Cancer biomarker and application thereof

Abstract

The present invention relates to a cancer biomarker and uses thereof, the cancer biomarker comprises at least one of the following genes: Ncl, Gpx2, Nkd1, Wif1, Notum, Axin2 and Prox1, and the cancer biomarker has the effect of predicting and diagnosing the risk level of cancer. The cancer biomarker can be used for preparing and screening a cancer diagnostic reagent, and is particularly suitable for a colon cancer diagnostic reagent, so that the condition of a patient after healing can be effectively monitored, the treatment reliability of the diagnostic reagent is effectively improved, the research and development period of the diagnostic reagent is shortened, and more effective medication and therapy selection suggestions can be provided for the rectal cancer patient; the treatment risk of tumor patients is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor molecular biology, and in particular to a cancer biomarker and a use thereof. Background Art

[0002] In 2023, the total number of new cancer cases and deaths worldwide is expected to be about 20 million, and about 9.7 million. Among them, the incidence of colorectal cancer ranks third among all cancers in the world, and the mortality rate ranks second in the world, causing serious social and economic burdens. Clinically, colon cancer is a malignant tumor. Many colorectal cancer patients are already in the late stage when diagnosed. Even with a combination of radiotherapy, chemotherapy and surgery (patients with stage III, IV or high-risk stage II colon cancer), there are still many problems such as easy recurrence and high incidence of complications after surgery; however, in the early stages of cancer, there is generally no distant metastasis of the tumor, and better results can be achieved through treatment; in terms of survival, the 5-year survival rate of stage I patients can reach more than 90%, while the survival rate of stage IV patients is only about 10%.

[0003] For disease research, biomarkers are certain characteristic biochemical indicators that can be objectively measured and evaluated. By measuring them, the biological process of the body can be known. Checking a disease-specific biomarker may play an important role in the identification, early diagnosis and prevention of the disease, and monitoring during the treatment process. Finding and discovering valuable biomarkers has become an important hotspot in current research. In view of this, discovering biomarkers for colon cancer is the technical problem that this application aims to solve. Summary of the invention

[0004] The purpose of the present invention is to overcome at least one of the above-mentioned technical defects and to provide a cancer biomarker and its use, which can realize the judgment of the treatment results of rectal cancer diagnostic agents, provide rectal cancer patients with more effective medication and therapy selection suggestions, and reduce the treatment risks of tumor patients.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The present invention provides a cancer biomarker, wherein the cancer biomarker comprises at least one of the following genes: Ncl, Gpx2, Nkd1, Wif1, Notum, Axin2 and Prox1, wherein the cancer biomarker has the function of predicting and diagnosing the cancer risk level.

[0007] Optionally, the cancer includes at least one of colon cancer, rectal cancer, and rectal adenocarcinoma.

[0008] In particular, the present application also provides a use of the cancer biomarker as described above in the preparation or screening of cancer diagnostic reagents.

[0009] Furthermore, the risk of cancer recurrence is determined based on the expression level of the cancer biomarker in the sample to be tested relative to the reference gene, which is used to prepare or screen cancer diagnostic reagents.

[0010] Optionally, the reference gene includes an internal reference gene and / or an external reference gene, and the internal reference gene includes at least one of a Gapdh gene, a Hprt gene, and an Actin gene.

[0011] Furthermore, the method for determining the risk of cancer recurrence based on the expression level of the cancer biomarker in the sample to be tested relative to the reference gene comprises the following working steps:

[0012] Calculate the relative expression RQ of the cancer biomarker relative to the reference gene, RQ = 2^ -ΔΔCt , wherein ΔΔCt=(the number of PCR cycles of the cancer biomarker as the target gene in the test sample-the number of PCR cycles of the reference gene)-(the number of PCR cycles of the cancer biomarker as the target gene in the control sample-the number of PCR cycles of the reference gene);

[0013] The risk value RISK is calculated according to the relative expression level RQ of each cancer biomarker, wherein the risk value RISK=[RQ (Ncl) *RQ (Gpx2) *RQ (Nkd1) *RQ (Wif1) *RQ (Notum) *RQ (Axin2) *RQ (Prox1) ] / α, α is a non-zero constant;

[0014] The risk value RISK is compared with a preset risk threshold RT. If the risk value RISK> the risk threshold RT, the risk of cancer recurrence is predicted to be high risk. If the risk value RISK≤the risk threshold RT, the risk of cancer recurrence is predicted to be low risk.

