Application of lncRNA HCG11, STAT3 and miR-450b-5p in diagnosis and treatment of thyroid cancer

Abstract

The application provides application of LncRNA HCG11, STAT3 and miR-450b-5p in diagnosis and treatment of thyroid cancer. Specifically, the application provides application of a marker of LncRNA HCG11, STAT3 and miR-450b-5p or a combination thereof in a risk of thyroid cancer, and further provides a risk assessment device for a detection reagent or a kit for assessing the risk of thyroid cancer. The application further provides a therapeutic method in which a LncRNA HCG11 expression promoter and a STAT3 expression promoter and / or a miR-450b-5p inhibitor are combined, and the therapy of the application has a significant therapeutic effect on thyroid cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of lncRNAs HCG11, STAT3, and miR-450b-5p in the diagnosis and treatment of thyroid cancer. Background Technology

[0002] Despite the glimmer of hope brought by new immunotherapies, the prognosis for advanced thyroid cancer (THCA) remains poor. Most patients are diagnosed after the optimal window for surgery has passed, and the cancer has already invaded and metastasized.

[0003] The pathogenic factors of THCA are complex, including environmental, genetic, disease, and their interactions. Compared with other common tumors, pathological research on 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 is a lack of reports on risk markers for thyroid cancer in this field, and more effective treatments for thyroid cancer need to be developed.

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

[0007] This invention provides a reagent combination and its application for assessing the risk and prognosis of thyroid cancer with high sensitivity and specificity.

[0008] In a first aspect of the invention, there is provided the use of a thyroid cancer risk marker or a detection reagent thereof for the preparation of a diagnostic reagent or kit, said diagnostic reagent or kit being used for (a) determining the risk of developing thyroid cancer; and / or (b) assessing the prognosis of thyroid cancer;

[0009] The diagnostic reagent is used to detect the level of the thyroid cancer risk marker in the sample to be tested, and the thyroid cancer risk marker includes: (A1) LncRNA HCG11.

[0010] In another preferred embodiment, the risk marker further includes any marker selected from the group consisting of: (B1) the gene, mRNA or protein of STAT3; (B2) miR-450b-5p; or a combination thereof.

[0011] In another preferred embodiment, the thyroid cancer risk markers are: (A1) genes, mRNAs, or proteins of LncRNA HCG11 and (B1) STAT3.

[0012] In another preferred embodiment, the sample to be tested is selected from the group consisting of thyroid cancer tissue, non-thyroid cancer tissue, or a combination thereof.

[0013] In another preferred embodiment, the thyroid cancer includes papillary thyroid carcinoma.

[0014] In another preferred embodiment, the thyroid cancer risk marker is selected from the markers in Table A below:

[0015] Table A

[0016] code name Gene name Upward / downward A1 LncRNA HCG11 Lower B1 STAT3 Lower B2 miR-450b-5p Increased.

[0017] In another preferred embodiment, when the biomarker selected from Table A is an upregulated biomarker, then when the level or expression level C1 of the biomarker is higher than the control reference value C0, it indicates that the subject has a high risk of developing thyroid cancer; when the biomarker selected from Table A is a downregulated biomarker, then when the level or expression level C1 of the biomarker is lower than the control reference value C0, it indicates that the subject has a high risk of developing thyroid cancer.

[0018] In another preferred embodiment, the risk of thyroid cancer is high when the level C1 of the risk marker LncRNA HCG11 is significantly lower than the control reference value C0.

[0019] In another preferred embodiment, the risk of thyroid cancer is high when the expression level of the risk marker STAT3, C1, is significantly lower than the control reference value, C0.

[0020] In another preferred embodiment, a high risk of thyroid cancer is indicated when the expression level C1 of the risk marker miR-450b-5p is significantly higher than the control reference value C0.

[0021] In a second aspect of the invention, a diagnostic reagent combination is provided, the diagnostic reagent combination comprising:

[0022] (a) LncRNA HCG11 detection reagent; and

[0023] (b) Detection reagents selected from the following group: STAT3 detection reagent; miR-450b-5p detection reagent; or combinations thereof.

[0024] In another preferred embodiment, the STAT3 detection reagent is used to detect the level of the STAT3 gene, mRNA, or protein.

[0025] In another preferred embodiment, the diagnostic reagent is used to detect the expression level of thyroid cancer risk markers in the sample to be tested.

[0026] In another preferred embodiment, the test sample is from a group selected from: thyroid cancer subjects, high-risk thyroid cancer subjects, subjects without thyroid cancer, or a combination thereof.

[0027] In a third aspect of the invention, a kit is provided containing a detection reagent for detecting the level of a thyroid cancer risk marker, wherein the thyroid cancer risk marker includes: (A1) LncRNAHCG11.

[0028] In another preferred embodiment, the kit contains the diagnostic reagent combination described in the second aspect of the invention.

