Marker probe combination and detection kit for evaluating risk of lung metastasis after liver transplantation of liver cancer

Abstract

The application discloses a kind of marker probes for evaluating the risk of lung metastasis after liver transplantation of liver cancer, including the probe for detecting MECOM, the probe for detecting MECOM is nucleotide sequence is one or more of SEQ ID NO.1-8.The application also discloses the application of the marker probes for evaluating the risk of lung metastasis after liver transplantation of liver cancer in preparation detection kit.The marker probes for evaluating the risk of lung metastasis after liver transplantation of liver cancer provided by the application realize the accurate detection of the expression level of MECOM of the patient with the risk of lung metastasis after liver transplantation of liver cancer;Meanwhile, combined with CTC typing detection, the overall positive coincidence rate reaches 97%, can be used to predict the recurrence risk and prognosis of the patient with the risk of lung metastasis after liver transplantation of liver cancer, to further provide new molecular basis for evaluating recurrence risk, predicting metastasis tendency and judging prognosis, with important clinical significance and application value.

Description

Technical Field

[0001] This invention belongs to the field of gene detection technology, specifically relating to a biomarker probe combination and detection kit for assessing the risk of lung metastasis after liver transplantation for liver cancer. Background Technology

[0002] Liver cancer is one of the most common malignant tumors worldwide, consistently ranking among the top in both incidence and mortality. Hepatocellular carcinoma constitutes the majority of primary liver cancers, and its development is often closely related to chronic liver disease and environmental factors. Despite advancements in surgical resection, liver transplantation, local ablation, and targeted therapy, the overall prognosis for liver cancer patients remains unsatisfactory. This is primarily due to high postoperative recurrence rates, difficulty in monitoring early metastasis, and a lack of precise prognostic tools. Clinically, current technologies largely rely on imaging examinations and serum biomarkers for monitoring and assessment. However, these methods have limited sensitivity and specificity in identifying early micrometastases, stratifying recurrence risk, and predicting individualized prognosis, failing to meet the needs of precision medicine. Particularly at the molecular level, there is a lack of stable and specific gene detection indicators and corresponding testing tools that can reflect liver cancer progression and the risk of recurrence and metastasis.

[0003] In recent years, molecular biology research has revealed a close relationship between the MECOM gene and the development of liver cancer, mainly in the following aspects:

[0004] Promoting the proliferation of liver cancer cells: The MECOM gene can regulate the expression of cell cycle-related proteins, enabling liver cancer cells to pass through cell cycle checkpoints more quickly and accelerate cell division, thereby promoting the proliferation of liver cancer cells. Studies have found that in liver cancer cell lines, after the expression of the MECOM gene is upregulated, the expression of proliferative proteins such as cyclin D1 increases, leading to an increase in the number of liver cancer cells.

[0005] Inhibiting apoptosis in liver cancer cells: The MECOM gene can inhibit the apoptosis process in liver cancer cells. It can regulate apoptosis-related signaling pathways, such as by inhibiting the activity of the caspase family, preventing the cascade reaction of apoptosis, allowing liver cancer cells to evade the body's immune surveillance and natural apoptosis mechanism, thus promoting the occurrence and development of liver cancer.

[0006] Induction of epithelial-mesenchymal transition (EMT): The MECOM gene can induce EMT in liver cancer cells. By activating related signaling pathways, it weakens the epithelial cell characteristics and enhances the mesenchymal cell characteristics of liver cancer cells, thereby giving them a stronger ability to migrate and invade. This process makes it easier for liver cancer cells to break through the basement membrane, invade surrounding tissues and blood vessels, and thus metastasize to distant sites.

[0007] Influence on the stemness of liver cancer cells: The MECOM gene is related to the maintenance of stemness in liver cancer cells; it can regulate liver cancer stem cell-related signaling pathways and markers, enabling liver cancer cells to maintain stem cell-like characteristics, such as self-renewal capacity and multi-lineage differentiation potential; these stem liver cancer cells have stronger resistance to traditional treatments such as chemotherapy and radiotherapy, which is one of the important reasons for liver cancer recurrence and metastasis.