[0015] Optionally, the value of α is obtained based on sequencing data of self-sequencing samples and sequencing data in the TCGA database, and the value range is 4.0 to 14.2.

[0016] Optionally, the PCR cycle number is obtained by real-time fluorescence quantitative PCR detection; the sample to be tested includes a tumor tissue sample; the control sample includes a normal tissue sample, and the normal tissue sample includes adjacent cancer tissue samples and other normal non-adjacent cancer tissue samples.

[0017] Compared with the prior art, the advantages of the cancer biomarkers and their uses in the patent application of the present invention are:

[0018] The cancer biomarkers of the present application can be used for the preparation and screening of cancer diagnostic reagents, and are particularly suitable for colon cancer diagnostic reagents. This can effectively monitor the patient's recovery, effectively improve the treatment reliability of diagnostic reagents, shorten the research and development cycle of diagnostic reagents, and provide rectal cancer patients with more effective medication and therapy selection recommendations, thereby reducing the treatment risks of tumor patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Hereinafter, some specific embodiments of the present invention will be described in detail in an exemplary and non-limiting manner with reference to the accompanying drawings. The same reference numerals in the accompanying drawings indicate the same or similar components or parts. It should be understood by those skilled in the art that these drawings are not necessarily drawn to scale. In the accompanying drawings:

[0020] Figure 1 is an expression level diagram of the gene expression of cancer biomarkers in normal colorectal tissue (or adjacent tissue to cancer) and colorectal cancer of colorectal cancer patients according to one embodiment of the present invention, Normal=51, Tumor=647;

[0021] Figure 2 This is an expression level diagram of the gene expression of a cancer biomarker in paired normal colorectal tissue (or adjacent cancer tissue) and colorectal cancer of colorectal cancer patients according to one embodiment of the present invention, Normal=50, Tumor=50. DETAILED DESCRIPTION

[0022] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0024] Embodiment 1:

[0025] "Biomarker" refers to a biochemical index that can mark changes or possible changes in the structure or function of a system, organ, tissue, cell or subcellular structure, and has a very wide range of uses. Biomarkers can be used to diagnose diseases, determine disease stages, or evaluate the safety and effectiveness of new drugs or new therapies in patients. In this article, "biomarker" and "molecular marker" can be used interchangeably.

[0026] The Ncl gene encodes a eukaryotic nucleolar phosphoprotein that is involved in the synthesis and maturation of ribosomes. It is mainly located in the dense fibrous region of the nucleolus. The human NCL gene consists of 14 exons and 13 introns, spanning approximately 11kb. Intron 11 of the Ncl gene encodes a small nucleolar RNA called U20.

[0027] The Gpx2 gene encodes glutathione peroxidase 2. This gene is a member of the glutathione peroxidase family and encodes a selenium-dependent glutathione peroxidase, one of two isozymes responsible for the majority of glutathione-dependent hydrogen peroxide reduction activity in the gastrointestinal epithelium.

[0028] The Nkd1 gene encodes the naked corneum 1 (NKD1) protein, a member of the naked corneum protein family that regulates the Wnt signaling pathway. Specific truncating NKD1 mutations found in DNA mismatch repair-deficient colon cancers disrupt Nkd1 / Dvl binding, suggesting that these mutations are responsible for enhanced Wnt signaling in a subset of human colon cancers, most of which are due to mutations in the adenomatous polyposis coli (APC), AXIN2, or rarely, β-catenin genes.

[0029] Wif1 gene edits Wnt inhibitory factor 1 protein, WIF1 is a lipid-binding protein that contains a WNT inhibitory factor (WIF) domain and five epidermal growth factor (EGF)-like domains that can bind to Wnt proteins and prevent them from triggering signal transduction.

