Application of POGZ and MAD2L2 in diagnosis and treatment of thyroid cancer

Abstract

The invention relates to application of POGZ and MAD2L2 in diagnosis and treatment of thyroid cancer. Specifically, the invention provides an application of POGZ and MAD2L2 markers or a combination thereof in the occurrence risk of thyroid cancer, and also provides a diagnostic reagent or kit and risk assessment equipment for assessing the occurrence risk of thyroid cancer. The invention also provides a treatment method of combining the POGZ expression promoter and the MAD2L2 expression promoter. The treatment method disclosed by the invention has a remarkable treatment effect on the thyroid cancer.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and clinical medicine, and specifically, to the application of POGZ and MAD2L2 in the diagnosis and treatment of thyroid cancer. Background Art

[0002] Approximately more than 40,000 patients die from thyroid cancer (THCA) globally every year, with approximately 590,000 new cases, accounting for 3.0% of all tumors and ranking 11th. Despite the glimmer of hope brought by new immunotherapies, the prognosis of advanced THCA remains poor. Moreover, most patients have missed the optimal surgical opportunity at the time of diagnosis and have invasion and metastasis.

[0003] The pathogenic factors of THCA are complex, including environment, genetics, diseases and their interactions. Compared with other common tumors, the pathological research of THCA is relatively lacking.

[0004] Therefore, it is very important to explore new diagnostic and therapeutic targets and the potential mechanisms of THCA development.

[0005] Currently, there are still no relevant reports on risk markers for the occurrence of thyroid cancer in this field, and more effective treatment methods for thyroid cancer also need to be developed.

[0006] Therefore, there is an urgent need in this field to develop risk markers for thyroid cancer with high sensitivity and specificity for clinical diagnosis, for the early diagnosis and timely intervention treatment of thyroid cancer; and there is a need for newer, more effective and more targeted drugs and methods for treating thyroid cancer. Summary of the Invention

[0007] The object of the present invention is to provide a reagent combination for judging the risk of occurrence and prognosis of thyroid cancer with high sensitivity and high specificity and its application.

[0008] In the first aspect of the present invention, there is provided the use of a gene, mRNA, cDNA, protein, or a detection reagent thereof of a thyroid cancer risk marker for preparing a diagnostic reagent or kit for (a) detecting the risk of occurrence of thyroid cancer; and / or (b) prognostic evaluation of thyroid cancer;

[0009] Wherein, the detection reagent is used to detect the level of the risk marker, and the thyroid cancer risk marker includes any marker selected from the following group, or a combination thereof:

[0010] (A1) POGZ; (B1) MAD2L2.

[0011] In another preferred example, the detection subject is selected from the group consisting of: thyroid cancer patients, high-risk subjects for thyroid cancer, subjects without thyroid cancer, or a combination thereof.

[0012] In a second aspect of the present invention, a kit is provided, the kit containing a detection reagent for detecting a gene, mRNA, cDNA, protein, or a combination thereof of a thyroid cancer risk marker.

[0013] Wherein, the thyroid cancer risk marker includes any marker selected from the following group, or a combination thereof:

[0014] (A1)POGZ; (B1)MAD2L2.

[0015] In another preferred embodiment, the diagnostic reagent includes:

[0016] (a) a specific antibody or specific binding molecule against the thyroid cancer risk marker; and / or

[0017] (b) primers or primer pairs, probes or chips (such as nucleic acid chips or protein chips) that specifically amplify the mRNA or cDNA of the thyroid cancer risk marker.

[0018] In another preferred embodiment, the detection reagent includes a primer pair or a probe, and the primer pair or probe is a primer pair or probe that specifically amplifies the mRNA or cDNA of the thyroid cancer risk marker.

[0019] In another preferred embodiment, the primer pair includes a primer pair for amplifying POGZ: SEQ ID NO:1, SEQ ID NO:2.

[0020] In another preferred embodiment, the thyroid cancer risk marker is of human origin.

[0021] In another preferred embodiment, the detection is for an in vitro sample.

[0022] In another preferred embodiment, the in vitro sample includes: a tissue sample.

[0023] In another preferred embodiment, the detection reagent is conjugated with or carries a detectable label.

[0024] In another preferred embodiment, the detectable label is selected from the following group: a chromophore, a chemiluminescent group, a fluorophore, an isotope, or an enzyme.

[0025] In another preferred embodiment, the antibody is a monoclonal antibody or a polyclonal antibody.

[0026] In another preferred embodiment, the detection reagent includes an antibody, a primer, a probe, a sequencing library, a nucleic acid chip (such as a DNA chip) or a protein chip.

[0027] In another preferred embodiment, the nucleic acid chip comprises a substrate and specific oligonucleotide probes spotted on the substrate, and the specific oligonucleotide probes comprise probes that specifically bind to the polynucleotide (mRNA or cDNA) of any one of the thyroid cancer risk markers.

[0028] In another preferred embodiment, the protein chip comprises a substrate and specific antibodies spotted on the substrate, and the specific antibodies comprise specific antibodies against the thyroid cancer risk markers.