[0008] Association with clinicopathological features: Clinical studies have shown that the expression level of the MECOM gene is closely related to clinicopathological features of liver cancer patients, such as tumor size, TNM stage, and vascular invasion. Liver cancer patients with high MECOM gene expression tend to have larger tumors, later stages, and are more prone to vascular invasion and distant metastasis, and their prognosis is relatively poor. The MECOM gene plays an important role in multiple stages of liver cancer occurrence, development, invasion, and metastasis. This suggests that the expression level of the MECOM gene may become a potential molecular marker for assessing the malignancy of liver cancer, predicting the risk of recurrence and metastasis, and improving patient prognosis.

[0009] However, the application of MECOM gene in the clinical detection of liver cancer is still in the research stage. There is a lack of stable, efficient and clinically applicable dedicated detection probes and matching reagents on the market, which makes it impossible to achieve rapid, sensitive and quantitative detection of MECOM mRNA expression levels in liver cancer tissue or peripheral blood. This limits the practical application of this molecular marker in clinical prognosis and personalized treatment guidance. Summary of the Invention

[0010] This invention aims to at least partially address one of the technical problems in related technologies. Therefore, the main objective of this invention is to provide a biomarker probe combination and detection kit for assessing the risk of lung metastasis after liver transplantation for liver cancer.

[0011] The objective of this invention is achieved through the following technical solution:

[0012] A biomarker probe for assessing the risk of lung metastasis and prognosis after liver transplantation for liver cancer is characterized by comprising a probe for detecting MECOM, wherein the probe for detecting MECOM has one or more nucleotide sequences of SEQ ID NO.1 to SEQ ID NO.8.

[0013] In some specific embodiments, a probe for detecting MECOM is included, wherein the probe for detecting MECOM is a combination of two nucleotide sequences, namely SEQ ID NO.3 and SEQ ID NO.6.

[0014] In some specific embodiments, a probe for detecting MECOM is included, wherein the probe for detecting MECOM is a combination of three nucleotide sequences: SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.7.

[0015] In some specific embodiments, the device further includes epithelial peripheral blood CTC-specific capture probes EpCAM, CK8, CK18, and CK19, with nucleotide sequences as shown in SEQ ID NO.9-SEQ ID NO.14, SEQ ID NO.15-SEQ ID NO.20, SEQ ID NO.21-SEQ ID NO.26, and SEQ ID NO.27-SEQ ID NO.32, respectively; it also includes mesenchymal peripheral blood CIC-specific capture probes Vimentin and Twist, with nucleotide sequences as shown in SEQ ID NO.33-SEQ ID NO.38 and SEQ ID NO.39-SEQ ID NO.44, respectively; and it also includes leukocyte phenotype-specific capture probe CD45, with nucleotide sequences as shown in SEQ ID NO.45-SEQ ID NO.50.

[0016] In some specific embodiments, the bDNA probes for EpCAM and CK8 / 18 / 19, whose sequences are shown in SEQ ID NO. 51-SEQ ID NO. 53; the bDNA probes for Vimentin and Twist, whose sequences are shown in SEQ ID NO. 54-SEQ ID NO. 56; and the bDNA probe for CD45, whose sequences are shown in SEQ ID NO. 57-SEQ ID NO. 59.

[0017] In some specific embodiments, the fluorescent dyes include AlexaFluor594 for labeling epithelial peripheral blood CTC-specific capture probes EpCAM, CK8, CK18, and CK19; AlexaFluor488 for labeling mesenchymal peripheral blood CTC-specific capture probes Vimentin and Twist; AlexaFluor750 for labeling leukocyte phenotype-specific capture probe CD45; and AlexaFluor647 for labeling MECOM probes.

[0018] As part of the same inventive concept, the present invention also provides the application of the probe in the preparation of a detection kit.

[0019] As part of the same inventive concept, the present invention also provides a kit for assessing the risk of lung metastasis and prognosis after liver transplantation for liver cancer, which includes the aforementioned probe for detecting MECOM.

[0020] As part of the same inventive concept, this invention also provides the application of the probe in the preparation of a kit for predicting the risk of lung metastasis and prognosis after liver transplantation for liver cancer.

[0021] As part of the same inventive concept, this invention also provides the application of the probe in constructing a risk assessment model for lung metastasis after liver transplantation for liver cancer.

[0022] Regarding the definition:

[0023] The term "detection" is used in its broadest sense herein to include both qualitative and quantitative measurements of target molecules. Detection includes simply identifying the presence of a target molecule in a sample as well as determining whether the target molecule is present in the sample at a detectable level.