[0029] In another preferred embodiment, the risk marker further includes any marker selected from the group consisting of: (B1) the gene, mRNA or protein of STAT3; (B2) miR-450b-5p; or a combination thereof.

[0030] In another preferred embodiment, the thyroid cancer risk markers are: (A1) genes, mRNAs, or proteins of LncRNA HCG11 and (B1) STAT3.

[0031] In another preferred embodiment, the detection reagent comprises:

[0032] (a) Specific antibodies or specific binding molecules against thyroid cancer risk markers; and / or

[0033] (b) Primers or primer pairs, probes or chips (such as nucleic acid chips or protein chips) that specifically amplify the mRNA or cDNA of thyroid cancer risk markers.

[0034] In another preferred embodiment, the detection reagent includes primer pairs or probes that specifically amplify the mRNA or cDNA of the thyroid cancer risk marker.

[0035] In another preferred embodiment, the primer pairs are selected from the group consisting of: primer pairs for amplifying LncRNA HCG11: SEQ ID NO: 1 and 2; and primer pairs for amplifying STAT3: SEQ ID NO: 5 and 6.

[0036] In another preferred embodiment, the primer pair further includes primers for amplifying miR-450b-5p: SEQ ID NO: 3 and 4.

[0037] In another preferred embodiment, the primer pair further includes a primer pair for amplifying the internal reference GAPDH: SEQ ID NO: 7 and 8.

[0038] In another preferred embodiment, the kit further includes a label or instruction manual that provides the following criteria: when the biomarker from Table A is an upregulated biomarker, then when the biomarker level or expression level C1 is higher than the control reference value C0, it indicates a high risk of thyroid cancer in the subject; when the biomarker from Table A is a downregulated biomarker, then when the biomarker level or expression level C1 is lower than the control reference value C0, it indicates a high risk of thyroid cancer in the subject.

[0039] In another preferred embodiment, the expression level C1a of the biomarker LncRNA HCG11 is compared with the control reference value C0a, and the expression level C1b of the biomarker STAT3 is compared with the control reference value C0b. If C1a and C1b are significantly higher than C0a and C0b, it indicates upregulation; if C1a and C1b are significantly lower than C0a and C0b, it indicates downregulation; if C1a and C1b are neither significantly higher than nor significantly lower than C0a and C0b, it indicates normal.

[0040] In another preferred embodiment, the expression level C1c of the marker miR-450b-5p is compared with the control reference value C0c. If C1c is significantly higher than C0c, it indicates upregulation; if C1c is significantly lower than C0c, it indicates downregulation; if C1c is neither significantly higher nor significantly lower than C0c, it indicates normal.

[0041] In another preferred embodiment, the upregulation index is, for the marker LncRNA HCG11, C1a / C0a ≥ 1.5, preferably ≥ 2; for the marker STAT3, C1b / C0b ≥ 1.5, preferably ≥ 2; and for the marker miR-450b-5p, C1c / C0c ≥ 1.5, preferably ≥ 2.

[0042] In another preferred embodiment, the downregulation index is as follows: for the biomarker LncRNA HCG11, C1a / C0a ≤ 2 / 3, preferably ≤ 1 / 2; for the biomarker STAT3, C1b / C0b ≤ 2 / 3, preferably ≤ 1 / 2; and for the biomarker miR-450b-5p, C1c / C0c ≤ 2 / 3, preferably ≤ 1 / 2.

[0043] In another preferred embodiment, the normal ratio refers to the following: for the biomarker LncRNA HCG11, C1a / C0a is between 0.67 and 1.5, preferably 0.7 and 1.3, and more preferably 0.8 and 1.2; for the biomarker STAT3, C1b / C0b is between 0.67 and 1.5, preferably 0.7 and 1.3, and more preferably 0.8 and 1.2; and for the biomarker miR-450b-5p, C1c / C0c is between 0.67 and 1.5, preferably 0.7 and 1.3, and more preferably 0.8 and 1.2.

[0044] In another preferred embodiment, the diagnosis is a diagnosis for an ex vivo sample.

[0045] In another preferred embodiment, the diagnostic reagent is used to detect the level of the risk marker in an ex vivo sample.

[0046] In another preferred embodiment, the ex vivo sample includes tissue samples, preferably cancer tissue samples and adjacent normal tissue samples.

[0047] In another preferred embodiment, the detection reagent is coupled with or carries a detectable marker.

[0048] In another preferred embodiment, the detectable marker is selected from the group consisting of chromophores, chemiluminescent groups, fluorophores, isotopes, or enzymes.

[0049] In another preferred embodiment, the antibody is a monoclonal antibody or a polyclonal antibody.

[0050] In another preferred embodiment, the judgment reagent includes antibodies, primers, probes, sequencing libraries, nucleic acid chips (such as DNA chips) or protein chips.

