Application of PIWIL1 and EVA1A in diagnosis and treatment of papillary thyroid carcinoma

Abstract

The invention provides application of PIWIL1 and EVA1A in diagnosis and treatment of papillary thyroid carcinoma. Specifically, the invention provides a combination of PIWIL1 and EVA1A diagnostic reagents and application thereof in preparation of a kit for judging the occurrence risk of papillary thyroid carcinoma and prognosis. The diagnostic reagent composition disclosed by the invention can be used for efficiently and quickly judging the prognosis condition of a papillary thyroid carcinoma patient. The invention further provides a treatment method combining the PIWIL1 inhibitor and the EVA1A inhibitor. 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 in particular to applications of PIWIL1 and EVA1A in the diagnosis and treatment of papillary thyroid cancer. Background Art

[0002] Papillary thyroid carcinoma (PTC) is the most common pathological subtype of thyroid malignancies. In the United States, PTC accounts for approximately 87% of diagnosed thyroid or endocrine tumors. Over the past decade, the incidence of PTC has increased by an average of 3.1% per year. Previous investigations have shown that lymph node metastasis (LNM) is the main mechanism of PTC spread, and nearly half of patients have LNM manifestations at diagnosis.

[0003] Furthermore, LNM in PTC has been shown to adversely affect prognosis, increase the risk of recurrence, and reduce long-term survival, especially in patients older than 45 years.

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

[0005] Currently, there is still a lack of reports on risk markers for papillary thyroid cancer in this field, and more effective treatments for thyroid cancer remain to be developed.

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

[0007] The purpose of the present invention is to provide a reagent combination and application thereof for judging the risk and prognosis of thyroid papillary carcinoma with high sensitivity and high specificity.

[0008] In a first aspect of the present invention, a diagnostic reagent combination is provided, the diagnostic reagent comprising:

[0009] (A) PIWIL1 detection reagent; and

[0010] (B) EVA1A detection reagent.

[0011] In another preferred embodiment, the diagnostic reagent is used to detect the expression level of a marker in a sample to be tested.

[0012] In another preferred embodiment, the sample to be tested is from a subject selected from the following group: a subject with papillary thyroid cancer, a subject at high risk of papillary thyroid cancer, a subject without papillary thyroid cancer, or a combination thereof.

[0013] In a second aspect of the present invention, there is provided a use of a diagnostic reagent combination for preparing a diagnostic kit, wherein the diagnostic kit is used for (a) performing a prognostic assessment on papillary thyroid carcinoma; and / or (b) diagnosing the risk of occurrence of papillary thyroid carcinoma;

[0014] Wherein, the diagnostic reagent combination is used to detect the level of risk markers, and the diagnostic reagent combination includes:

[0015] (A) PIWIL1 detection reagent; and

[0016] (B) EVA1A detection reagent.

[0017] In another preferred embodiment, the kit further comprises a label or instructions, which provide the prognostic criteria in Table A below:

[0018] Table A

[0019]

[0020] In another preferred embodiment, the expression level C1a of the marker PIWIL1 is compared with the control reference value C0a, and the expression level C1b of the marker EVA1A is compared with the control reference value C0b. If C1a or C1b is significantly higher than C0a or C0b, it indicates up-regulation; if C1a or C1b is significantly lower than C0a or C0b, it indicates down-regulation; if C1a or C1b is neither significantly higher than nor significantly lower than C0a or C0b, it indicates normal;

[0021] In another preferred embodiment, the up-regulation means that, for the marker PIWIL1, C1a / C0a≥1.5, preferably ≥2; for the marker EVA1A, C1b / C0b≥1.5, preferably ≥2.

[0022] In another preferred embodiment, the down-regulation means that, for the marker PIWIL1, C1a / C0a≤2 / 3, preferably ≤1 / 2; for the marker EVA1A, C1b / C0b≤2 / 3, preferably ≤1 / 2.

[0023] In another preferred embodiment, the normal value refers to that, for the marker PIWIL1, C1a / C0a is between 0.67-1.5, preferably 0.7-1.3, more preferably 0.8-1.2; for the marker EVA1A, C1b / C0b is between 0.67-1.5, preferably 0.7-1.3, more preferably 0.8-1.2.

[0024] In another preferred embodiment, the diagnosis is for an in vitro sample.

[0025] In another preferred embodiment, the diagnostic reagent is used to detect the level of the risk marker in an in vitro sample.

[0026] In another preferred embodiment, the in vitro sample includes a tissue sample, preferably a cancer tissue sample and a paracancerous tissue sample.

[0027] In another preferred embodiment, the detection reagent is coupled with or carries a detectable label.

[0028] 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.

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

[0030] In another preferred embodiment, the determination reagent includes an antibody, a primer, a probe, a sequencing library, a nucleic acid chip (such as a DNA chip) or a protein chip.

[0031] In another preferred embodiment, the nucleic acid chip includes a substrate and specific oligonucleotide probes spotted on the substrate, and the specific oligonucleotide probes include probes that specifically bind to any polynucleotide (mRNA or cDNA) of the papillary thyroid carcinoma risk marker.

[0032] 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 papillary thyroid carcinoma risk markers.

[0033] In another preferred embodiment, the antibody is a monoclonal antibody or a polyclonal antibody.

[0034] In another preferred embodiment, the reagents include primers, probes, gRNA or a combination thereof, more preferably primer pairs or probes for PCR, qPCR, or RT-PCR.