[0024] As used herein, the term "biomarker" refers to an indicator that can be detected in a sample, such as predictive, diagnostic, and / or prognostic indicators. Biomarkers can serve as indicators of a specific subtype of disease or condition (e.g., cancer) characterized by certain molecular, pathological, histological, and / or clinical features. In some embodiments, the biomarker is a gene. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and / or RNA), polynucleotide copy number alterations (e.g., DNA copy number), peptides, peptide and polynucleotide modifications (e.g., post-translational modifications), carbohydrates, and / or glycolipid-based molecular markers.

[0025] The term “expression level” refers to the amount of a biomarker in a biological sample. “Expression” generally refers to the process by which information (e.g., genetically encoded and / or epigenetically inherited) is transformed into structures that are present and function in the cell. Thus, as used herein, “expression” can refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of a polypeptide). Fragments of transcribed polynucleotides, translated polypeptides, or polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of a polypeptide) should also be considered expressed, whether they originate from transcripts generated by alternative splicing or degraded transcripts, or from post-translational processing of polypeptides, such as through proteolysis.

[0026] As used herein, the term “sample” refers to a composition obtained or derived from a subject and / or individual of interest that contains cells and / or other molecular entities to be characterized and / or identified, for example, based on physical, biochemical, chemical, and / or physiological characteristics. For example, the phrase “disease sample” or variations thereof refers to any sample obtained from a subject of interest that is expected to or is known to contain cells and / or molecular entities to be characterized. Samples include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, and tissue culture fluids, tissue extracts such as homogenized tissue, cell extracts, and combinations thereof.

[0027] As used herein, “treatment” refers to a clinical intervention that attempts to alter the natural course of the disease in the individual being treated, and may be implemented during the course of clinicopathological development. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and eliminating or improving prognosis.

[0028] Predicting prognosis refers to predicting the process or outcome of a patient's condition, but does not mean that the process or outcome can be predicted with 100% accuracy. Predicting prognosis means determining whether the likelihood of certain processes or outcomes has increased, not determining the likelihood of certain processes or outcomes occurring by comparing them to a situation where they do not occur.

[0029] Compared with the prior art, the present invention has at least the following advantages:

[0030] The biomarker probe provided by this invention for assessing the risk of lung metastasis after liver transplantation for hepatocellular carcinoma (HCC) was tested on patients with lung metastasis after liver transplantation for HCC. The experiment showed that it accurately detected the expression level of MECOM in patients with lung metastasis after liver transplantation for HCC. When combined with CTC typing, the overall positive concordance rate reached 97%. It can be used to predict the recurrence risk and prognosis of lung metastasis after liver transplantation for HCC, thus providing new molecular evidence for assessing recurrence risk, predicting metastasis tendency and judging prognosis. It has important clinical significance and application value. Attached Figure Description

[0031] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0032] Figure 1The image shown is a microscopic examination under a fluorescence microscope of a liver cancer patient with lung metastasis after liver transplantation, obtained by the detection methods of Examples 2 and 3 using the detection kit of Example 5 of this invention (high expression of MECOM).

[0033] Figure 2 The image shown is a microscopic examination of a liver cancer patient with lung metastasis after liver transplantation, obtained by the detection methods of Examples 2 and 3 using the detection kit of Example 5 of this invention (low expression of MECOM).

[0034] Figure 3 The image shown is a microscopic examination (expressed in MECOM) of a liver cancer patient with lung metastasis after liver transplantation, obtained by the detection methods of Examples 2 and 3 using the detection kit of Example 5 of this invention.

[0035] In the figure, red fluorescent dots represent epithelial markers of peripheral blood CTCs; EpCAM, CK8, CK18 and CK19 gene expression; green fluorescent dots represent the expression of Vimentin and Twist genes, which are mesenchymal markers of peripheral blood CTCs; purple fluorescent dots represent the expression of MECOM gene, which is a marker of peripheral blood CTCs; and white signal dots represent the expression of CD45 gene, a marker of leukocytes. Detailed Implementation

[0036] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are merely descriptive and not limiting, and should not be construed as limiting the scope of protection of the present invention.

[0037] When a quantity, concentration, or other value or parameter is described as a range, preferred range, or preferred upper and lower limits, it should be understood that it is equivalent to specifically disclosing any range by combining any pair of upper or preferred values ​​with any lower or preferred values, regardless of whether the range is specifically disclosed. Unless otherwise stated, the numerical range values ​​listed herein include the endpoints of the range and all integers and fractions within that range.