[0051] In another preferred embodiment, the nucleic acid chip includes a substrate and specific oligonucleotide probes spotted on the substrate, the specific oligonucleotide probes including probes that specifically bind to the polynucleotides (mRNA or cDNA) of any of the said papillary thyroid carcinoma risk markers.

[0052] In another preferred embodiment, the protein chip includes a substrate and specific antibodies spotted on the substrate, the specific antibodies including specific antibodies against the risk markers of papillary thyroid carcinoma.

[0053] In another preferred embodiment, the antibody is a monoclonal antibody or a polyclonal antibody.

[0054] In another preferred embodiment, the reagents include primers, probes, gRNA, or combinations thereof, more preferably primer pairs or probes for PCR, qPCR, or RT-PCR.

[0055] In another preferred embodiment, the risk markers for papillary thyroid carcinoma can be detected by sequencing, PCR, or a combination thereof.

[0056] In another preferred embodiment, the detection of the thyroid cancer risk marker can be quantitative.

[0057] In another preferred embodiment, the diagnostic reagent is used to detect the expression level of a marker in the sample to be tested.

[0058] In another preferred embodiment, the sample to be tested is derived from a human or a non-human mammal.

[0059] In another preferred embodiment, the sample to be tested is derived from a human.

[0060] In another preferred embodiment, the test sample is from a group selected from: thyroid cancer subjects, high-risk thyroid cancer subjects, subjects without thyroid cancer, or a combination thereof.

[0061] In a fourth aspect of the invention, a risk assessment device for thyroid cancer is provided, the device comprising:

[0062] (a) An input module configured to input risk biomarker data from a tissue of a test subject; wherein the risk biomarkers include: (A1) LncRNA HCG11;

[0063] (b) An evaluation module configured to compare the expression level C1 of an input adenocarcinoma risk marker with a control reference value C0 to obtain an evaluation result; wherein the evaluation includes:

[0064] (1) When a certain marker is upregulated, if its expression level C1 is higher than the control reference value C0, it indicates that the subject has a high risk of thyroid cancer; otherwise, it indicates that the risk of thyroid cancer is not high.

[0065] (2) When a biomarker is downregulated, a lower expression level (C1) below the control reference value (C0) indicates a high risk of thyroid cancer in the subject; conversely, a higher expression level (C1) indicates a lower risk of thyroid cancer.

[0066] (c) Output module, which is used to output the evaluation results.

[0067] In another preferred embodiment, the risk marker further includes any marker selected from the group consisting of: (B1) the gene, mRNA or protein of STAT3; (B2) miR-450b-5p.

[0068] In another preferred embodiment, the device further includes a detection module for detecting the level of the prognostic risk marker.

[0069] In another preferred embodiment, the detection module is selected from the group consisting of: ELISA analyzer, PCR sequencer, sequencer, or combinations thereof.

[0070] In another preferred embodiment, the device further includes a control module for controlling the operation of each module.

[0071] In a fifth aspect of the invention, an active ingredient combination is provided, the active ingredient combination comprising:

[0072] (Z1) The first active ingredient, wherein the first active ingredient is a LncRNA HCG11 expression promoter; and

[0073] (Z2) The second active ingredient is a STAT3 expression promoter.

[0074] In another preferred embodiment, the active ingredient further includes:

[0075] (Z3) The third active ingredient is a miR-450b-5p inhibitor.

[0076] In a sixth aspect of the invention, there is provided the use of an active ingredient combination in the preparation of a pharmaceutical composition for treating thyroid cancer, said active ingredient combination comprising:

[0077] (Z1) The first active ingredient, wherein the first active ingredient is a LncRNA HCG11 expression promoter; and

[0078] (Z2) The second active ingredient is a STAT3 expression promoter.

[0079] In another preferred embodiment, the active ingredient further includes:

[0080] (Z3) The third active ingredient is a miR-450b-5p inhibitor.

[0081] In a seventh aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0082] (Z1) First active ingredient, wherein the first active ingredient is an LncRNA HCG11 expression promoter;

[0083] (Z2) A second active ingredient, wherein the second active ingredient is a STAT3 expression promoter; and

[0084] (Z4) Pharmaceutically acceptable carrier.

[0085] In another preferred embodiment, the pharmaceutical composition further comprises:

[0086] (Z3) The third active ingredient is a miR-450b-5p inhibitor.

[0087] In an eighth aspect of the present invention, a detection method is provided, comprising the steps of:

[0088] (a) Provide a sample to be tested;

[0089] (b) The level of the thyroid cancer risk marker in the test sample is recorded as C1; and

[0090] (c) Compare the levels of the thyroid cancer risk markers to the control reference value C0;

[0091] The thyroid cancer risk markers mentioned above include:

[0092] (A1)LncRNA HCG11;

[0093] If the levels of thyroid cancer risk markers in a subject meet the following criteria, it indicates a high risk of developing thyroid cancer:

[0094] (1) When a certain biomarker is an upregulated biomarker in Table A, its expression level C1 is significantly higher than the control reference value C0, which indicates that the subject has a high risk of thyroid cancer; otherwise, it indicates that the risk of thyroid cancer is not high.