[0029] In another preferred embodiment, the antibody is a monoclonal antibody or a polyclonal antibody.

[0030] In another preferred embodiment, the kit contains genes, mRNAs, cDNAs and / or proteins of thyroid cancer risk markers as control products or quality control products.

[0031] In another preferred embodiment, the reagent comprises primers, probes, gRNAs or combinations thereof, and more preferably primer pairs or probes for PCR, qPCR, RT-PCR.

[0032] In another preferred embodiment, the detection of the thyroid cancer risk marker can be carried out by the following methods: sequencing, PCR, or a combination thereof.

[0033] In another preferred embodiment, the detection of the thyroid cancer risk marker can be quantitatively detected.

[0034] In another preferred embodiment, the kit further comprises a label or an instruction manual, and the label or the instruction manual indicates that the kit is used for (a) diagnosing the occurrence risk of thyroid, and / or (b) prognostic evaluation of thyroid cancer.

[0035] In the third aspect of the present invention, a detection method is provided, comprising the steps of:

[0036] (a) Providing a test sample;

[0037] (b) Detecting the level of the thyroid cancer risk marker in the test sample and recording it as C1; and

[0038] (c) Comparing the level of the thyroid cancer risk marker with a control reference value C0;

[0039] Wherein, the thyroid cancer risk marker comprises any marker selected from the following group, or a combination thereof:

[0040] (A1) POGZ; (B1) MAD2L2;

[0041] If the level of the thyroid cancer risk marker of the test subject meets the following conditions, it indicates that the test subject has a high risk of thyroid cancer occurrence:

[0042] When the expression level C1 of a certain marker is significantly lower than C0, it indicates a high risk of thyroid cancer in the subject; conversely, it indicates a low risk of thyroid cancer.

[0043] In another preferred embodiment, the test sample is selected from: thyroid cancer tissue, non-thyroid cancer tissue, or a combination thereof.

[0044] In another preferred embodiment, the detection method is non-diagnostic and non-therapeutic.

[0045] In another preferred embodiment, the detection method is an in vitro method.

[0046] In a fourth aspect of the present invention, there is provided a device for assessing the risk of thyroid cancer, the device comprising:

[0047] (a) An input module for inputting data of thyroid cancer risk markers of a certain test subject;

[0048] Wherein, the risk markers include any marker selected from the following group, or a combination thereof:

[0049] (A1) POGZ; (B1) MAD2L2;

[0050] (b) A processing module for processing the thyroid cancer risk marker data and giving a risk assessment result, wherein the processing includes: when the expression level C1 of a certain marker is significantly lower than the control reference value C0, it indicates a high risk of thyroid cancer in the subject; conversely, it indicates a low risk of thyroid cancer in the subject; and

[0051] (c) An output module for outputting the evaluation result.

[0052] In another preferred embodiment, the device further comprises (d) a storage module for storing data including: comparison result values, control reference values.

[0053] In another preferred embodiment, the device further comprises (e) a control module for controlling the operation of each module.

[0054] In a fifth aspect of the present invention, there is provided an active ingredient combination selected from the following group:

[0055] (Z1) A first active ingredient, which is a POGZ expression promoter; and

[0056] (Z2) A second active ingredient, which is a MAD2L2 expression promoter.

[0057] In the sixth aspect of the present invention, there is provided the use of an active ingredient combination in the preparation of a pharmaceutical composition for treating thyroid cancer, wherein the active ingredient combination comprises:

[0058] (Z1) A first active ingredient, which is a POGZ expression promoter; and

[0059] (Z2) A second active ingredient, which is a MAD2L2 expression promoter.

[0060] In the seventh aspect of the present invention, there is provided a pharmaceutical composition, which contains:

[0061] Any active ingredient selected from the following group, or a combination thereof:

[0062] (Z1) A first active ingredient, which is a POGZ expression promoter,

[0063] (Z2) A second active ingredient, which is a MAD2L2 expression promoter; and

[0064] (Z3) A pharmaceutically acceptable carrier.

[0065] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0066] Figure 1 It shows that POGZ is down-regulated in THCA and interacts with MAD2L2: (A) Expression characteristics of POGZ in 33 cancer types; (B) POGZ 5 levels in THCA and normal tissues in GEPIA; (C-D) Expression characteristics of POGZ in normal HTori-3 cells and THCA cell lines, *P, ***P < 0.05, 0.001 vs. normal or HTori-3; (E) Protein-based POGZ function and interacting protein analysis; (F) Co-IP confirmed the interaction between POGZ and MAD2L2.

[0067] Figure 2 It shows the construction of THCA cells with overexpressed POGZ and silenced MAD2L2: (A-B) Construction of overexpressed POGZ in KTC-1 and CAL-62 cells; (C-D) Construction of silenced MAD2L2 in KTC-1 and CAL-62 cells. ***P < 0.001 vs. NC.

[0068] Figure 3Shown is that POGZ inhibits THCA cell proliferation through MAD2L2: (A-B) Effects of POGZ overexpression and / or MAD2L2 silencing on cell viability by CCK-8; (C-D) Effects of POGZ overexpression and / or MAD2L2 silencing on cell proliferation by colony formation assay, ***P<0.001 vs. NC+siNC; ##P<0.01 vs. POGZ+siNC; ^^^P<0.001 vs. NC+siMAD2L2.