[0030] Notum gene edits palmitoleyl protein carboxylesterase, which is involved in the negative regulation of Wnt signaling pathway and protein depalmitoylation, and can regulate Fas cell surface death receptor-mediated apoptosis in colon adenocarcinoma through the Wnt signaling pathway.

[0031] Axin2 gene editing Axis-like protein (AXIL) is also called axis inhibitory protein 2 (AXIN2) or conductin. AXIN2 may play an important role in regulating the stability of β-catenin in the Wnt signaling pathway. In mice, conductin organizes a multiprotein complex of APC (adenomatous polyposis coli), β-catenin, glycogen synthase kinase 3-β and conductin, leading to β-catenin degradation.

[0032] Prox1 gene editing encodes Prospero-homeobox1. The Prox1 gene is essential for the development of multiple tissues. The activity of Prox1 is necessary and sufficient to determine the lymphatic endothelial cell fate of endothelial progenitor cells located in embryonic veins.

[0033] This embodiment describes a cancer biomarker, wherein the cancer biomarker comprises at least one of the following genes: Ncl, Gpx2, Nkd1, Wif1, Notum, Axin2 and Prox1, wherein the cancer biomarker has the function of predicting and diagnosing the cancer risk level, and is specifically used for preparing or screening cancer diagnostic reagents.

[0034] The gene sequences of the cancer biomarkers are as follows:

[0035]

[0036]

[0037] Specifically, the cancer includes at least one of colorectal cancers such as colon cancer, rectal cancer, and rectal adenocarcinoma.

[0038] Figure 1 is an expression level diagram of the gene expression of cancer biomarkers in normal colorectal tissue (or adjacent tissue to cancer) and colorectal cancer of colorectal cancer patients according to one embodiment of the present invention, Normal=51, Tumor=647;

[0039] Figure 2 This is an expression level diagram of the gene expression of a cancer biomarker in paired normal colorectal tissue (or adjacent cancer tissue) and colorectal cancer of colorectal cancer patients according to one embodiment of the present invention, Normal=50, Tumor=50.

[0040] Embodiment 2:

[0041] This example describes the use of a cancer biomarker as described in Example 1 in preparing or screening cancer diagnostic reagents.

[0042] Furthermore, the risk of cancer recurrence is determined based on the expression level of the cancer biomarker in the sample to be tested relative to the reference gene, which is used to prepare or screen cancer diagnostic reagents.

[0043] The reference gene includes a gene constitutively and stably expressed in the cell. The reference gene includes an internal reference gene and / or an external reference gene, and the internal reference gene includes at least one of the Gapdh gene, the Hprt gene, and the Actin gene.

[0044] The method for determining the risk of cancer recurrence based on the expression level of the cancer biomarker in the sample to be tested relative to the reference gene comprises the following working steps:

[0045] Calculate the relative expression RQ of the cancer biomarker relative to the reference gene, RQ = 2^ -ΔΔCt, wherein ΔΔCt=(the number of PCR cycles of the cancer biomarker as the target gene in the test sample-the number of PCR cycles of the reference gene)-(the number of PCR cycles of the cancer biomarker as the target gene in the control sample-the number of PCR cycles of the reference gene), the PCR cycle number is obtained by real-time fluorescence quantitative PCR detection; the test sample includes a tumor tissue sample; the control sample includes a normal tissue sample, and the normal tissue sample includes a paracancerous tissue sample and other normal non-paracancerous tissue samples;

[0046] The risk value RISK is calculated according to the relative expression level RQ of each cancer biomarker, wherein the risk value RISK=[RQ (Ncl) *RQ (Gpx2) *RQ (Nkd1) *RQ (Wif1) *RQ (Notum) *RQ (Axin2) *RQ (Prox1) ] / α, α is a non-zero constant. The value of α is obtained based on the sequencing data of the self-sequenced samples and the sequencing data in the TCGA database, and the value range is 4.0 to 14.2 (the prediction accuracy of the α value can be confirmed by a positive control experiment; the accuracy of risk prediction can be optimized by continuously adjusting the value of α);

[0047] The risk value RISK is compared with a preset risk threshold RT. If the risk value RISK> the risk threshold RT, the risk of cancer recurrence is predicted to be high risk. If the risk value RISK≤the risk threshold RT, the risk of cancer recurrence is predicted to be low risk.