[0035] In another preferred embodiment, the detection of the papillary thyroid carcinoma risk marker can be performed by the following methods: sequencing, PCR, or a combination thereof.

[0036] In another preferred embodiment, the detection of the thyroid cancer risk marker can be quantitative.

[0037] In another preferred embodiment, the diagnostic reagent is used to detect the expression level of a marker in a sample to be tested.

[0038] In another preferred embodiment, the sample to be tested comes from a human or a non-human mammal.

[0039] In another preferred embodiment, the sample to be tested comes from a human.

[0040] In another preferred embodiment, the sample to be tested is from a subject selected from the following group: a subject with papillary thyroid cancer, a subject at high risk of papillary thyroid cancer, a subject without papillary thyroid cancer, or a combination thereof.

[0041] In a third aspect of the present invention, a prognosis assessment device for papillary thyroid carcinoma is provided, the assessment device comprising:

[0042] (a) an input module, the input module is used to input risk marker data in a certain test object tissue;

[0043] Wherein, the risk markers are PIWIL1 and EVA1A;

[0044] (b) a processing module, the processing module is used to process the prognostic risk marker data of papillary thyroid carcinoma and provide a prognostic risk assessment value, wherein the processing includes: comparing the expression levels of the input markers C1a and C1b with the control reference values ​​C0a and C0b, if C1a or C1b is significantly higher than C0a or C0b, it is indicated as up-regulated; if C1a or C1b is significantly lower than C0a or C0b, it is indicated as down-regulated; if C1a or C1b is neither significantly higher than nor significantly lower than C0a or C0b, it is indicated as normal; and substituting the comparison result into the judgment standard of Table A to obtain the high or low prognostic risk of the subject;

[0045] Wherein, the table A is as follows:

[0046] Table A

[0047]

[0048] and

[0049] (c) An output module, which is used to output the evaluation results.

[0050] In another preferred example, the device further comprises a detection module, and the detection module is used to detect the level of the prognostic risk marker.

[0051] In another preferred embodiment, the detection module is selected from the following group: ELISA analyzer, PCR sequencer, sequencer, or a combination thereof.

[0052] In another preferred example, the device further includes a control module, and the control module is used to control the operation of each module.

[0053] In a fourth aspect of the present invention, there is provided an active ingredient combination, the active ingredient combination comprising:

[0054] (Z1) a first active ingredient, wherein the first active ingredient is a PIWIL1 inhibitor; and

[0055] (Z2) A second active ingredient, wherein the second active ingredient is an EVA1A inhibitor.

[0056] In a fifth aspect of the present invention, there is provided a use of an active ingredient combination in preparing a pharmaceutical composition for treating papillary thyroid carcinoma, the active ingredient combination comprising:

[0057] (Z1) a first active ingredient, wherein the first active ingredient is a PIWIL1 inhibitor; and

[0058] (Z2) A second active ingredient, wherein the second active ingredient is an EVA1A inhibitor.

[0059] In a sixth aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0060] (Z1) a first active ingredient, wherein the first active ingredient is a PIWIL1 inhibitor;

[0061] (Z2) a second active ingredient, wherein the second active ingredient is an EVA1A inhibitor; and

[0062] (Z3) a pharmaceutically acceptable carrier.

[0063] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 The results showed that PIWIL1 was highly expressed in PTC tissues and positively correlated with EVA1A: (A) Expression of PIWIL1 in various cancer types (Cancer Genome Atlas Database) mRNA levels of (B) PIWIL1 and (C) EVA1A in PTC clinical specimens (n=20) and adjacent tissues (n=20); GAPDH was used for normalization; (D) Linear regression and correlation analysis of PIWIL1 mRNA level and EVA1A mRNA level in PTC clinical specimens; (E) Representative images of PIWIL1 protein blot; (F) Representative images of EVA1A protein blot; (G) Relative changes in PIWIL1 and EVA1A protein levels in PTC and adjacent tissues; Each experiment was performed three times and the data are expressed as mean ± SD; PIWIL1, piRNA binding protein 1; EVA1A, Eva-1 homolog A; PTC, papillary thyroid carcinoma; #: p < 0.05.

[0065] Figure 2Figure 3 Overexpression of PIWIL1 promoted the expression of EVA1A in TPC-1 cells: (A) mRNA levels of EVA1A and (B) PIWIL1 in various PTC cell lines (NTHY-ORI3-1, TPC-1, KTC-3, WRO, PDTC-1, SW579, and FTC-133); GAPDH was used for normalization; (C) mRNA levels of PIWIL1 in TPC-1 cells after EVA1A overexpression or knockdown; GAPDH was used for normalization; (D) mRNA levels of PIWIL1 in TPC-1 cells after PIWIL1 overexpression or knockdown (A) PIWIL1 mRNA level in TPC-1 cells; GAPDH was used for normalization; (E) EVA1A mRNA level in TPC-1 cells when EVA1A was overexpressed or knocked down; GAPDH was used for normalization; (F) EVA1A mRNA level in TPC-1 cells when PIWIL1 was overexpressed or knocked down; GAPDH was used for normalization; Each experiment was performed three times and the data are presented as the mean ± SD; PIWIL1, piRNA binding protein 1; EVA1A, Eva-1 homolog A; PTC, papillary thyroid carcinoma; #: p < 0.05; N = 9.