[0069] Figure 4 Shown is that POGZ inhibits THCA cell invasion and tumor growth in nude mice through MAD2L2: (A-C) Effects of POGZ overexpression and / or MAD2L2 silencing on cell invasion by Transwell assay; (D) Establishment of THCA xenograft tumor model with THCA cells with POGZ overexpression and / or MAD2L2 silencing; (E-F) Effects of POGZ overexpression and / or MAD2L2 silencing on tumor volume and weight, ***P<0.001 vs. NC+siNC; ##P<0.01 vs. POGZ+siNC; ^^^P<0.001 vs. NC+siMAD2L2. Detailed implementation manners

[0070] After extensive and in-depth research, the present inventors unexpectedly discovered for the first time the thyroid cancer risk markers POGZ and MAD2L2 and the close interaction relationship between them. And accordingly developed diagnostic reagents / kits and devices for judging the occurrence risk and prognosis of thyroid cancer. Using POGZ and / or MAD2L2 as diagnostic markers, the occurrence risk and prognosis of papillary thyroid carcinoma can be diagnosed and evaluated with high sensitivity and high specificity.

[0071] The present inventors also found that using the reagent combination of the present invention (POGZ expression promoter and MAD2L2 expression promoter) can effectively inhibit the development of papillary thyroid carcinoma. Based on this, the present invention was completed.

[0072] Terms

[0073] As used herein, the term "sample" or "specimen" refers to a material specifically associated with a subject from which specific information related to the subject can be determined, calculated or inferred. The sample can be wholly or partially composed of biological materials from the subject.

[0074] As used herein, the term "expression" includes the production of mRNA from a gene or gene portion, and includes the production of a protein encoded by the RNA or gene portion, and further includes the appearance of a detectable substance associated with expression. For example, cDNA, the binding of a binding ligand (such as an antibody) to a gene or other oligonucleotide, protein, or protein fragment, and the chromogenic portion of the binding ligand are all included within the scope of the term "expression". Thus, an increase in the half dot density on an immunoblot such as a Western blot is also within the scope of the term "expression" based on biological molecules.

[0075] As used herein, the term "reference value" or "control reference value" refers to a value that is statistically relevant to a particular result when compared to the analysis result. In a preferred embodiment, the reference value is determined by comparing the mRNA expression and / or protein expression of thyroid papillary carcinoma risk markers and performing a statistical analysis. Some such studies are shown in the Examples section herein. However, studies from the literature and the user experience of the methods disclosed herein can also be used to produce or adjust the reference value. The reference value can also be determined by considering circumstances and results that are particularly relevant to the patient's ethnicity, medical history, genetics, age, and other factors.

[0076] POGZ

[0077] POGZ (Pogo transposable element with ZNF domain) is a component of the centromere and is involved in chromosome condensation, mitosis, and DNA repair. Its neurobiological function as an autism-related protein is well known.

[0078] However, recent studies have revealed its cancer regulatory role. For example, POGZ is upregulated in osteosarcoma tissues and is associated with poor prognosis. Elevated POGZ can promote the proliferation of docetaxel-resistant prostate cancer cells and inhibit apoptosis. However, the role of POGZ in THCA remains to be determined.

[0079] MAD2L2

[0080] MAD2L2 is not only involved in spindle assembly and mitosis but also in the inhibition of DNA repair. In colorectal cancer, MAD2L2 inhibits cell proliferation and migration by regulating the ubiquitination modification of the NCOA3 protein. MAD2L2 also blocks DNA end repair and hinders tumor progression.

[0081] However, some studies have also shown that silencing MAD2L2 can promote tumor cell apoptosis. This indicates that different tumors may have different effects.

[0082] Thyroid cancer risk marker

[0083] As used herein, the terms "thyroid cancer risk marker of the present invention" and "risk marker of the present invention" are used interchangeably and refer to "POGZ, MAD2L2, or a combination thereof".

[0084] In the present invention, the terms "thyroid cancer risk marker gene" and "polynucleotide of thyroid cancer risk marker" are used interchangeably and both refer to the nucleotide sequence of any thyroid cancer risk marker shown in POGZ, MAD2L2, or a combination thereof.

[0085] It should be understood that nucleotide substitutions in codons are acceptable when encoding the same amino acid. Additionally, it should be understood that nucleotide changes are also acceptable when conservative amino acid substitutions are produced by nucleotide substitutions.

[0086] In the case where information on the thyroid cancer risk marker is obtained, a nucleic acid sequence encoding it can be constructed based on it, and specific probes can be designed according to the nucleotide sequence. The full-length nucleotide sequence or its fragment can usually be obtained by PCR amplification, recombination, or artificial synthesis methods. For the PCR amplification method, primers can be designed according to the nucleotide sequence of the thyroid cancer risk marker disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art can be used as a template to amplify the relevant sequence. When the sequence is long, it is often necessary to perform PCR amplification two or more times, and then splice the fragments amplified each time together in the correct order.