[0095] (2) When a certain marker is a downregulated marker in Table A, if its expression level C1 is significantly lower than the control reference value C0, it indicates that the subject has a high risk of thyroid cancer; otherwise, it indicates that the risk of thyroid cancer is not high.

[0096] In another preferred embodiment, the thyroid cancer risk markers further include:

[0097] (B) Any biomarker selected from the following groups, or a combination thereof: (B1) the gene, mRNA or protein of STAT3; (B2) miR-450b-5p; or a combination thereof.

[0098] In another preferred embodiment, the detection method is non-diagnostic and non-therapeutic.

[0099] In another preferred embodiment, the detection method is an in vitro method.

[0100] In another preferred embodiment, the sample to be tested is selected from: thyroid cancer tissue, non-thyroid cancer tissue, or a combination thereof.

[0101] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0102] Figure 1 The following diagram illustrates the regulation of LncRNA HCG11 expression by STAT3 in thyroid cancer tissues and cells: (A) STAT3 expression in thyroid cancer tissues and adjacent normal tissues; (B) Correlation between STAT3 expression and LncRNA HCG11 expression in thyroid cancer tissues and adjacent normal tissues; (C) Schematic diagram of LncRNA HCG11 promoter fragments, ranging from -2,000 to -1,795 to -1,638 to -1,339 to -568 to -140 to -56 to 0, with these promoter fragments cloned upstream of the firefly luciferase reporter gene in the pGL 3-basic vector; (D) Transcriptional activity analysis of potential LncRNA HCG11 promoter fragments in 293T cells; (E) Expression of LncRNA HCG11 in TPC-1 cells or WRO cells after STAT3 knockout, normalized using GAPDH; (FG) ChIP assay in TPC-1 cells or WRO cells, followed by quantitative PCR amplification of LncRNA. Binding site 2 within the HCG11 promoter region. Genomic DNA input was 1%; (H)STAT3 regulates miR-450b-5p expression; (I)STAT3 regulates TAS2R14 expression. Error bars represent the mean ± SEM of three independent experiments. **p<0.01.

[0103] Figure 2 The direct interaction between lncRNA HCG11 and miR-450b-5p was demonstrated: (A) ENCORI / starBase datasets predicted lncRNAs that may bind to miR-450b-5p; (B) PCR analysis of lncRNAs NOP14-AS1, PURPL, and HCG11 in NTHY-ORI 3-1, TPC-1, or 7WRO cell lines, normalized with GAPDH; (CE) Expression of miR-450b-5p in NTHY-ORI 3-1, TPC-1, or WRO cells after knockdown of lncRNAs NOP14-AS1, PURPL, and HCG11, normalized with GAPDH; (FH) Dual-luciferase reporter gene analysis confirmed the direct binding between miR-450b-5p and the 3'UTR region of lncRNA HCG11. Error bars represent the mean ± SEM of three independent experiments. **p < 0.01.

[0104] Figure 3 The following data demonstrates the targeting of lncRNA HCG11 to miR-450b-5p in thyroid cancer cells: (AB) Cell viability of TPC-1 cells overexpressing lncRNA HCG11 or WRO cells knocked down with lncRNA HCG11 after incubation with miR-450b-5p or anti-miR-450b-5p; (CD) Colony formation of TPC-1 cells overexpressing lncRNA HCG11 or WRO cells knocked down with lncRNA HCG11 after incubation with miR-450b-5p or anti-miR-450b-5p; (EG) Colony formation of TPC-1 cells overexpressing lncRNA HCG11 or with lncRNA knockout after incubation with miR-450b-5p or anti-miR-450b-5p. Apoptosis of HCG11-inducing WRO cells; wound healing assay of TPC-1 cells overexpressing lncRNA HCG11 or WRO cells knocked down with lncRNA HCG11 after incubation with miR-450b-5p or anti-miR-450b-5p.

[0105] Figure 4 Low expression of LncRNA HCG11 in thyroid cancer tissues is shown: (A) UALCAN dataset showing LncRNA HCG11 in thyroid cancer cases (n=512) and controls (n=337); (B) Expression of LncRNA HCG11 in thyroid cancer tissues at different stages; (C) Expression of LncRNA HCG11 in thyroid cancer tissues between men and women; (D) Expression of LncRNA HCG11 in thyroid cancer tissues of different age groups; (E) Expression of LncRNA HCG11 in different subtypes of thyroid cancer tissues; (F) Prognosis of patients with low and high expression of LncRNA HCG11; (G) Expression of LncRNA HCG11 in thyroid cancer tissues and adjacent normal tissues; (HI) Nuclear grade separation of LncRNA HCG11 in TPC-1 and WRO cells. Error bars represent the mean ± SEM of three independent experiments. *p<0.05. Detailed Implementation