[0066] Figure 3 Figure 3 shows that overexpression of PIWIL1 promoted the expression of EVA1A in KTC-3 cells: (A) EVA1A or (B) PIWIL1 mRNA level in TPC-1 cells when PIWIL1 was overexpressed or knocked down; GAPDH was used for normalization; (C) EVA1A or (D) EVA1A RNA level in TPC-1 cells when PIWIL1 was overexpressed or knocked down; GAPDH was used for normalization; each experiment was performed three times and the data are expressed as mean ± SD; PIWIL1, piRNA binding protein 1; EVA1A, Eva-1 homolog A. #: p < 0.05; N = 9.

[0067] Figure 4 Figure 4 shows that PIWIL1 and EVA1A promote the proliferation of papillary thyroid carcinoma cells in vitro: the viability of TPC-1 cells upon overexpression or knockdown of (A) EVA1A or (B) PIWIL1; the viability of KTC-3 cells upon overexpression or knockdown of (C) EVA1A or (D) PIWIL1; each experiment was performed three times and the data are presented as mean ± SD; PIWIL1, piRNA binding protein 1; EVA1A, Eva-1 homolog A. #: p < 0.05; N = 9.

[0068] Figure 5Figure 4 shows that PIWIL1 and EVA1A increased the cell cycle activity of papillary thyroid carcinoma cells in vitro: (A) The percentage of TPC-1 cells in each stage of the cell cycle after overexpression or knockdown of EVA1A or (B) PIWIL1; (C) The percentage of KTC-3 cells in each stage of the cell cycle after overexpression or knockdown of EVA1A or (D) PIWIL1; The cell cycle activity of TPC-1 cells after overexpression or knockdown of (E) EVA1A or (F) PIWIL1 was analyzed by flow cytometry; Each experiment was performed three times and the data are expressed as mean ± SD; PIWIL1, piRNA binding protein 1; EVA1A, Eva-1 homolog A. #: p < 0.05; N = 9.

[0069] Figure 6 Figure 4 shows that PIWIL1 and EVA1A inhibit apoptosis of papillary thyroid carcinoma cells in vitro: (A) apoptosis of TPC-1 cells after overexpression or knockdown of EVA1A or (B) PIWIL1; (C) apoptosis of KTC-3 cells after overexpression or knockdown of EVA1A or (D) PIWIL1; apoptosis of TPC-1 cells after overexpression or knockdown of (E) EVA1A or (F) PIWIL1 was analyzed by flow cytometry; each experiment was performed three times and the data are expressed as mean ± SD; PIWIL1, piRNA binding protein 1; EVA1A, Eva-1 homolog A. #: p < 0.05; N = 9.

[0070] Figure 7 Figure 4 shows that PIWIL1 and EVA1A promote the invasion of papillary thyroid carcinoma cells in vitro: (A) Transwell cell counts of TPC-1 cells when EVA1A or (B) PIWIL1 was overexpressed or knocked down; (C) Transwell cell counts of KTC-3 cells when EVA1A or (D) PIWIL1C was overexpressed or knocked down; (E) Transwell invasion assay was used to analyze the invasion of TPC-1 cells after EVA1A overexpression or knockdown; (F) Transwell invasion assay was used to analyze the invasion of TPC-1 cells after PIWIL1 was overexpressed or knocked down; each experiment was performed three times and the data are expressed as mean ± SD; PIWIL1, piRNA binding protein 1; EVA1A, Eva-1 homolog A. #: p < 0.05; N = 9.

[0071] Figure 8Figure 1 shows that PIWIL1 promotes proliferation and apoptosis of papillary thyroid carcinoma via EVA1A in vitro and in vivo: (A) (B) Survival rate and apoptosis of TPC-1 cells when EVA1A is overexpressed and PIWIL1 is knocked down or PIWIL1 is overexpressed and EVA1A is knocked down; (C) (D) Survival rate and (D) apoptosis of KTC-3 cells when EVA1A is overexpressed and PIWIL1 is knocked down or PIWIL1 is overexpressed and EVA1A is knocked down; (E) TPC-1 cells injected with EVA1A overexpressing and PIWIL1 knocked down Images of xenograft tumors from animal models after injection of cells or TPP-1 cells overexpressing PIWIL1 and EVA1A knockout; (F) Tumor size of animal models after injection of TPC-1 cells overexpressing EVA1A and PIWIL1 knockout, or TPP-1 cells overexpressing PIWIL1 and EVA1A knockout; Each experiment was performed three times and the data are presented as mean ± SD; PIWIL1, piRNA binding protein 1; EVA1A, Eva-1 homolog A. #: p < 0.05; N = 9. DETAILED DESCRIPTION

[0072] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that the papillary thyroid carcinoma (PTC) risk markers PIWIL1 and EVA1A have a close interactive relationship, and accordingly developed diagnostic reagents / kits and equipment for judging the risk and prognosis of papillary thyroid carcinoma. The combination of PIWIL1 detection reagent and EVA1A detection reagent can be used for diagnosis, which can diagnose and evaluate the risk and prognosis of papillary thyroid carcinoma with high sensitivity and specificity.

[0073] The present inventors have also found that the use of the inhibitor combination of the present invention (PIWIL1 inhibitor and EVA1A inhibitor) can effectively inhibit the development of papillary thyroid carcinoma. Based on this, the present invention was completed.

[0074] the term

[0075] As used herein, the term "sample" or "specimen" refers to material specifically associated with a subject from which specific information about the subject can be determined, calculated, or inferred. A sample may consist in whole or in part of biological material from a subject.