[0087] Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombination methods. This is usually to clone it into a vector, then transfer it into cells, and then isolate the relevant sequence from the proliferated host cells by conventional methods.

[0088] In addition, the relevant sequence can also be synthesized by artificial synthesis methods, especially when the fragment length is short. Usually, a very long fragment can be obtained by first synthesizing multiple small fragments and then ligating them.

[0089] Currently, it is already possible to completely obtain the DNA sequence encoding the protein (or its fragment, derivative) of the present invention by chemical synthesis. Then this DNA sequence can be introduced into various existing DNA molecules (such as vectors) and cells known in the art.

[0090] By conventional recombinant DNA techniques, the polynucleotide sequence of the present invention can be used to express or produce recombinant thyroid cancer risk markers.

[0091] Detection methods

[0092] Based on the fact that the expression level of the thyroid cancer risk marker POGZ is decreased in tissues of thyroid cancer patients, and the expression level of MAD2L2 regulated by POGZ is also decreased in tissues, the present invention also provides corresponding methods for diagnosing the onset risk of thyroid cancer.

[0093] The present invention relates to diagnostic test methods for quantitatively and locally detecting the levels of human thyroid cancer risk markers. These tests are well known in the art. The levels of thyroid cancer risk markers detected in the tests can be used for diagnosing (including assisting in diagnosis) the risk of thyroid cancer occurrence and / or evaluating the prognosis of thyroid cancer.

[0094] A preferred method is to quantitatively detect thyroid cancer risk markers.

[0095] Preferably, a method for detecting whether there is a thyroid cancer risk marker in a sample is to detect it using a specific antigen, which includes: contacting the sample with a specific antibody of the antigen protein; observing whether an antibody complex is formed, and the formation of an antibody complex indicates the presence of a thyroid cancer risk marker in the sample.

[0096] The thyroid cancer risk markers of the present invention can be used for the diagnosis of thyroid cancer. The antigen proteins of the thyroid cancer risk markers can be immobilized on a protein chip for detecting the thyroid cancer risk markers in a sample.

[0097] Based on the research of the present invention, the levels of the thyroid cancer risk markers of the present invention are significantly increased in thyroid cancer patients. Therefore, the thyroid cancer risk markers of the present invention can be used as markers for detecting or diagnosing (especially assisting in diagnosis and / or early diagnosis) the risk of thyroid cancer occurrence. During detection, when the thyroid cancer risk marker is an up-regulated marker and the ratio (C1 / C0) of the marker level C1 to the corresponding level C0 in the normal population is ≥1.5, preferably ≥2, more preferably ≥3; when the thyroid cancer risk marker is a down-regulated marker and the ratio (C1 / C0) of the corresponding level C0 in the normal population to the marker level C1 is ≥1.5, preferably ≥2, more preferably ≥3; then it can be regarded as an increased risk of thyroid cancer occurrence.

[0098] Detection kit

[0099] Based on the correlation between the thyroid cancer risk markers of the present invention and the risk of thyroid cancer occurrence and prognosis, therefore, the thyroid cancer risk markers of the present invention can be used as diagnostic markers for thyroid cancer occurrence and / or markers for evaluating the prognosis of thyroid cancer.

[0100] The thyroid cancer risk markers provided by the present invention include any marker selected from the following group, or a combination thereof:

[0101] (A1) POGZ; (B1) MAD2L2.

[0102] The present invention also provides a kit for diagnosing the occurrence of thyroid cancer. The kit contains a detection reagent for detecting the thyroid cancer risk marker of the present invention. Preferably, the kit contains the antigen of the thyroid cancer risk marker of the present invention, or its active fragment.

[0103] In another preferred embodiment, the kit further includes a label or an instruction manual, which indicates that the kit is used for diagnosing the risk of thyroid cancer occurrence and / or evaluating the prognosis of thyroid cancer.

[0104] Pharmaceutical compositions and methods of administration

[0105] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity in humans and / or animals and is acceptable to humans and / or animals.

[0106] As used herein, the components of the term "pharmaceutically acceptable" are suitable for humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, substances with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier used for administering a therapeutic agent, including various excipients and diluents.

[0107] The pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and combinations thereof. Usually, the pharmaceutical preparation should be matched with the administration method. The dosage form of the pharmaceutical composition of the present invention is an injection, an oral preparation (tablet, capsule, oral liquid), a transdermal agent, a sustained-release agent. For example, it is prepared by a conventional method with physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition should be manufactured under aseptic conditions.

[0108] The effective amount of the active ingredient described in the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by those of ordinary skill in the art according to various factors (such as through clinical trials). Such factors include but are not limited to: the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, the route of administration, etc. Usually, when the active ingredient of the present invention is administered at a dose of about 0.00001 mg - 50 mg / kg animal body weight (preferably 0.0001 mg - 10 mg / kg animal body weight) per day, satisfactory effects can be obtained. For example, due to the urgent requirements of the treatment condition, several separate doses may be administered per day, or the dose may be proportionally reduced.