[0038] Unless otherwise stated, all percentages, parts, ratios, etc. in this document are by weight.

[0039] The materials, methods, and embodiments described herein are exemplary and should not be construed as limiting unless otherwise stated.

[0040] Example 1

[0041] This embodiment provides a biomarker probe for assessing the risk of lung metastasis after liver transplantation for liver cancer, including a probe for detecting MECOM. The nucleotide sequence of the probe for detecting MECOM is as follows:

[0042] Table 1. Nucleotide sequences of probes used to detect MECOM

[0043]

[0044] The probes used for detecting MECOM were designed primarily using the online Primer5 software, which itself selects eight specific probe sequences through sequence alignment. The probe sequences were synthesized by Invitrogen.

[0045] Example 2

[0046] This embodiment provides a method for detecting peripheral blood CTC typing in assessing the risk of lung metastasis after liver transplantation for hepatocellular carcinoma. The kit used specifically includes the following components:

[0047] Table 2. Components of the kit for detecting CTC subtypes in the risk of lung metastasis after liver transplantation for hepatocellular carcinoma.

[0048]

[0049] The specific testing method includes the following steps:

[0050] S1, enrichment of circulating tumor cells;

[0051] 1) Collect 5 ml of peripheral blood sample from the patient using an EDTA anticoagulant blood collection tube, invert and mix well, add 15 ml of red blood cell lysis buffer (SurExam Inc. USA), mix well, and let stand at room temperature for 30 min to lyse the red blood cells;

[0052] The red blood cell lysis buffer formulation is: 154 mM NH4Cl, 10 mM KHCO3 and 0.1 mM EDTA;

[0053] 2) Centrifuge at 500g for 5 minutes to remove the supernatant from the blood sample;

[0054] 3) Cells were resuspended in PBS buffer (Wuhan Boster Biological Engineering Co., Ltd., catalog number AR0030) for cell precipitation;

[0055] 4) Fix the remaining cell pellet with 4% formaldehyde for 8 minutes;

[0056] 5) Transfer the fixed cells to a filter tube containing a filter membrane (BD Biosciences, USA, 8 μM), and use a vacuum filtration pump to filter the cells onto the filter membrane;

[0057] 6) After filtration, the filter membrane sample was fixed at room temperature for 1 hour using 4% formaldehyde.

[0058] In this embodiment, a filter membrane with a pore size of 8 μM is used, and epithelial tumor cells are separated according to the membrane filtration method (ISET method), which effectively removes leukocytes and retains tumor cells.

[0059] S2 and CTC typing identification:

[0060] 1) Wash the fixed filter membrane samples three times with PBS buffer and place them in a 24-well plate;

[0061] 2) Add 0.1 mg / ml proteinase K (Sigma, St. Louis, USA, CAS No.: 39450-01-6) for treatment, and let stand at room temperature for 1 hour to increase cell membrane permeability;

[0062] 3) Wash three times with PBS buffer, then add the specific capture probe:

[0063] ① Epithelial biomarker probes EpCAM, CK8, CK18, and CK19;

[0064] ② Interstitial biomarker probes Vimentin and Twist;

[0065] Probes ① and ② were hybridized with the leukocyte marker CD45 (probe sequences are shown in Table 3) and the hybridization reaction was carried out at 40℃ for 3 h. Unbound specific capture probes were washed three times with 1000 μl of elution buffer. Elution buffer formulation: 0.1×SSC (Sigma, St. Louis, USA).

[0066] 4) Add 100 μl of pre-amplification solution.

[0067] Pre-amplification buffer formulation: 30% horse serum, 1.5% sodium dodecyl sulfate (Sigma, St. Louis, USA), 3 mM Tris-HCl (pH 8.0) (Sigma, St. Louis, USA), 0.5 fmol pre-amplification probe (sequence shown in Table 4), incubated at 40℃ for 30 min to perform signal amplification probe reaction;

[0068] 5) Cool the membrane: Elute three times with 1000 μl of elution buffer (0.1×SSC), then incubate with 100 μl of amplification solution and 1 fmol of pre-amplified probe (sequences shown in Table 4) at 40 °C for 30 min;