[0076] As used herein, the term "expression" includes the production of mRNA from a gene or gene portion, and includes the production of proteins encoded by the RNA or gene portion, and also includes the appearance of detection substances related to expression. For example, cDNA, the binding of binding ligands (such as antibodies) to genes or other oligonucleotides, proteins or protein fragments, and the chromogenic portion of the binding ligands are all included within the scope of the term "expression". Therefore, the increase in half-point density on an immunoblot such as a Western blot is also within the scope of the term "expression" based on biological molecules.

[0077] As used herein, the term "reference value" or "control reference value" refers to a value that is statistically correlated with a particular result when compared to the results of an analysis. In a preferred embodiment, the reference value is determined based on comparing the mRNA expression and / or protein expression of papillary thyroid cancer risk markers and performing statistical analysis. Some such studies are shown in the Examples section of this article. However, studies from the literature and user experience of the methods disclosed herein can also be used to produce or adjust reference values. Reference values ​​can also be determined by considering situations and results that are particularly relevant to the patient's ethnic group, medical history, genetics, age, and other factors.

[0078] PIWIL1

[0079] Argonaute proteins of the PIWIL subfamily play a central role in reproductive function by binding piRNAs in the testis. However, recent evidence suggests that these proteins may also be involved in other diseases, particularly cancer.

[0080] piRNA-binding protein 1 (PIWIL1) is the most extensively studied protein in the PIWI protein family. It has been implicated in regulating various physiological and pathological processes, including DNA damage response, cell cycle progression, apoptosis, cell proliferation, and tight junction formation. In particular, it has been shown to play an important role in regulating the progression of several cancers. However, the contribution of PIWIL1 to the pathogenesis of papillary thyroid carcinoma (PTC) remains to be elucidated.

[0081] EVA1A

[0082] EVA1A (Eva-1 homolog A) functions as a lysosomal and endoplasmic reticulum-associated protein that regulates cell proliferation and apoptosis. Downregulation of EVA1A has been observed in clinical samples of HCC and non-small cell lung cancer, where it inhibits tumor cell growth through autophagy and apoptosis.

[0083] In contrast, analysis of The Cancer Genome Atlas (TCGA) database revealed that EVA1A was significantly upregulated in thyroid cancer tissues (P < 0.0001), although no association between EVA1A and PIWIL1 in thyroid cancer has been revealed so far.

[0084] Several studies have shown that EVA1A is associated with tumorigenesis. Ren et al. demonstrated that microRNA-125b inhibited EVA1A-mediated autophagy and reversed oxaliplatin resistance in hepatocellular carcinoma. The differential expression of EVA1A in pancreatic cancer tissues suggests that it is involved in the progression of pancreatic cancer.

[0085] Papillary thyroid cancer risk markers

[0086] As used herein, the terms "risk marker for papillary thyroid cancer of the present invention", "risk marker for papillary thyroid cancer of the present invention" and "risk marker of the present invention" are used interchangeably to refer to "PIWIL1, EVA1A, or a combination thereof".

[0087] In the present invention, the terms "papillary thyroid cancer risk marker gene" and "papillary thyroid cancer risk marker polynucleotide" are used interchangeably, and both refer to the nucleotide sequence of any papillary thyroid cancer risk marker shown in PIWIL1, EVA1A, or a combination thereof.

[0088] It should be understood that when encoding the same amino acid, the substitution of nucleotides in the codon is acceptable. It should also be understood that when conservative amino acid substitutions are produced by nucleotide substitutions, the change of nucleotides is also acceptable.

[0089] In the case of obtaining information on risk markers for papillary thyroid carcinoma, a nucleic acid sequence encoding it can be constructed based thereon, and a specific probe can be designed based on the nucleotide sequence. The full-length nucleotide sequence or a fragment thereof can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed based on the nucleotide sequence of the risk marker for papillary thyroid carcinoma 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 is used as a template to amplify the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the fragments amplified each time are spliced ​​together in the correct order.

[0090] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0091] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0092] At present, the DNA sequence encoding the protein of the present invention (or its fragment, derivative) can be obtained completely by chemical synthesis. Then the DNA sequence can be introduced into various existing DNA molecules (such as vectors) and cells known in the art.

[0093] The polynucleotide sequence of the present invention can be used to express or produce recombinant papillary thyroid cancer risk markers using conventional recombinant DNA techniques.

[0094] Detection Methods

[0095] Based on the fact that the expression levels of PIWIL1 and EVA1A, risk markers for papillary thyroid cancer, are increased in tissues of patients with papillary thyroid cancer, the present invention also provides a corresponding method for diagnosing the risk of occurrence and prognosis of papillary thyroid cancer.

[0096] The present invention relates to a diagnostic test method for quantitatively and positionally detecting the level of risk markers for human papillary thyroid carcinoma. These tests are well known in the art. The level of risk markers for papillary thyroid carcinoma detected in the test can be used to diagnose (including auxiliary diagnosis) the risk of papillary thyroid carcinoma, and / or the prognosis evaluation of papillary thyroid carcinoma.

[0097] A preferred method is to perform quantitative detection of papillary thyroid cancer risk markers.

[0098] Preferably, a method for detecting whether a papillary thyroid cancer risk marker is present in a sample is to use specific antigens for detection, which includes: contacting the sample with an antigen protein-specific antibody; and observing whether an antibody complex is formed, wherein the formation of an antibody complex indicates the presence of a papillary thyroid cancer risk marker in the sample.

[0099] The papillary thyroid cancer risk marker of the present invention can be used for the diagnosis of papillary thyroid cancer. The antigen protein of the papillary thyroid cancer risk marker can be fixed on a protein chip to detect the papillary thyroid cancer risk marker in a sample.