[0106] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time the promotion or inhibition mechanism of thyroid cancer mediated by the STAT3 / LncRNA HCG11 / miR-450b-5p signaling axis. The research also demonstrates a close interaction between thyroid cancer risk markers such as LncRNA HCG11, STAT3, and miR-450b-5p and thyroid cancer. Correspondingly, diagnostic reagents / kits and devices for assessing the risk and prognosis of thyroid cancer have been developed. Diagnosis using a combination of LncRNA HCG11 detection reagents, STAT3 detection reagents, and / or miR-450b-5p detection reagents can provide highly sensitive and specific diagnosis and assessment of the risk and prognosis of thyroid cancer. Based on this, the present invention was completed.

[0107] The experimental results of this invention show that LncRNA HCG11 is expressed at low levels in thyroid cancer tissues, while STAT3 regulates the expression of LncRNA HCG11 in thyroid cancer tissues. A high risk of thyroid cancer is indicated by significantly decreased expression of LncRNA HCG11, and / or significantly decreased expression of STAT3, and / or significantly increased expression of miR-450b-5p.

[0108] The experimental results of this invention also show that knocking down STAT3 leads to downregulation of LncRNA HCG11 expression, indicating that STAT3 regulates the expression of LncRNA HCG11, and the levels of STAT3 and LncRNA HCG11 are positively correlated.

[0109] the term

[0110] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0111] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101.

[0112] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0113] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0114] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0115] The term "sample" or "sample" as used in this text refers to material specifically associated with a subject from which specific information relating to the subject can be identified, calculated, or inferred. A sample may consist wholly or partially of biological material from the subject.

[0116] As used herein, the term "expression" includes the production of mRNA from a gene or a gene segment, and includes the production of proteins encoded by the RNA or gene segment, as well as the presence of detection substances associated with expression. For example, the binding of cDNA, binding ligands (such as antibodies) to genes or other oligonucleotides, proteins, or protein fragments, and the chromogenic portion of the binding ligand are all included within the scope of the term "expression." Therefore, an increase in the density of half-points on an immunoblot such as a Western blot also falls within the scope of the biologically molecular-based term "expression."

[0117] As used herein, the term "reference value" or "control reference value" refers to a value that is statistically relevant to a particular outcome when compared with the results of an analysis. In a preferred embodiment, the reference value is determined based on a statistical analysis of the mRNA expression and / or protein expression of a comparative risk marker for papillary thyroid carcinoma. Some such studies are shown in the Examples section of this document. However, studies from the literature and user experience with the methods disclosed herein can also be used to produce or adjust reference values. Reference values ​​can also be determined by considering circumstances and outcomes that are particularly relevant to the patient's ethnicity, medical history, genetics, age, and other factors.

[0118] STAT3

[0119] STAT3 is a latent transcription factor in the cytoplasm that is activated in response to extracellular signals (such as cytokines and growth factors) by binding to polypeptide receptors on the cell surface. After activation, STAT3 undergoes tyrosine phosphorylation, dimerization, and translocation to the nucleus, where it binds to specific DNA sequences to regulate the transcription of downstream target genes.

[0120] LncRNA HCG11

[0121] LncRNA HCG11 (long non-coding RNA HCG11) is a non-coding RNA molecule that plays a regulatory role in various cancers. In this study, it was identified as a tumor suppressor in thyroid cancer, inhibiting tumor progression by regulating the miR-450b-5p / TAS2R14 signaling axis. STAT3, as an upstream transcription factor, directly binds to the promoter region of LncRNA HCG11 (the -568nt and -140nt fragments). Figure 1 STAT3 expression is positively correlated with the level of LncRNA HCG11. STAT3 knockdown leads to downregulation of LncRNA HCG11, thereby relieving the inhibition of miR-450b-5p.

[0122] miR-450b-5p

[0123] miR-450b-5p belongs to the microRNA (miRNA) family, a class of small non-coding RNAs approximately 22 nucleotides in length that regulate the expression of target genes by binding to their 3'UTR. This invention demonstrates that miR-450b-5p is an oncogenic miRNA highly expressed in thyroid cancer, promoting malignant tumor behavior by directly inhibiting TAS2R14.

[0124] Thyroid cancer risk markers

[0125] 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 STAT3, LncRNA HCG11, miR-450b-5p, or combinations thereof.

[0126] In this invention, the terms "thyroid cancer risk marker gene" and "thyroid cancer risk marker polynucleotide" are used interchangeably and both refer to the nucleotide sequence of any of the thyroid cancer risk markers shown in STAT3, LncRNA HCG11, miR-450b-5p, or combinations thereof.

[0127] It should be understood that nucleotide substitutions in a codon are acceptable when encoding the same amino acid. Furthermore, it should be understood that nucleotide substitutions are also acceptable when they result in conserved amino acid substitutions.