[0069] Amplification solution formulation: 30% horse serum, 1.5% sodium dodecyl sulfate and 3mM Tris-HCl (pH 8.0);

[0070] 6) Add three labeled fluorescent proteins (sequences shown in Table 4), namely the fluorescent dyes Alexa Fluor 594 (for labeling epithelial biomarker probes EpCAM, CK8, CK18 and CK19), Alexa Fluor 488 (for labeling mesenchymal biomarker probes Vimentin and Twist), and Alexa Fluor 750 (for labeling leukocyte marker CD45), and incubate at 40°C for 30 min;

[0071] 7) Elute with 0.1×SSC, then stain the cell nuclei with DAPI (Sigma, St. Louis, USA) for 5 min, and observe the samples under 100x oil immersion using an automated fluorescence scanning microscope.

[0072] In the microscopic examination, red punctate fluorescence represents the expression of epithelial markers of peripheral blood circulating tumor cells: EpCAM, CK8, CK18 and CK19 genes; green punctate fluorescence represents the expression of mesenchymal markers of peripheral blood circulating tumor cells: Vimentin and Twist genes; white signal dots represent the expression of the leukocyte marker CD45 gene; in the test results, if the number of fluorescent signal dots is greater than 7, it is judged as positive.

[0073] Table 3 Nucleic acid probe sequences

[0074]

[0075] Table 4. Sequences of bDNA signal amplification probes

[0076]

[0077] Example 3

[0078] This embodiment provides a method for detecting the expression level of peripheral blood CTCMECOM mRNA using a biomarker probe to assess the risk of lung metastasis after liver transplantation for hepatocellular carcinoma. The kit used specifically includes the following components, as shown in Table 5:

[0079] Table 5. Components of the reagent kit for detecting risk markers of lung metastasis after liver transplantation in liver cancer.

[0080]

[0081] The detection method is basically the same as the CTC typing detection method, except that the probe used is a MECOM probe, and it is labeled with the fluorescent dye Alexa Fluor 647, which is purple. Specifically:

[0082] S1, enrichment of circulating tumor cells;

[0083] Same as implementation 2;

[0084] S2 and MECOM mRNA expression levels:

[0085] 1) Wash the fixed filter membrane samples three times with PBS buffer and place them in a 24-well plate;

[0086] 2) Add 0.1 mg / ml proteinase K (Sigma, St. Louis, USA, CAS No.: 39450-01-6) for treatment, and let stand at room temperature for 1 hour to increase cell membrane permeability;

[0087] 3) Wash three times with PBS buffer, add MECOM probe (MECOM probe sequence is SEQ ID NO.3) for hybridization, and incubate at 40℃ for 3 h; wash three times with 1000 μl elution buffer if no specific capture probe is bound; elution buffer formulation: 0.1×SSC (Sigma, St. Louis, USA);

[0088] 4) Add 100 μl of pre-amplification solution

[0089] Pre-amplification buffer formulation: 30% horse serum, 1.5% sodium dodecyl sulfate (Sigma, St. Louis, USA), 3 mM Tris-HCl (pH 8.0) (Sigma, St. Louis, USA), 0.5 fmol pre-amplification probe (sequence shown in Table 4), incubated at 40℃ for 30 min to perform signal amplification probe reaction;

[0090] 5) Cool the membrane: Elute three times with 1000 μl of elution buffer (0.1×SSC), then incubate with 100 μl of amplification solution and 1 fmol of pre-amplified probe (sequences shown in Table 3) at 40 °C for 30 min;

[0091] Amplification solution formulation: 30% horse serum, 1.5% sodium dodecyl sulfate and 3mM Tris-HCl (pH 8.0);

[0092] 6) Add the fluorescent dye Alexa Fluor 647 (marked as purple) and incubate at 40°C for 30 min;

[0093] 7) Elute with 0.1×SSC, then stain the cell nuclei with DAPI (Sigma, St. Louis, USA) for 5 min, and observe the samples under 100x oil immersion using an automated fluorescence scanning microscope.

[0094] Purple signal dots represent MECOM gene expression; MECOM expression levels are classified as high, medium, and low based on the number of signal dots: if there are ≥ 1 cell in each of the three categories of low expression / medium expression / high expression in the test results, it is considered positive.