[0109] The pharmaceutically acceptable carriers described in the present invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein - antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The selection of the carrier should match the administration route, which is well - known to those of ordinary skill in the art.

[0110] In the present invention, the expression vector can be directly administered to a subject, or the expression vector can be formulated into a pharmaceutical combination with a pharmaceutically acceptable carrier and then administered. The administration includes intravenous injection.

[0111] Treatment method

[0112] The present invention also provides a method for treating thyroid cancer with low expression of POGZ and / or MAD2L2, that is, administering a safe and effective amount of the pharmaceutical composition of the present invention to a subject in need, thereby treating thyroid cancer with low expression of POGZ and / or MAD2L2.

[0113] Generally, "thyroid cancer with low expression of POGZ" means that in the tumor, the expression level E1 of POGZ has a significant difference compared with the amount E0 of POGZ in adjacent - cancerous tissue or normal tissue. Preferably, the "low expression" means E0≥1.5E1, more preferably E0≥2E1; "thyroid cancer with low expression of MAD2L2" means that in the tumor, the expression level E1 of MAD2L2 has a significant difference compared with the amount E0 of MAD2L2 in adjacent - cancerous tissue or normal tissue. Preferably, the "low expression" means E0≥1.5E1, more preferably E0≥2E1;

[0114] Whether POGZ and / or MAD2L2 are low - expressed in tumor tissues can be detected by conventional methods.

[0115] The main advantages of the present invention:

[0116] (a) The risk markers of the present invention can efficiently and accurately predict the occurrence risk of thyroid cancer.

[0117] (b) The present invention provides gene targets for the development of thyroid cancer treatment drugs.

[0118] (c) The risk marker genes of the present invention can be used for gene therapy by artificial vector delivery.

[0119] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0120] Materials and Methods

[0121] Bioinformatics Analysis

[0122] The GEPIA tool was used to analyze THCA cases in the TCGA database. A total of 512 tumor and 337 normal tissue samples were included for differential analysis. Pearson's test was used to examine the correlation between LINC01355 and POGZ in THCA tissues. The String tool was used to perform protein-protein interaction (PPI) dissection on POGZ.

[0123] Cell Culture

[0124] Human normal thyroid cells HTori-3 (YS4003C, Yoji, Shanghai, China) and THCA cells ACT-1 (YB-H1078, Science, China), KTC-1 (AC340144, ATCC), TTA1 (YB-H1077, Science, China) and CAL-62 (CL-0618, Pricilla, China) were preserved in DMEM medium (Gibco, USA) supplemented with 10% fetal bovine serum. The cells were cultured in an incubator at 37 °C, 95% humidity, and 5% CO.

[0125] Co-Immunoprecipitation (Co-IP)

[0126] After removing the culture medium, the cells were washed 3 times with PBS, and then an appropriate amount of lysis buffer was added on ice for 1 hour. Centrifuge at 1200 rpm for 15 minutes to remove the precipitate. The supernatant was divided into a positive control group, a negative control group, and a test group. The positive control group was directly frozen for standby use, the negative control group was added with IgG, and the test group was added with IP antibodies (POGZ, ab249423; MAD2L2, ab180579, Abcam, USA), and then incubated at 4°C overnight. Then, 40 μL (Thermo Fisher) magnetic beads were added to each group, rotated at 4°C for 1 hour, and then the magnetic beads were washed 5 times with pre-cooled PBS, protein loading buffer was added, and Western blot was used to detect the target protein.

[0127] Cell transfection

[0128] KTC-1 and CAL-62 cells, which have relatively low POGZ expression, were transfected to overexpress POGZ and / or silence MAD2L2.

[0129] On this basis, KTC-1 and CAL-62 group 1 were formed: NC+siNC, POGZ+siNC, NC+siMAD2L2 and POGZ+siMAD2L2. pcDNA 3.1POGZ, siMAD2L2 and the corresponding NC / siNC were from GenePharma Co., Ltd. (China).

[0130] 50 pmol (0.67 μg) of plasmid was diluted in 25 μL serum-free DMEM as reagent A, and 1 μL Entranter TM -R4000 (Engreen) was mixed with 24 μL serum-free DMEM for 25 min as reagent B, 25 μL reagent A was fully mixed with 25 μL reagent B (pipetted 10 times) and allowed to stand for 15 min as transfection complex. Cells in 0.45 mL complete medium were transfected with 50 μL transfection complex, and NC / siNC plasmid was used as control.

[0131] RT-qPCR

[0132] Add 400 μL of Trizol (Thermo Fisher, USA) to the cells or tissues in a 1.5 mL sterile RNase-free EP tube. After grinding the cells or tissues into a homogenate, add 600 μL of Trizol, mix for 5 min, centrifuge for 5 min (4 °C, 12,000 × g), and discard the pellet. Mix Trizol and chloroform in a ratio of 1:2 and add it to the centrifugation product of the previous step, then shake it up and down for 15 s. Then add isopropanol to precipitate the RNA in the aqueous phase. Carefully wash the collected RNA pellets with 75% ethanol to remove impurities. Dissolve the RNA in DEPC water and store it at -80 °C.