[0128] Once information about thyroid cancer risk markers is available, the nucleic acid sequence encoding it can be constructed, and specific probes can be designed based on the nucleotide sequence. The full-length nucleotide sequence or fragments thereof can typically be obtained using PCR amplification, recombinant methods, or synthetic methods. For PCR amplification, primers can be designed based on the nucleotide sequence of the thyroid cancer risk markers disclosed in this invention, especially the open reading frame sequence, and the relevant sequence can be amplified using a commercially available cDNA library or a cDNA library prepared according to conventional methods known to those skilled in the art. When the sequence is long, two or more PCR amplifications are often required, and then the fragments amplified from each amplification are spliced ​​together in the correct order.

[0129] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0130] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0131] Currently, the DNA sequence encoding the protein of this invention (or its fragments, derivatives) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art.

[0132] Using conventional recombinant DNA technology, the polynucleotide sequence of this invention can be used to express or produce recombinant thyroid cancer risk markers.

[0133] Detection methods

[0134] Based on the fact that the expression levels of the thyroid cancer risk marker STAT3 and the lncRNA HCG11 are reduced in thyroid cancer tissues, this invention also provides corresponding diagnostic methods for thyroid cancer risk.

[0135] This invention relates to diagnostic test methods for the quantitative and localized detection of risk markers for human thyroid cancer. These tests are well known in the art. The levels of thyroid cancer risk markers detected in these tests can be used to diagnose (including as an adjunct to diagnosis) the risk of developing thyroid cancer and / or to assess the prognosis of thyroid cancer.

[0136] A preferred method is to quantitatively detect thyroid cancer risk markers.

[0137] Preferably, a method for detecting the presence of thyroid cancer risk markers in a sample is to use a specific antigen for detection, which includes: contacting the sample with a specific antibody against the antigen protein; observing whether an antibody complex is formed, and the formation of an antibody complex indicates the presence of thyroid cancer risk markers in the sample.

[0138] The thyroid cancer risk biomarkers of this invention can be used for the diagnosis of thyroid cancer. The antigen proteins of the thyroid cancer risk biomarkers can be immobilized on a protein chip for detection of thyroid cancer risk biomarkers in samples.

[0139] Based on the research of this invention, the levels of the thyroid cancer risk markers of this invention are significantly decreased in patients with thyroid cancer. Therefore, the thyroid cancer risk markers of this invention can be used as markers for detecting or diagnosing (especially for auxiliary diagnosis and / or early diagnosis) the risk of thyroid cancer. During detection, when the thyroid cancer risk marker is upregulated and the ratio of marker level C1 to the corresponding level C0 in the normal population (C1 / C0) is ≥1.5, preferably ≥2, more preferably ≥3; when the thyroid cancer risk marker is downregulated and the ratio of the corresponding level C0 in the normal population to the marker level C1 (C1 / C0) is ≥1.5, preferably ≥2, more preferably ≥3, both are considered as an increased risk of thyroid cancer.

[0140] Test kit

[0141] Based on the correlation between the thyroid cancer risk biomarkers of this invention and the risk and prognosis of thyroid cancer, the thyroid cancer risk biomarkers of this invention can be used as diagnostic biomarkers for the occurrence of thyroid cancer and / or prognostic biomarkers for thyroid cancer.

[0142] The thyroid cancer risk markers provided by this invention include:

[0143] (A) LncRNA HCG11; and

[0144] (B) Any biomarker selected from the following groups, or a combination thereof: (B1) the gene, mRNA or protein of STAT3; (B2) miR-450b-5p; or a combination thereof.

[0145] The present invention also provides a kit for diagnosing thyroid cancer, the kit containing a detection reagent for detecting the thyroid cancer risk marker of the present invention. Preferably, the kit contains an antigen of the thyroid cancer risk marker of the present invention, or an active fragment thereof.

[0146] In another preferred embodiment, the kit further includes a label or instructions indicating that the kit is used to diagnose the risk of thyroid cancer and / or evaluate the prognosis of thyroid cancer.

[0147] Pharmaceutical Composition and Administration

[0148] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0149] As used herein, the term "pharmaceuticalally acceptable" refers to a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of a therapeutic agent, including various excipients and diluents.

[0150] The pharmaceutical compositions of the present invention contain 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 solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, pharmaceutical formulations should be matched to the route of administration; the dosage forms of the pharmaceutical compositions of the present invention are injections, oral formulations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. They are prepared, for example, using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. The pharmaceutical compositions are preferably manufactured under aseptic conditions.

[0151] The effective amount of the active ingredient described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. Generally, satisfactory results are obtained when the active ingredient of this invention is administered daily at a dose of approximately 0.00001 mg to 50 mg / kg animal body weight (preferably 0.0001 mg to 10 mg / kg animal body weight). For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0152] The pharmaceutically acceptable carriers described in this invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched to the route of administration, as is well known to those skilled in the art.