[0095] Table 6. Classification of MECOM expression levels (high, medium, and low)

[0096]

[0097] Example 4

[0098] This embodiment provides a method for detecting the expression level of peripheral blood CTCMECOM mRNA using a biomarker probe to assess the risk of lung metastasis after liver transplantation for liver cancer. The detection method is basically the same as that in Example 3, except that the MECOM probe is a combination of the two nucleotide sequences listed in Table 1, namely SEQ ID NO.3 and SEQ ID NO.6.

[0099] Example 5

[0100] This embodiment provides a reagent kit for detecting biomarkers of lung metastasis risk and prognosis prediction after liver transplantation for liver cancer. The kit specifically includes the following components:

[0101] Table 7. Components of the reagent kit for detecting risk markers of lung metastasis after liver transplantation in liver cancer.

[0102]

[0103] The detection method of this kit is a combined detection of CTC typing and MECOM mRNA expression level. The specific detection method is as follows:

[0104] The CTC typing detection method is the same as in Example 2;

[0105] The detection method for MECOM mRNA expression level is the same as in Example 3 (Example 4 or Example 5).

[0106] Effect verification

[0107] 1) Reliability Test 1

[0108] This application uses Example 3 (Group 3) as an example, and replaces the probe sequence of the MECOM probe in Example 3 with SEQ ID NO.1 (Group 1), SEQ ID NO.2 (Group 2), SEQ ID NO.4 (Group 4), SEQ ID NO.5 (Group 5), SEQ ID NO.6 (Group 6), SEQ ID NO.7 (Group 7), and SEQ ID NO.8 (Group 8). Blood samples from 200 patients with liver cancer and lung metastasis after liver transplantation were tested, and the statistical results are as follows:

[0109] Table 9. Detection results of blood samples from 200 patients with lung metastases after liver transplantation for hepatocellular carcinoma.

[0110]

[0111] The data in the table show that the positive rate of the MECOM probe with sequence SEQ ID NO.1 is 25.5%, the positive rate of the MECOM probe with sequence SEQ ID NO.2 is 36%, the positive rate of the MECOM probe with sequence SEQ ID NO.3 is 36.5%, the positive rate of the MECOM probe with sequence SEQ ID NO.4 is 28.5%, the positive rate of the MECOM probe with sequence SEQ ID NO.5 is 32%, the positive rate of the MECOM probe with sequence SEQ ID NO.6 is 41%, the positive rate of the MECOM probe with sequence SEQ ID NO.7 is 32%, and the positive rate of the MECOM probe with sequence SEQ ID NO.8 is 29%. In summary, the MECOM probes with nucleotide sequences SEQ ID NO.1 to SEQ ID NO.8 disclosed in this application can be used to predict the recurrence risk and prognosis of patients with lung metastases after liver transplantation for liver cancer.

[0112] 3) Reliability verification

[0113] The application utilizes the detection methods described in Examples 2 to 5 to conduct tests on blood samples from 200 patients with liver cancer who have undergone liver transplantation and subsequently developed lung metastases. The statistical results are as follows:

[0114] Table 10. Detection results of blood samples from 200 patients with liver cancer and lung metastasis after liver transplantation.

[0115]

[0116] The data in the table show that the positive concordance rate of the detection method using CTC typing alone (Example 2) is 87.5%; the positive concordance rate of the detection method using CTC MECOM mRNA expression level alone (Example 3) is 36.5%; while the positive concordance rate of the detection method using MECOM probes combining SEQ ID NO.3 and SEQ ID NO.6 is 89.5%. By comparing the positive concordance rates of Example 3 and Example 5, it can be seen that the MECOM probes combining SEQ ID NO.3 and SEQ ID NO.6 can synergistically enhance the expression level of CTC MECOM mRNA, providing key technical support for the accurate and stable assessment of the prognostic risk of lung metastasis after liver transplantation for liver cancer.

[0117] Furthermore, as shown in the table, the combined detection method for CTC subtyping and the detection method for CTC MECOM mRNA expression levels achieved an overall positive concordance rate of 97%, which can be used to predict the recurrence risk and prognosis of patients with liver cancer and lung metastases after liver transplantation. The microscopic images under a fluorescence microscope of patients with liver cancer and lung metastases after liver transplantation exhibiting high MECOM expression, low MECOM expression, and moderate MECOM expression, using the kit from Example 5, are shown below. Figure 1 and Figure 3 As shown, this invention is the first to discover the role of combined MECOM and CTC detection in assessing postoperative recurrence and metastasis in patients with liver cancer who have undergone liver transplantation and subsequently developed lung metastases.