[0133] Reverse transcribe the RNA into cDNA using the PrimeScript RT Reagent kit (RR047A, Takara) under the conditions: 37 °C / 15 min; perform qPCR amplification with SYBR Green reagent (Takara, Japan) under the following conditions: 94 °C, 3 min; 94 °C, 15 s; 58 °C, 20 s; 72 °C, 30 s; 40 cycles. Normalize the expression of POGZ to GAPDH using the 2 -ΔΔCt method. The primers (5' end to 3' end) are designed as shown in Table 1.

[0134] Table 1

[0135]

[0136]

[0137] Immunoblotting

[0138] Use 100 μL of lysis buffer + 1 μL of protease inhibitor + 1 μL of PMSF as the lysis buffer, remove the culture medium, and lyse the cells or tissues after washing. After centrifugation (12,000 × g, 4 °C, 15 min), collect the supernatant into another 1.5 mL tube. Take 2.5 μL of the sample and 22.5 μL of triple distilled water, and measure the protein concentration using BCA. Separate using 8% - 12% SDS-PAGE.

[0139] Cut the gel according to the label instructions and the position of the target band, and soak the eluted gel in the transfer buffer for 15 minutes. After washing, add 5 mL of milk powder blocking solution and gently shake it overnight at 4 °C. Add rabbit anti-primary antibodies POGZ (1:1000, ab167408, Abcam, USA) and MAD2L2 (1:1000, ab180579) (4 °C, overnight) respectively.

[0140] Then, incubate with the secondary antibody (1:2000, ab6721) at 37 °C for 2 h. Use an ECL kit (Amersham Biote) and IPP6.0 for blot visualization, and obtain the relative expression level with the internal reference GAPDH.

[0141] CCK-8 assay

[0142] Add 100 μL of cell suspension (5×10 4 / mL) to the wells of a 96-well plate. After 24 h, 48 h, and 72 h, add 10 μL of CCK-8 solution respectively. Gently mix on an orbital shaker at 37 °C for 1 minute to ensure uniform mixing. Then, carry out a 2-hour dehydrogenation reaction. Use an Elx808 microplate reader (25-315S, Lonza, USA) to detect the optical density (OD) value at a wavelength of 450 nm.

[0143] Colony formation assay

[0144] Resuspend the cells by digestion, and evenly inoculate 200 cells into the culture medium. Then, culture for 14 days under the above conditions. According to the actual situation, the culture medium needs to be changed every 2 - 3 days.

[0145] At this time, macroscopic colonies appear. Discard the supernatant and carefully wash twice with PBS. Take out 4% PFA after fixation for 15 minutes. Add Giemsa and keep for 20 minutes, then slowly wash off the staining solution and air dry.

[0146] Invert the plate, cover it with a grid transparent film, and count the number of colonies with more than 20 cells.

[0147] Transwell assay

[0148] Add Matrigel (diluted 1:8, Corning, USA) to the upper chamber and incubate at 37 °C for 30 min. Add 600 μL of complete medium (20% FBS) to the lower chamber of a 24-well plate - Transwell device. Culture the cells (5×10 4 / mL) in serum-free medium at 37 °C for 24 h for starvation treatment.

[0149] After digestion, add 100 μL of cell suspension (5×10 4 / mL) to the hydrated Transwell chamber. After 24 h, wash off the non-invasive cells. Stain the cells infiltrating into the lower chamber with 0.1% crystal violet at room temperature for 20 minutes, and then fix with 95% ethanol.

[0150] Randomly select 5 fields of view in a ×400-fold field of view to count the number of invading cells.

[0151] Statistical analysis

[0152] All experiments were independently performed three times. Data were expressed as mean ± SD, and statistical analysis was performed using one-way analysis of variance (ANOVA) and Tukey's multiple comparison test (GraphPad Prism version 7.0). The difference between two groups was analyzed using the T-test. A P value < 0.05 was considered statistically significant.

[0153] Example 1. Results of bioinformatics analysis

[0154] POGZ was downregulated in THCA and interacted with MAD2L2

[0155] Through database analysis, the present invention found that POGZ showed differential expression in different tumors and was lowly expressed in some tumors ( Figure 1 A).

[0156] The level of POGZ in THCA tissues was lower than that in normal thyroid tissues ( Figure 1 B).

[0157] In the 4 THCA cell lines in this study, the mRNA and protein levels of POGZ were lower than those in the normal cell line HTori-3 (P < 0.001) ( Figure 1 C-1D); among them, the CAL-62 cell line had the lowest mRNA and protein levels of POGZ, and the KTC-1 cell line had the second lowest levels of POGZ mRNA and protein. Therefore, they could be used as preferred cell lines for cell experiments. It also indicated that POGZ was downregulated in THCA.

[0158] To preliminarily explore the role and mechanism of POGZ in THCA, the present invention used String for PPI analysis and found a total of 5 proteins: MAD2L2, CBX1, CB5, PSIP1, and CHAMP1 ( Figure 1 E).