[0153] In this invention, the expression vector can be applied directly to the target, or the expression vector can be combined with a pharmaceutically acceptable carrier to form a drug for administration. Administration includes intravenous injection.

[0154] Treatment

[0155] The present invention also provides a method for treating thyroid cancer with low expression of STAT3 and / or LncRNA HCG11, namely, applying a safe and effective amount of the pharmaceutical composition of the present invention to the desired subject to treat thyroid cancer with low expression of STAT3 and / or LncRNA HCG11.

[0156] Generally, thyroid cancer with low STAT3 expression refers to a tumor where the STAT3 expression level (E1) is significantly different from the STAT3 level (E0) in adjacent or normal tissues. Preferably, "low expression" means E0 ≥ 1.5E1, and more preferably E0 ≥ 2E1. Thyroid cancer with low lncRNA HCG11 expression refers to a tumor where the lncRNA HCG11 expression level (E1) is significantly different from the lncRNA HCG11 level (E0) in adjacent or normal tissues. Preferably, "low expression" means E0 ≥ 1.5E1, and more preferably E0 ≥ 2E1. Whether STAT3 and / or lncRNA HCG11 are lowly expressed in tumor tissue can be detected using conventional methods.

[0157] The main advantages of this invention include:

[0158] (a) The risk markers of the present invention can efficiently and accurately predict the risk of developing thyroid cancer.

[0159] (b) This invention provides gene targets for the development of drugs for the treatment of thyroid cancer.

[0160] (c) The RNA of the present invention (e.g., LncRNA HCG11) can be chemically synthesized and modified to become a therapeutic nucleic acid drug.

[0161] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally 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 as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0162] Materials and methods

[0163] Bioinformatics Analysis

[0164] mRNA expression data and clinical data from 337 normal samples and 512 thyroid cancer samples were obtained from The Cancer Genome Atlas (TCGA) database through the UALCAN dataset portal. Differential analysis was performed to identify differentially expressed lncRNAs and mRNAs, with selection criteria of |logFC|>1.5 and padj<0.05. TargetScan was used to predict target miRNAs, and the interaction between miR-450b-5p and lncRNA HCG11 was explored using the ENCORI platform. STAT3 expression data in various cancers were extracted from the GEPIA dataset.

[0165] Cell culture

[0166] Normal thyroid epithelial cell line NTHY-ORI 3-1 and thyroid cancer cell lines TPC-1, KTC-1, WRO, B-CPAP, SW579, and FTC-133 were used. Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, using DMEM medium, with the medium changed every 48 hours.

[0167] Cell transfection

[0168] Using LipoFiter transfection reagent (Beyotime, China), miR-450b-5p mimic, LncRNAHCG11 mimic, pcDNA3.1-TAS2R14 plasmid, and blank pcDNA3.1 plasmid (OE-NC) were transfected. STAT3 shRNA was cloned into the hU6-MCS-CBh-gcGFP-IRES-puromycin lentiviral vector (Genechem, China), and lentiviral packaging was performed in HEK293T cells (Jet Bio-Filtration, China). Cells were then selected for 10 days using 0.5 mg / L puromycin (Sigma, USA).

[0169] Cell viability assay

[0170] Cell viability was assessed using a CCK-8 assay kit (Abcam, UK). Cells were cultured at a concentration of 6 × 10⁶ cells / year. 3 The cells were seeded at a density of 1 cell per well in 96-well plates. After culturing for 24 hours, CCK-8 reagent was added, and the plates were incubated for another 3 hours. The optical density (OD) value was then measured at 450 nm using a microplate reader (PerkinElmer, USA).

[0171] Scratch test

[0172] Cells were seeded into six-well plates. When the cells reached 80% confluence, a 200 μL pipette tip was used to make a small incision. The cells were washed twice with PBS to remove detached cells, and fresh culture medium was added. After culturing for 24 hours, cell migration was recorded by photographing.

[0173] Cell cycle and apoptosis analysis

[0174] Using a cell cycle assay kit (Abcam, UK), cells were transfected for 24 hours, fixed overnight with 95% ethanol, and then stained with 50 μg / mL propidium iodide (PI) in the dark for 30 minutes. Cell cycle distribution was then detected by flow cytometry.

[0175] Cell migration analysis

[0176] Using a Transwell chamber (8 μm pore size, Corning, USA), 1 × 10⁵ cells were suspended in serum-free DMEM and evenly seeded into the upper chamber. The lower chamber was filled with culture medium containing 20% ​​fetal bovine serum. After 30 hours, the cells were fixed with methanol, stained with 0.5% crystal violet, and the cells in the lower chamber were counted under a microscope.