[0048] Experimental example

[0049] The reagents used included: RNAse-free 1.5 ml EP tube; chloroform; isopropanol;

[0050] RNAse-free; water washing solution (75% V / V ethanol prepared with RNAse-free water); reverse transcription kit; Q-PCR kit.

[0051] Here’s how to do it:

[0052] (1) Sample preparation: For patients with multiple colorectal cancer foci, a tumor piece (30-50 mg / piece) was collected from each foci, with a total of 3-4 foci. For patients with less than 3 colorectal cancer foci, 3 tumor pieces (30-50 mg / piece) were randomly collected; 3-4 points of control tissue were randomly collected simultaneously.

[0053] (2) Use sterile scissors to cut the tumor and control samples into small pieces, add 1000 μL of Trizol lysis buffer, grind the tissue fragments until there is no obvious granular tissue, repeatedly pipette and mix several times, and let it stand at room temperature for 5 minutes.

[0054] (3) Add 200 μL of chloroform to the tissue lysis buffer, invert several times to mix the liquid, and let it stand at room temperature for 5 minutes.

[0055] (4) The tissue lysate was centrifuged at 12,000 × g at 4°C for 20 min.

[0056] (5) After centrifugation, use a pipette to transfer 300 μL of the supernatant into a new RNAse-free 1.5 mL EP tube.

[0057] (6) Add an equal volume of isopropanol to the supernatant, invert several times to mix the liquid, and let it stand at room temperature for 10 minutes.

[0058] (7) Centrifuge the above liquid at 4°C and 12,000×g for 10 minutes.

[0059] (8) Discard the supernatant. A white precipitate will be visible at the bottom of the tube. Add 500 μl of washing solution to the precipitate and shake the test tube three times to wash the precipitate.

[0060] (9) Centrifuge at 4°C, 5000 × g for 5 min.

[0061] (10) Discard the supernatant and place the test tube with the lid open in a fume hood for 10 minutes to dry.

[0062] (11) Add 50 μL of RNAse-free water.

[0063] (12) Measure the RNA concentration and place in an ice box or a 4°C refrigerator until use.

[0064] The above steps may also be adjusted in a manner well known to technicians.

[0065] (13) For the same patient or the same batch of tests, use the same mass of RNA (total RNA) for reverse transcription (the template amount can be 500 ng to 1000 ng).

[0066] (14) Add the RNA to be tested to the reverse transcription kit mixture (10 μL / tube), and add RNAse-free water to make up to 20 μL. The volume of RNAse-free water added (μL) = 10-RNA mass (ng) / RNA concentration (ng / μL).

[0067] (15) Perform reverse transcription reaction in a PCR instrument under the following reaction conditions:

[0068] 30℃ 10min 42℃ 30min 85℃ 5min 4℃ 5min

[0069] (16) Q-PCR reaction

[0070] Prepare the following reaction system: 1 μL of the reverse transcription reaction solution obtained in step 15, 1 μL of the primer mixture, 0.5 μL of the mixture, and 8.5 μL of the Q-PCR reaction mixture. Set up 3 replicates for each reaction.

[0071] The Q-PCR reaction process is as follows:

[0072]

[0073] (17) Calculation of -ΔΔCt

[0074] RQ=2^ -ΔΔCt

[0075] ΔCt = tumor sample (CRC) target gene PCR cycle number - internal reference PCR cycle number.

[0076] ΔΔCt = [number of PCR cycles of target gene in tumor sample (CRC) - number of PCR cycles of internal reference] - [number of PCR cycles of target gene in control sample (Normal) - number of PCR cycles of internal reference].

[0077] (18) Risk value:

[0078] Risk=[RQ (Ncl) *RQ (Gpx2) *RQ (Nkd1) RQ (Wif1) *RQ (Notum) *RQ (Axin2) *RQ (Prox1) ] / α.