[0100] Based on the research of the present invention, the level of the thyroid papillary carcinoma risk marker of the present invention is significantly increased in patients with thyroid papillary carcinoma. Therefore, the thyroid papillary carcinoma risk marker of the present invention can be used as a marker for detecting or diagnosing (especially auxiliary diagnosis and / or early diagnosis) the risk of thyroid papillary carcinoma.

[0101] During the test, when the papillary thyroid cancer risk marker is an up-regulated marker and the ratio of the marker level C1 to the corresponding level C0 in the normal population (C1 / C0) is ≥1.5, preferably ≥2, and more preferably ≥3; it can be regarded as an increased risk of papillary thyroid cancer. When the papillary thyroid cancer risk marker is a down-regulated marker, and the ratio of the corresponding level C0 in the normal population to the marker level C1 (C1 / C0) is ≥1.5, preferably ≥2, and more preferably ≥3; it can be regarded as an increased risk of papillary thyroid cancer.

[0102] Detection Kits

[0103] Based on the correlation between the papillary thyroid carcinoma risk marker of the present invention and the risk and prognosis of papillary thyroid carcinoma, the papillary thyroid carcinoma risk marker of the present invention can be used as a diagnostic marker for the occurrence of papillary thyroid carcinoma and / or a prognosis assessment marker for papillary thyroid carcinoma.

[0104] The papillary thyroid cancer risk markers provided by the present invention include PIWIL1 and EVA1A.

[0105] The invention also provides a kit for diagnosing the occurrence of papillary thyroid carcinoma, the kit comprising a detection reagent, the detection reagent being used to detect the papillary thyroid carcinoma risk marker of the present invention. Preferably, the kit comprises an antigen of the papillary thyroid carcinoma risk marker of the present invention, or an active fragment thereof.

[0106] In another preferred embodiment, the kit further comprises a label or instructions, wherein the label or instructions indicate that the kit is used for diagnosing the risk of papillary thyroid carcinoma and / or evaluating the prognosis of papillary thyroid carcinoma.

[0107] Pharmaceutical compositions and methods of administration

[0108] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0109] As used herein, the term "pharmaceutically acceptable" ingredients are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), i.e., substances with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

[0110] 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, glucose, water, glycerol, ethanol, and combinations thereof. Usually, the pharmaceutical preparation should match the mode of administration. The dosage form of the pharmaceutical composition of the present invention is injection, oral preparation (tablet, capsule, oral liquid), transdermal agent, sustained release agent. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.

[0111] The effective amount of the active ingredient of the present invention may vary with the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: 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 body weight, the patient's immune status, the route of administration, etc. Generally, 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 results can be obtained. For example, depending on the urgency of the treatment condition, several divided doses may be administered per day, or the dose may be reduced proportionally.

[0112] The pharmaceutically acceptable carrier of the present invention includes (but is not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should match the mode of administration, which are well known to those of ordinary skill in the art.

[0113] In the present invention, the expression vector can be directly administered to the subject, or the expression vector can be prepared into a drug combination with a pharmaceutically acceptable carrier and then administered. The administration includes intravenous injection.

[0114] Treatment

[0115] The present invention also provides a method for treating papillary thyroid carcinoma with high expression of PIWIL1 or EVA1A, that is, administering a safe and effective amount of the pharmaceutical composition of the present invention to a desired subject, thereby treating papillary thyroid carcinoma with high expression of PIWIL1 or EVA1A.

[0116] Generally, "PIWIL1 high expression or EVA1A high expression papillary thyroid carcinoma" means that in the tumor, the expression level E1 of PIWIL1 or EVA1A is significantly different from the level E0 of PIWIL1 or EVA1A in adjacent cancer tissue or normal tissue. Preferably, the "high expression" refers to E1≥1.5E0, and more preferably E1≥2E0.

[0117] The main advantages of the present invention include:

[0118] (a) The diagnostic reagent combination of the present invention can determine the risk of occurrence and prognosis of papillary thyroid cancer with high sensitivity and higher accuracy.

[0119] (b) The pharmaceutical composition of the present invention can effectively inhibit the development of papillary thyroid carcinoma.

[0120] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed 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 under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0121] Materials and methods

[0122] Study subjects

[0123] The present invention collected 20 pairs of papillary thyroid carcinoma (PTC) tissues and adjacent normal tissues.

[0124] The experimental cells of the present invention are human thyroid cancer cell lines (NTHY-ORI 3-1, TPC-1, KTC-3, WRO, SW579 and FTC-133), which were purchased from the American Type Culture Collection.

[0125] The in vivo animal experiment of the present invention uses nude mice (BALB / c nude mice; male, 4 weeks old), which are obtained from a company (Shanghai Xipuboke Experimental Animal Co., Ltd.) and cultured under non-pathogenic conditions.

[0126] TCGA database

[0127] The present invention collected data of 468 PTC patients, which showed complete clinicopathological characteristics, including age, gender, LNM, tumor size, clinical stage and histological type. The data were obtained from the TCGA data portal (https: / / tcga-data.nci.nih.gov / docs / publications / tcga / ) for further analysis.