[0159] Through protein function annotation, it was found that MAD2L2 had similar functions to POGZ and was involved in the regulation of mitosis and DNA repair.

[0160] In addition, the PPI between POGZ and MAD2L2 was confirmed in the CAL-62 and KTC-1 cell lines by Co-IP. As Figure 1 shown in F, there was a positive correlation between POGZ and MAD2L2.

[0161] Example 2. Cell experiments

[0162] 2.1 POGZ inhibits the proliferation of THCA cells through MAD2L2

[0163] To clarify the effects of POGZ and MAD2L2 on THCA cells, the present invention constructed THCA cells with overexpressed POGZ and silenced MAD2L2, respectively.

[0164] Among them, the CAL-62 cell line and KTC-1 cell line preferred in Example 1 were selected as THCA cells. And their overexpression or silencing efficiency ( Figure 2 A-2D) was evaluated.

[0165] For this purpose, the present invention divided CAL-62 and KTC-1 cells into NC+siNC, POGZ+siNC (POGZ overexpression), NC+siMAD2L2 (MAD2L2 silencing), and POGZ+siMAD2L2 (POGZ overexpression + MAD2L2 silencing).

[0166] First, the proliferation viability of each group of cells was evaluated, and the results were as Figure 3 shown in A-3B. The elevation of POGZ inhibited cell viability, and the reduction of MAD2L2 promoted cell viability; the silencing of MAD2L2 blocked the inhibitory effect of POGZ on cell viability.

[0167] The clonogenicity results of each group were as Figure 3 shown in C-3E. Compared with the NC+siNC group, the clonogenicity of the POGZ+siNC group decreased, and the clonogenicity of the NC+siMAD2L2 group increased; the clone formation level of the POGZ+siMAD2L2 group was higher than that of the POGZ+siNC group and lower than that of the NC+siMAD2L2 group.

[0168] The experimental results indicate that the mechanism by which POGZ inhibits proliferation is inseparable from MAD2L2.

[0169] 2.2 POGZ inhibits THCA cell invasion through MAD2L2

[0170] The present invention also studied the effects of POGZ and MAD2L2 on invasion.

[0171] The results showed that the invasion level of the POGZ+siNC group was lower than that of the NC+siNC group, and the invasion level of the NC+siMAD2L2 group was higher than that of the NC+siNC group. The invasion ability of the POGZ+siMAD2L2 group was higher than that of the POGZ+siNC group and lower than that of the NC+siMAD2L2 group ( Figure 4 A-4B). The silencing of MAD2L2 blocked the inhibitory effect of POGZ on invasion, indicating that the mechanism by which POGZ inhibits invasion is inseparable from MAD2L2 ( Figure 4 A-4C).

[0172] Example 3. Xenograft experiment

[0173] Effects of POGZ and MAD2L2 on Nude Mice Bearing THCA Tumors

[0174] 3.1 Experimental Methods

[0175] BALB / C nude mice (4 weeks old, male, Vital River (Charles River) Co., Ltd., China) were used to evaluate cell tumorigenesis. The breeding environment was 24 ± 1 °C and the relative humidity was 60 ± 5%.

[0176] First, 5×10 6 CAL-62 cells transfected with pcDNA 3.1-POGZ and / or siMAD2L2 were resuspended in 200 μL of PBS respectively. Then they were injected into the axillary region of nude mice at one time. After 4 weeks, euthanasia was performed, and the tumors were collected and weighed.

[0177] When the tumor diameter was greater than 2 cm (not participating in this experiment), the animals were euthanized. The experimental protocol involving animals was approved by the Ethics Committee of Gongli Hospital.

[0178] 3.2 Experimental Results

[0179] Models were obtained by subcutaneous injection of cells in the NC+siNC, POGZ+siNC, NC+siMAD2L2, and POGZ+siMAD2L2 groups as shown in Figure 4 Figure D.

[0180] The experimental results are shown in Figure 4 Figures E-4F. Compared with the siNC group, the tumor volume and mass in the POGZ+siNC group decreased, while the tumor volume and mass in the NC+siMAD2L2 group increased. The tumor volume and mass in the POGZ+siMAD2L2 group were higher than those in the POGZ+siNC group and lower than those in the NC+siMAD2L2 group. The silencing of MAD2L2 weakened the inhibitory effect of POGZ on the tumorigenesis of THCA cells in vivo, indicating that the mechanism by which POGZ inhibits THCA in vivo is also inseparable from MAD2L2.

[0181] Discussion

[0182] The incidence rate of THCA is approximately 15 / 100,000 person-years, and the 5-year survival rate in China is approximately 84.3%. Although the malignancy of THCA is relatively low, there are still about 5% of locally advanced THCA patients with problems of inoperability and high local recurrence rate, which are the main reasons for the increase in THCA mortality. Due to the lack of understanding of the occurrence and development mechanisms of THCA, there is no effective treatment method.