[0118] In this invention, central cytokines (CTCs) are a crucial component of liquid biopsy. They closely resemble tissue biopsies in morphology, origin, and composition. Therefore, CTCs can be repeatedly obtained from peripheral blood, and detecting the MECOM mRNA level in CTCs allows for dynamic monitoring of the risk of recurrence and metastasis in patients with hepatocellular carcinoma (HCC) at risk of lung metastasis after liver transplantation. This invention utilizes a designed MECOM probe and employs nanomembrane filtration combined with mRNA in situ hybridization to detect the MECOM mRNA level in peripheral blood CTCs from HCC patients at risk of lung metastasis after liver transplantation. The results showed that a positive result for both MECOM and CTC detection indicated a high risk of recurrence and metastasis, while a single positive result for either MECOM or CTC detection indicated a low risk. Therefore, the combined MECOM and CTC detection method provided by this invention can accurately assess the postoperative risk of recurrence and metastasis in HCC patients at risk of lung metastasis after liver transplantation, as well as prognosis and treatment outcomes.

[0119] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A biomarker probe for assessing the risk of lung metastasis after liver transplantation for hepatocellular carcinoma, characterized in that, Includes probes for detecting MECOM, wherein the probes for detecting MECOM have one or more nucleotide sequences of SEQ ID NO.1 to SEQ ID NO.

8.

2. The biomarker probe for assessing the risk of lung metastasis after liver transplantation for liver cancer according to claim 1, characterized in that, It includes probes for detecting MECOM, which are combinations of nucleotide sequences SEQ ID NO.3 and SEQ ID NO.

6.

3. The biomarker probe for assessing the risk of lung metastasis after liver transplantation for liver cancer according to claim 1, characterized in that, It includes probes for detecting MECOM, which are combinations of three nucleotide sequences: SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.

7.

4. The use of the probe according to any one of claims 1-3 in the preparation of a detection kit.

5. A kit for assessing the risk of lung metastasis after liver transplantation for hepatocellular carcinoma, characterized in that, Includes the probe for detecting MECOM as described in claim 1.

6. The kit for assessing the risk of lung metastasis after liver transplantation for hepatocellular carcinoma according to claim 5, characterized in that, It also includes epithelial peripheral blood CTC-specific capture probes EpCAM, CK8, CK18, and CK19, with nucleotide sequences as shown in ISEQ ID NO.9-SEQ ID NO.14, SEQ ID NO.15-SEQ ID NO.20, SEQ ID NO.21-SEQ ID NO.26, and SEQ ID NO.27-SEQ ID NO.32, respectively; it also includes mesenchymal peripheral blood CIC-specific capture probes Vimentin and Twist, with nucleotide sequences as shown in ISEQ ID NO.33-SEQ ID NO.38 and SEQ ID NO.39-SEQ ID NO.44, respectively; and it also includes leukocyte phenotype-specific capture probe CD45, with nucleotide sequences as shown in SEQ ID NO.45-SEQ ID NO.

50.

7. The kit for assessing the risk of lung metastasis after liver transplantation for hepatocellular carcinoma according to claim 5, characterized in that, It also includes bDNA probes for EpCAM and CK8 / 18 / 19, the sequences of which are shown in SEQ ID NO.51-SEQ ID NO.53; bDNA probes for Vimentin and Twist, the sequences of which are shown in SEQ ID NO.54-SEQ ID NO.56; bDNA probes for CD45, the sequences of which are shown in SEQ ID NO.57-SEQ ID NO.59; and fluorescent dyes AlexaFluor594 for labeling epithelial peripheral blood CTC-specific capture probes EpCAM, CK8, CK18, and CK19, AlexaFluor488 for labeling mesenchymal peripheral blood CTC-specific capture probes Vimentin and Twist, AlexaFluor750 for labeling leukocyte phenotype-specific capture probe CD45, and AlexaFluor647 for labeling MECOM probes.

8. The use of the probe according to any one of claims 5-7 in the preparation of a kit for predicting the risk of lung metastasis after liver transplantation for hepatocellular carcinoma.

9. The application of the probe according to any one of claims 5-7 in constructing a risk assessment model for lung metastasis after liver transplantation for hepatocellular carcinoma.

Images