[0128] Western blotting

[0129] Total protein was extracted from PTC and adjacent normal tissues by lysis with RIPA buffer (Sigma, USA). The quantity and quality of protein were evaluated using the BCA assay (Beyotime, China). Equal amounts of protein were loaded onto 12% gels (Bio-Rad, USA) and transferred to PVDF membranes (GE Healthcare, USA). The membranes were incubated in skim milk (Beyotime, China) for 1 hour and then incubated overnight at 4hC with primary antibodies against PIWIL1, EVA1A or GAPDH (Sigma, USA). Subsequently, the membranes were washed with TBST and exposed to secondary antibodies (Sigma, USA) for 2 hours at room temperature. Finally, the blots were visualized using ECL luminescent reagents (Absin Bioscience Inc., China Co., Ltd.) and the band intensity was quantified using Image-Pro Plus 6.0 (Media Control netics, Inc.).

[0130] Cell culture

[0131] Human thyroid cancer cell lines (NTHY-ORI 3-1, TPC-1, KTC-3, WRO, SW579 and FTC-133) were purchased from the American Type Culture Collection. Cells were cultured in high-glucose DMEM (Millipore, Sigma) supplemented with 20% FBS (Gibco; Thermo Fisher Scientific, Inc.). Cells were placed in T-75 flasks (Corning, USA) and cultured in a 5% CO2 atmosphere at 37°C.

[0132] Apoptosis assay

[0133] The apoptosis rate of cells was evaluated using the Annexin V-FITC apoptosis detection kit (Nanjing Kechuang Biotechnology Co., Ltd.). The cells were collected and suspended in 5 × 105 cells / ml of binding buffer. Next, the cells were labeled with Annexin V-FITC (10 μl) and PI (5 μl) and used Cell apoptosis was analyzed by flow cytometry (BD Biosciences). Flow cytometry data were analyzed using Flowjo V10.0.7 software.

[0134] Cell cycle assay

[0135] TPC-1 or KTC-3 cells were trypsinized and then centrifuged at 400 x g for 10 min at 37 °C. After washing twice with PBS, the cells were fixed with 75% ethanol at -20 °C overnight. Before testing, the cells were washed twice with PBS and then stained with 500 μl of PI staining solution. The stained cells were fixed for 20 min at room temperature in the dark. Cell cycle analysis was performed by flow cytometry (BD Biosciences), and the results were analyzed using Flowjo V10.0.7 software.

[0136] Cell viability assay

[0137] The viability of TPC-1 or KTC-3 cells was assessed using the Cell Counting Kit (CCK)-8 assay (Abcam) according to the manufacturer's instructions. 5 × 10 cells were used as the suspension for each group. 4 Cells were seeded into 96-well plates at a concentration of 10 cells / ml and cultured in high-glucose DMEM for 24 hours. The culture medium was then replaced with a mixture of 90 μl fresh culture medium and 5 μl CCK-8 solution. The 96-well plate was cultured at 37hC for 2 hours, and the optical density of each well was measured at 450 nm using a microplate reader (BioTek Instruments, Inc.).

[0138] Transwell invasion assay

[0139] Cells were cultured in growth medium and 2×104 cells were seeded in transwell chambers (Corning, Inc.). Matrigel (Millipore, Sigma) was used to pre-coat the membrane. The bottom of the chamber was filled with RPMI-1640 medium containing 5% serum as an inducer. After 48 hours, non-invaded cells were removed using a cotton swab. The invading cells at the bottom of the chamber were fixed with 8% paraformaldehyde and observed under an optical microscope (Zeiss, Germany) after staining with 0.1% crystal violet.

[0140] Reverse transcription quantitative PCR (RT-qPCR)

[0141] Follow the manufacturer's instructions for use (Millipore, Sigma) isolated total RNA of cells. In ABIPRISM 7500 real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.), reverse transcription (RT) was performed using SYBR PrimeScript RT-PCR kit (Shanghai Yesen Biotechnology Co., Ltd.). Each reaction was performed three times. The data obtained were analyzed using the 2-ΔΔCt method, and the expression of the gene was standardized by using GAPDH as a loading control. The qPCR primers used are listed in Table 1.

[0142] Table 1 Primers used for polymerase chain reaction

[0143]

[0144]

[0145] Small interfering RNA (siRNA)

[0146] To inhibit the expression of EVA1A or PIWIL1, TPC-1 or KTC-3 cells cultured to 75-85% confluency were transfected with specific siRNA (100 nM) or treated with 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) to disrupt siRNA (100 nM). After culturing for 6 hours at 37°C and 5% CO2, growth medium containing 10% FBS (Gibco; Thermo-Fisher Scientific, Inc.) was added. Cells were harvested 48 hours after transfection for RNA extraction. These siRNAs were obtained from Genomedicech (Shanghai, China).

[0147] Lentiviral transfection

[0148] EVA1A or PIWIL1 overexpression was induced by lentiviral vectors obtained from GeneChem, Inc. Negative control vectors and specific lentiviral vectors were also obtained from the same source. TPC-1 or KTC-3 cells were cultured and seeded into 12-well plates and then used 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) and the corresponding lentiviral vector were incubated. The cells were then cultured for 72 hours, with the medium replaced every 6 hours.

[0149] Animal experiments

[0150] Nude mice (BALB / c nude mice; male, 4 weeks old) were obtained from the company (Shanghai Xipuboke Experimental Animal Co., Ltd.) and cultured under pathogen-free conditions. The mice were randomly divided into five groups (N=6), and the experiment was repeated three times. To induce the development of xenograft tumors, TPC-1 cells (2×107 cells / ml, 200μl) were injected subcutaneously. The volume and weight of the tumor were measured every 7 days until the end of the 42-day study period when the mice were euthanized. Tumor nodules were collected to assess tumor size. The tissue was frozen in liquid nitrogen for further analysis. All animal studies were performed after approval by the Punan Hospital Animal Care Committee (PNHEA2021001).