[0183] So far, a large number of studies have focused on the neuromodulatory function of POGZ and its related diseases. However, as an important component of the centromere, POGZ is not only involved in mitosis but also related to DNA repair. These two points are closely related to tumors. However, whether POGZ is involved in the occurrence and development of THCA is still unclear. Therefore, the present invention first conducted bioinformatics analysis and found that POGZ was downregulated in THCA tissues and was also significantly reduced in cells. To explore the mechanism of action of POGZ, the present invention conducted protein interaction analysis and a total of 5 proteins were found: MAD2L2, CBX5, CBX1, PSIPI, and CHAMP1( Figure 1 E).

[0184] Through annotation, it was found that the function of MAD2L2 was similar to that of POGZ. Through the Co-IP experiment of the present invention, the binding relationship between MAD2L2 and POGZ was confirmed in cells( Figure 1 F).

[0185] In this study, THCA cell models with overexpression of POGZ and / or silencing of MAD2L2 were constructed respectively, and it was found that POGZ inhibited both proliferation and motility, and this tumor-suppressing effect of POGZ was reversed by silencing of MAD2L2( Figure 3-4 ). At the animal level, silencing of MAD2L2 also blocked the inhibitory effect of POGZ on tumorigenesis( Figure 4 D-4F). This indicates that the inhibitory effect of POGZ on THCA is inseparable from MAD2L2.

[0186] The clinical value of POGZ and MAD2L2 in THCA remains to be studied in a larger sample size in the future. The molecular mechanism of the interaction between POGZ and MAD2L2 and its impact on THCA still need further research.

[0187] In this study, the present invention focused on the tumor-suppressing effect of the classical neuromodulatory gene POGZ. Through the theoretical prediction and experimental verification by combining cell and nude mouse models, the present invention found that at the protein level, POGZ restricted the growth of THCA by interacting with MAD2L2. The significance of POGZ in THCA may provide inspiration for the research of POGZ and contribute to the diagnosis and treatment of THCA.

[0188] All the documents mentioned in the present invention are cited in this application as references, just as if each document was cited separately as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Use of a gene, mRNA, cDNA, protein of a thyroid cancer risk marker, or a detection reagent thereof, characterized in that, For preparing a diagnostic reagent or kit, which is used for (a) detecting the occurrence risk of thyroid cancer; and / or (b) prognostic evaluation of thyroid cancer; Wherein, the detection reagent is used for detecting the level of the risk marker, and the thyroid cancer risk marker includes any marker selected from the following group, or a combination thereof: (A1) POGZ; (B1) MAD2L2.

2. The use according to claim 1, characterized in that, The detection subject is selected from the following group of subjects: thyroid cancer patients, high-risk subjects for thyroid cancer, subjects without thyroid cancer, or a combination thereof.

3. A kit, characterized in that, The kit contains a detection reagent, which is used for detecting the gene, mRNA, cDNA, protein, or a combination thereof of the thyroid cancer risk marker, Wherein, the thyroid cancer risk marker includes any marker selected from the following group, or a combination thereof: (A1) POGZ; (B1) MAD2L2.

4. The kit according to claim 3, characterized in that, The detection reagent includes a primer pair or a probe, and the primer pair or the probe is a primer pair or a probe for specifically amplifying the mRNA or cDNA of the thyroid cancer risk marker.

5. The kit according to claim 4, wherein, The primer pair includes a primer pair for amplifying POGZ: SEQ ID NO:1, SEQ ID NO:

2.

6. The kit according to claim 3, characterized in that, The kit further includes a label or an instruction manual, and the label or the instruction manual indicates that the kit is used for (a) diagnosing the occurrence risk of thyroid cancer, and / or (b) prognostic evaluation of thyroid cancer.

7. A device for assessing the risk of thyroid cancer, characterized in that, The device includes: (a) An input module, which is used for inputting the thyroid cancer risk marker data of a certain detection subject; Wherein, the risk marker includes any marker selected from the following group, or a combination thereof: (A1) POGZ; (B1) MAD2L2; (b) A processing module, which is used for processing the thyroid cancer risk marker data and giving an occurrence risk assessment result. Wherein, the processing includes: when the expression level C1 of a certain marker is significantly lower than the control reference value C0, it indicates that the thyroid cancer occurrence risk of the subject is high; otherwise, it indicates that the thyroid cancer occurrence risk of the subject is not high; and (c) An output module, which is used for outputting the evaluation result.

8. An active ingredient combination, characterized in that, The active ingredient combination includes: (Z1) A first active ingredient, and the first active ingredient is a POGZ expression promoter; and (Z2) A second active ingredient, and the second active ingredient is a MAD2L2 expression promoter.

9. Use of a combination of active ingredients in the preparation of a pharmaceutical composition for treating thyroid cancer, characterized in that, The active ingredient combination includes: (Z1) A first active ingredient, and the first active ingredient is a POGZ expression promoter; and (Z2) A second active ingredient, and the second active ingredient is a MAD2L2 expression promoter.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: Any active ingredient selected from the following group, or a combination thereof: (Z1) A first active ingredient, and the first active ingredient is a POGZ expression promoter, (Z2) A second active ingredient, and the second active ingredient is a MAD2L2 expression promoter; and (Z3) A pharmaceutically acceptable carrier.

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