC hypermethylation will not be enhanced; when DNMT1 is not highly expressed and does not perform its primary protein function, 5mC hypermethylation may be enhanced due to the action of other methyltransferases; therefore, when DNMT1 is highly expressed but 5mC hypermethylation is not enhanced, the relapse risk can be considered medium. Since protein activity may be inhibited, 5mC hypermethylation will be enhanced when the inhibitory factor is removed. When DNMT1 is not highly expressed but 5mC hypermethylation is enhanced, this enhancement is detached from the core factor DNMT1 and is considered low-risk for relapse.

[0080] 5. Result Output The results of the data analysis module (4) are output to the result output module (5) for final result output. Upon receiving the judgment result from module (4): if the patient's tumor tissue shows simultaneous upregulation of DNMT1 and 5mC protein levels, i.e., the quantitative results of modules (1) and (2) are both positive, then the patient is assessed as a high-risk group for recurrence after thermal ablation of liver cancer, and the result output is "high-risk group for recurrence after thermal ablation of liver cancer"; if the DNMT1 result is positive and the 5mC result is negative, then the patient is assessed as a medium-risk group for recurrence after thermal ablation of liver cancer, and the result output is "medium-risk group for recurrence after thermal ablation of liver cancer"; if the DNMT1 result is negative and the 5mC result is positive, then the patient is assessed as a low-risk group for recurrence after thermal ablation of liver cancer, and the result output is "low-risk group for recurrence after thermal ablation of liver cancer"; if the quantitative results of modules (1) and (2) are both negative, then the patient is assessed as a relatively low-risk group for recurrence, and the result output is "low-risk group for recurrence after thermal ablation of liver cancer".

[0081] The sublethal heat stress (41-50°C) during thermal ablation triggers the direct binding of heat shock protein 90α (HSP90α) to DNMT1. This binding stabilizes DNMT1 through ubiquitin-specific protease 7 (USP7)-mediated deubiquitination, preventing its degradation and leading to its accumulation in the cell nucleus. Upregulated DNMT1 then catalyzes hypermethylation of DNA across the entire genome (including key regions such as promoters, CGI islands, and the genome body). This epigenetic remodeling is the core mechanism driving residual hepatocellular carcinoma cells to acquire stronger proliferative and metastatic capabilities, ultimately leading to clinical recurrence. Therefore, detecting the key molecule DNMT1 and its functional product (DNA hypermethylation at 5mC) in this pathway can directly reflect whether this malignant progression mechanism is activated, thereby enabling accurate prediction of recurrence risk.

[0082] This embodiment also provides a test kit for post-ablation typing or prognostic assessment of liver cancer. The kit detects the protein expression level of DNMT1 and the 5mC state of DNA hypermethylation in the sample to perform post-ablation typing and prognostic assessment of liver cancer.

[0083] The kit contains reagents required for detecting the protein expression level of DNMT1. The reagents are primers for detecting the expression level of DNA methyltransferase 1, including an upstream primer: 5′-AGAACGGTGCTCATGCTTACA-3′; and a downstream primer: 5′-CTCTACGGGCTTCACTTCTTG-3′.

[0084] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of a reagent for detecting DNA methyltransferase 1 expression levels in the preparation of products for diagnosing recurrence after thermal ablation of liver cancer, characterized in that, The gene ID of the DNA methyltransferase 1 in NCBI is 1786.

2. The application of reagents for detecting DNA methyltransferase 1 expression levels in the preparation of detection kits for post-ablation typing or prognostic assessment of liver cancer, characterized in that... The gene ID of the DNA methyltransferase 1 in NCBI is 1786.

3. The application of a formulation that knocks down DNA methyltransferase 1 expression in the preparation of a product that blocks heat stress-induced enhanced growth and metastasis of liver cancer tumors; characterized in that, The formulation that knocks down DNA methyltransferase 1 expression is a recombinant vector, recombinant bacteria, or recombinant virus containing a recombinant vector that knocks down DNA methyltransferase 1 expression; the gene ID of DNA methyltransferase 1 in NCBI is 1786.

4. A diagnostic kit for liver cancer after thermal ablation, characterized in that, Contains reagents for detecting DNA methyltransferase 1 expression levels.

5. The reagent kit according to claim 4, characterized in that, The reagent is a primer for detecting the expression level of DNA methyltransferase 1, containing an upstream primer: 5′-AGAACGGTGCTCATGCTTACA-3′; and a downstream primer: 5′-CTCTACGGGCTTCACTTCTTG-3′.

6. A detection and analysis system for post-ablation classification or prognostic assessment of liver cancer, characterized in that, It includes (1) a quantitative detection module for DNMT1 expression, (2) a quantitative detection module for DNA hypermethylation 5mC, (3) a data input module, (4) a data analysis and judgment module and (5) a result output module.

7. The analysis system according to claim 6, characterized in that, The quantitative detection module (1) and module (2) quantitatively detect the expression level of DNMT1 or 5mC in the sample, and transmit the measured expression level value to the data analysis and judgment module (4) through the data input module (3) for data integration and analysis, and output the result through the result output module (5) according to its standard judgment.

8. The analysis system according to claim 7, characterized in that, The quantitative detection modules (1) and (2) stain paraffin sections of tumor tissue using immunohistochemistry and perform comprehensive scoring of the percentage of positive cells.

9. The analysis system according to claim 8, characterized in that, Data analysis and judgment are performed in module (4); Positive determination criteria: By comparing with known negative and positive controls, if significant DNMT1 or 5mC specific staining appears in the nuclei of tumor cells in the test sample, and the combined score of its staining intensity and the percentage of positive cells is significantly higher than that of the negative control, then it is determined that the DNMT1 or 5mC level is upregulated; specifically, the IHC score of DNMT1 or 5mC is defined as a high-risk threshold of more than 1.5 times the average level of the non-ablation group, and the result is judged as positive. Negative interpretation criteria: If the IHC score of DNMT1 or 5mC is not significantly different from that of the non-ablation group or is less than 1.5 times the standard deviation of the average level of the non-ablation group, the result is judged as negative. Output the results of the (4) data analysis module to the (5) result output module and output the classification or prognostic assessment results: If the judgment result of module (4) is received: the patient's tumor tissue has both DNMT1 and 5mC protein levels upregulated, that is, the quantitative results of modules (1) and (2) are both positive, then the patient is assessed as a high-risk group for recurrence after thermal ablation of liver cancer, and the result output is a high-risk group for recurrence after thermal ablation of liver cancer. If the DNMT1 result is positive and the 5mC result is negative, the patient is assessed as being in the intermediate-risk group for recurrence after thermal ablation of liver cancer, and the result output is: intermediate-risk group for recurrence after thermal ablation of liver cancer. If the DNMT1 result is negative and the 5mC result is positive, the patient is assessed as a low-risk group for recurrence after thermal ablation of liver cancer, and the result output is "low-risk group for recurrence after thermal ablation of liver cancer". If the quantitative results of modules (1) and (2) are both negative, the patient is assessed as a relatively low-risk group for recurrence, and the output is a low-risk group for recurrence after thermal ablation of liver cancer.

10. Application of reagents for detecting the expression level of DNA methyltransferase 1 and whole-genome DNA hypermethylation at 5mC in the preparation of products for assessing the risk of recurrence after thermal ablation of liver cancer.

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