[0151] Statistical analysis

[0152] Data analysis was performed using SPSS 21.0 software (IBM Corp.), and linear regression and correlation analysis were performed to determine the relationship between EVA1A mRNA and PIWIL1 mRNA. Data are presented as mean ± SD, and each experiment was repeated at least three times. A p value less than 0.05 was considered statistically significant.

[0153] Example 1 PIWIL1 is highly expressed in PTC tissues and positively correlated with EVA1A

[0154] The expression of PIWIL1 in various tumors was evaluated using the TCGA database.

[0155] The results showed that a significant increase in expression was observed in PTC compared with normal thyroid tissue (Figure 1A).

[0156] RT-qPCR validation of PIWIL1 expression in clinical specimens (n ​​= 20) was consistent with TCGA data ( Figure 1 B).

[0157] In addition, EVA1A mRNA levels were upregulated in PTC tissues (n = 20) ( Figure 1 C), a positive correlation was observed between EVA1A and PIWIL1 expression in PTC tissues ( Figure 1 D).

[0158] Compared with adjacent tissues, the protein levels of PIWIL1 and EVA1A were significantly increased in PTC tissues ( Figure 1 EG).

[0159] Example 2 PIWIL1 induces the expression of EVA1A in PTC cells

[0160] The present inventors evaluated the mRNA levels of EVA1A and PIWIL1 in various thyroid cancer cell lines (NTHY-OR13-1, TPC-1, KTC-3, WRO, PDTC-1, SW579 and FTC-133). Only TPC-1 and KTC-3 cells showed significant upregulation of mRNA levels of PIWIL1 and EVA1A ( Figure 2 A and B). Therefore, the inventors selected TPC-1 and KTC-3 cells for further experiments.

[0161] Cell test results showed that the expression of PIWIL1 did not change significantly in EVA1A knockdown or overexpression TPC-1 cells ( Figure 2 C and E).

[0162] In contrast, PIWIL1 can regulate the expression of EVA1A in TPC-1 cells ( Figure 2 D and F).

[0163] Furthermore, RT-qPCR analysis of KTC-3 cells confirmed these results ( Figure 3 AD).

[0164] In conclusion, the inventors' data indicate that overexpression of PIWIL1 promotes the expression of EVA1A in PTC cells.

[0165] Example 3 Effects of PIWIL1 and EVA1A on PTC cells

[0166] PIWIL1 and EVA1A promote PTC cell proliferation

[0167] The proliferation of PTC cell lines (TPC-1 and KTC-3) was assessed by cell viability assay.

[0168] Compared with the control groups (PTC cells untreated or treated with siRNA or lentiviral control), overexpression of PIWIL1 or EVA1A in TPC-1 cells resulted in a positive increase in cell viability, whereas knockdown of PIWIL1 and EVA1A resulted in a decrease in cell viability ( Figure 4 A and B).

[0169] These findings were also confirmed by cell viability assay in KTC-3 cells ( Figure 4 C and D).

[0170] Taken together, the results indicate that upregulation of PIWIL1 or EVA1A enhances the proliferation of PTC cells in vitro.

[0171] PIWIL1 and EVA1A enhance the cell cycle activity of PTC cells

[0172] The effects of PIWIL1 and EVA1A on the cell cycle of PTC cells were evaluated.

[0173] Overexpression of either gene in TPC-1 cells increased the percentage of cells in the G2 / M phase, whereas downregulation resulted in cell cycle arrest (Fig. 5A, B, and E). Similar results were observed in KTC-3 cells ( Figure 5 C, D and F).

[0174] These findings suggest that overexpression of PIWIL1 or EVA1A enhances cell cycle activity in PTC cells in vitro ( Figure 5 AF).

[0175] PIWIL1 and EVA1A inhibit apoptosis of PTC cells

[0176] Apoptosis assays showed that overexpression of PIWIL1 or EVA1A blocked apoptosis of TPC-1 cells in vitro. Conversely, knockdown of PIWIL1 or EVA1A enhanced apoptosis of TPC-1 cells ( Figure 6 A, B and E).

[0177] Similar observations were made in KTC-3 cells ( Figure 6 C, D, and F), which collectively indicate that PIWIL1 or EVA1A overexpression inhibits apoptosis of PTC cells in vitro.

[0178] PIWIL1 and EVA1A promote the invasion of PTC cells

[0179] Transwell invasion assays showed that overexpression of PIWIL1 or EVA1A exacerbated the invasion of TPC-1 cells, whereas their downregulation attenuated this invasion ( Figure 7 A, B, and E). These findings were further confirmed by Transwell invasion assays performed on KTC-3 cells ( Figure 7 C, D and F).

[0180] Therefore, the results indicated that overexpression of PIWIL1 or EVA1A enhanced the invasion of PTC cells.

[0181] Example 4 PIWIL1 promotes PTC cell proliferation and apoptosis in vitro and in vivo via EVA1A

[0182] 4.1 In vitro experiments

[0183] The relationship between PIWIL1 and EVA1A was further explored by manipulating their expression levels in TPC-1 and KTC-3 cells. Specifically, overexpression of PIWIL1 and knockdown of EVA1A, or overexpression of EVA1A and knockdown of PIWIL1, was performed in TPC-1 and KTC-3 cells. Cell viability and apoptosis assays were used to evaluate the effects of these manipulations on proliferation and apoptosis.