[0079] (19) Determination method

[0080] If Risk>50, it is a high risk, indicating that the prognosis is not optimistic and the tumor condition should be closely monitored; Risk=20-50 is a medium risk and active reexamination is required; if Risk≤20, it is a low risk.

[0081] Figure 1 This is an expression level diagram of the gene expression of cancer biomarkers in normal colorectal tissue (or adjacent tissue) and colorectal cancer of colorectal cancer patients according to one embodiment of the present invention, Normal = 51, Tumor = 647. Normal refers to adjacent normal tissue, 51 represents adjacent tissue from the same 51 patients; Tumor refers to cancer tissue, 647 represents cancer tissue from the same 647 patients.

[0082] Figure 2This is an expression level diagram of the gene expression of cancer biomarkers in paired normal colorectal tissues (or adjacent tissues) and colorectal cancer of colorectal cancer patients according to one embodiment of the present invention, Normal = 50, Tumor = 50. Normal refers to adjacent normal tissues, 50 represents adjacent tissues from the same 50 patients; Tumor refers to cancer tissues, 50 represents cancer tissues from the same 50 patients.

[0083] It is not difficult to find from the experimental examples that the cancer biomarkers of the present application can be used for the preparation and screening of cancer diagnostic reagents, especially for colon cancer diagnostic reagents. This can effectively improve the treatment reliability of diagnostic reagents, shorten the research and development cycle of diagnostic reagents, and provide rectal cancer patients with more effective medication and therapy selection recommendations, thereby reducing the treatment risks of tumor patients.

[0084] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the contents of the present invention and implement them accordingly. They cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A cancer biomarker, characterized in that The cancer biomarker comprises at least one of the following genes: Ncl, Gpx2, Nkd1, Wif1, Notum, Axin2 and Prox1, wherein the cancer biomarker has the function of predicting and diagnosing the cancer risk level.

2. The cancer biomarker according to claim 1, characterized in that The cancer includes at least one of colon cancer, rectal cancer, and rectal adenocarcinoma.

3. Use of the cancer biomarker according to claim 1 or 2 in preparing or screening cancer diagnostic reagents.

4. The use according to claim 3, characterized in that The risk of cancer recurrence is determined based on the expression level of the cancer biomarker in the sample to be tested relative to the reference gene, which is used to prepare or screen cancer diagnostic reagents.

5. The use according to claim 4, characterized in that The reference gene includes an internal reference gene and / or an external reference gene, and the internal reference gene includes at least one of a Gapdh gene, a Hprt gene, and an Actin gene.

6. The use according to claim 4 or 5, characterized in that: The method for determining the risk of cancer recurrence based on the expression level of the cancer biomarker in the sample to be tested relative to the reference gene comprises the following working steps: Calculate the relative expression RQ of the cancer biomarker relative to the reference gene, RQ = 2^ -ΔΔCt , wherein ΔΔCt=(the number of PCR cycles of the cancer biomarker as the target gene in the test sample-the number of PCR cycles of the reference gene)-(the number of PCR cycles of the cancer biomarker as the target gene in the control sample-the number of PCR cycles of the reference gene); The risk value RISK is calculated according to the relative expression level RQ of each cancer biomarker, wherein the risk value RISK=[RQ (Ncl) *RQ (Gpx2) *RQ (Nkd1) *RQ (Wif1) *RQ (Notum) *RQ (Axin2) *RQ (Prox1) ] / α, α is a non-zero constant; The risk value RISK is compared with a preset risk threshold RT. If the risk value RISK> the risk threshold RT, the risk of cancer recurrence is predicted to be high risk. If the risk value RISK≤the risk threshold RT, the risk of cancer recurrence is predicted to be low risk.

7. The use according to claim 6, characterized in that The value of α is obtained based on the sequencing data of the self-sequencing samples and the sequencing data in the TCGA database, and the value range is 4.0 to 14.

2.

8. The use according to claim 6, characterized in that The PCR cycle number is obtained by real-time fluorescence quantitative PCR detection; the sample to be tested includes a tumor tissue sample; the control sample includes a normal tissue sample, and the normal tissue sample includes adjacent cancer tissue samples and other normal non-adjacent cancer tissue samples.

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