[0184] Results in TPC-1 ( Figure 8 A and B) and KTC-3( Figure 8 C and D) Cells showed that PIWIL1 promoted PTC cell proliferation and apoptosis through EVA1A in vitro.

[0185] 4.2 In vivo experiments

[0186] The present inventors verified the in vivo functions of PIWIL1 and EVA1A by establishing a xenograft tumor mouse model.

[0187] The inventors' data showed that tumors overexpressing PIWIL1 showed an increase in size, which was attenuated upon EVA1A knockdown.

[0188] In contrast, tumors with reduced PIWIL1 expression exhibited a reduction in size, which was blocked by EVA1A overexpression.

[0189] These findings support the idea that PIWIL1 promotes PTC via EVA1A in vivo ( Figure 8 E and F).

[0190] In conclusion, the results of the present invention indicate that PIWIL1 promotes the proliferation and apoptosis of PTC cells through EVA1A in vitro and in vivo, thereby exacerbating PTC.

[0191] Based on the above experiments of the present invention, since PIWIL1 and EVA1A are closely related and PIWIL1 promotes the occurrence and progression of papillary thyroid carcinoma (PTC) through EVA1A, the prognosis can be judged based on the following judgment criteria (or see Table A), and an optimized personalized treatment plan can be adopted accordingly:

[0192] (a) When the expression levels of both PIWIL1 and EVA1A are upregulated, or one is upregulated and the other is normal, the prognosis of papillary thyroid carcinoma (PTC) is poor (or the risk of developing papillary thyroid carcinoma is high);

[0193] (b) When the expression levels of both PIWIL1 and EVA1A are normal (compared with the normal population), or one is downregulated and the other is normal, the prognosis of papillary thyroid carcinoma (PTC) is good (or the risk of developing papillary thyroid carcinoma is low);

[0194] (c) When the expression level of PIWIL1 is upregulated and the expression level of EVA1A is downregulated, the prognosis of papillary thyroid carcinoma (PTC) is good (or the risk of papillary thyroid carcinoma is low);

[0195] (d) When the expression level of PIWIL1 is downregulated and the expression level of EVA1A is upregulated, the prognosis of papillary thyroid carcinoma (PTC) is better (or the risk of papillary thyroid carcinoma is lower).

[0196] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. 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 claims attached to this application.

Claims

1. A diagnostic reagent combination, It is characterized in that The diagnostic reagent combination comprises: (A) PIWIL1 detection reagent; and (B) EVA1A detection reagent.

2. The diagnostic reagent combination according to claim 1, It is characterized in that The diagnostic reagent is used to detect the expression level of the marker in the sample to be tested.

3. The diagnostic reagent combination according to claim 1, It is characterized in that The sample to be tested is from a subject selected from the group consisting of a subject with papillary thyroid cancer, a subject at high risk of papillary thyroid cancer, a subject without papillary thyroid cancer, or a combination thereof.

4. Use of a diagnostic reagent combination, It is characterized in that Used for preparing a diagnostic kit, wherein the diagnostic kit is used for (a) performing prognostic assessment on papillary thyroid carcinoma; and / or (b) diagnosing the risk of occurrence of papillary thyroid carcinoma; Wherein, the diagnostic reagent combination is used to detect the level of risk markers, and the diagnostic reagent combination includes: (A) PIWIL1 detection reagent; and (B) EVA1A detection reagent.

5. The use according to claim 4, It is characterized in that The diagnostic reagent is used to detect the level of the risk marker in an ex vivo sample.

6. The use according to claim 4, It is characterized in that The kit also includes a label or instructions, which provide the prognostic criteria of Table A below: Table A 7. A prognostic assessment device for papillary thyroid carcinoma, It is characterized in that The evaluation equipment comprises: (a) an input module, the input module being used to input data of prognostic risk markers in a tissue of a test subject; Wherein, the prognostic risk markers are PIWIL1 and EVA1A; (b) a processing module, the processing module is used to process the prognostic risk marker data of papillary thyroid carcinoma and provide a prognostic risk assessment value, wherein the processing includes: comparing the expression levels of the input markers C1a and C1b with the control reference values ​​C0a and C0b, if C1a or C1b is significantly higher than C0a or C0b, it is indicated as up-regulated; if C1a or C1b is significantly lower than C0a or C0b, it is indicated as down-regulated; if C1a or C1b is neither significantly higher than nor significantly lower than C0a or C0b, it is indicated as normal; and substituting the comparison result into the judgment standard of Table A to obtain the high or low prognostic risk of the subject; Wherein, the table A is as follows: Table A and (c) An output module, which is used to output the evaluation results.

8. An active ingredient combination, It is characterized in that The active ingredient combination includes: (Z1) a first active ingredient, wherein the first active ingredient is a PIWIL1 inhibitor; and (Z2) A second active ingredient, wherein the second active ingredient is an EVA1A inhibitor.

9. Use of an active ingredient combination in the preparation of a pharmaceutical composition for treating papillary thyroid carcinoma, It is characterized in that The active ingredient combination includes: (Z1) a first active ingredient, wherein the first active ingredient is a PIWIL1 inhibitor; and (Z2) A second active ingredient, wherein the second active ingredient is an EVA1A inhibitor.

10. A pharmaceutical composition, It is characterized in that The pharmaceutical composition contains: (Z1) a first active ingredient, wherein the first active ingredient is a PIWIL1 inhibitor; (Z2) a second active ingredient, wherein the second active ingredient is an EVA1A inhibitor; and (Z3) a pharmaceutically acceptable carrier.

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