[0177] RNA extraction and quantitative PCR analysis

[0178] RNA was extracted using Trizol reagent (Life Technologies, USA), and cDNA was synthesized using a cDNA synthesis kit (Vazyme, China). qPCR was performed using the SsoFast EvaGreen Supermix system (Bio-Rad, USA). All gene expression data were normalized using GAPDH as an internal control. Primer sequences are shown in Table 1.

[0179] Table 1

[0180]

[0181] Immunoimprinting

[0182] Proteins were extracted using RIPA buffer (Beyotime, China), and protein concentrations were determined using a BCA kit (Beyotime, China). SDS-PAGE and protein transfer were performed using a 12% polyacrylamide gel (Bio-Rad, USA) and a 0.45-μm PVDF membrane (GE Healthcare, USA). Blocking was performed with 5% skim milk powder, followed by overnight incubation with primary antibody (e.g., anti-POGZ antibody, 1:1000, ab167408, Abcam, USA), and then incubation for 2 hours with secondary antibody (e.g., HRP-conjugated secondary antibody, 1:2000, ab6721). Detection was performed using a chemiluminescence imaging system (Bio-Rad, USA), with GAPDH as an internal control.

[0183] Microarray analysis

[0184] Total RNA was extracted from paired tissue samples from six thyroid cancer patients and purified using Trizol reagent (Invitrogen, USA) and the RNeasy Mini Kit (Qiagen, USA). Biotin-labeled cRNA was synthesized using the HiScript IV 1st Strand cDNASynthesis Kit (Vazyme, China) for microarray hybridization. Microarray analysis was performed according to the methods described in the literature to identify differentially expressed miRNAs.

[0185] Biotin-coupled miRNA pull-down assay

[0186] Biotin was conjugated to the 3' end of miR-450b-5p for specific pull-down of miRNA-related molecules. One × 10⁶ TPC-1 or WRO cells were transfected with 100 pmol of biotin-labeled miR-450b-5p (Bi-miR-450b-5p) or a corresponding negative control using Lipofectamine 2000 (Thermo Fisher, USA). After 48 hours, cells were collected, resuspended in cryolysis buffer, and pull-down assays were performed using streptavidin magnetic beads. Real-time polymerase chain reaction (PCR) was used to quantify the enrichment of target RNA.

[0187] Chromatin Immunoprecipitation (ChIP) Assay

[0188] Using Millipore EZ Magna ChIP TM Kit (Millipore, USA). TPC-1 and WRO cell lines were treated with 1% formaldehyde solution for 10 minutes, then incubated overnight with STAT3-specific antibody (Sigma-Aldrich, Shanghai, China) or control IgG antibody (Sigma-Aldrich, Shanghai, China) and Protein A / G agarose beads for immunoprecipitation. The amount of extracted DNA was determined using quantitative PCR. LncRNA HCG11 primers are shown in Table 1.

[0189] Statistical analysis

[0190] SPSS software (version 17.0, SPSS Inc., Chicago, USA) was used. One-way ANOVA was performed on the quantitative data, followed by Dunnett's post-hoc test for multiple comparisons to determine statistically significant differences between groups. Results are expressed as mean ± standard deviation (SD), and a statistical significance threshold of less than 0.05 was set.

[0191] Example 1: STAT3 is expressed at low levels in THCA tissues and is positively correlated with LncRNA HCG11.

[0192] 1.1 Data Analysis

[0193] In this embodiment, the inventors used RNA sequencing data from the GEPIA dataset (including 512,397 thyroid cancer cases and 337 controls) to analyze and evaluate the expression of STAT3 in various tumors.

[0194] The results showed that STAT3 expression was significantly reduced in THCA compared with normal thyroid tissue (data not shown). Furthermore, correlation and regression analyses revealed a significant positive correlation between STAT3 mRNA levels and LncRNA HCG11 expression in thyroid cancer tissue (p<0.0001) (data not shown), indicating that LncRNA HCG11 expression was also significantly reduced in thyroid cancer tissue.

[0195] This correlation was further validated in a PCR-based analysis of paired thyroid cancer and adjacent normal tissues. Low STAT3 expression was found, and it was moderately correlated with LncRNA HCG 11 levels in the collected tissues (p = 0.0002). Figure 1 AB).

[0196] 1.2 Cell validation

[0197] In TPC-1 and WRO cells, siRNA-mediated STAT3 knockout led to decreased expression of LncRNA HCG11. Figure 1 E) indicates a regulatory link between STAT3 and LncRNA HCG11 in thyroid cancer. When STAT3 levels decrease, the expression level of LncRNA HCG11 also decreases.

[0198] Example 2: Study on the molecular mechanism of downregulation of lncRNA HCG11 in THCA tissues

[0199] To elucidate the molecular mechanism of LncRNA HCG11 downregulation, a detailed promoter analysis was performed in this study. Specifically, the promoter sequence of human LncRNA HCG11 was retrieved from the UCSC genome browser. A series of luciferase activity assays were performed using TPC-1 cells to identify the proximal promoter region of LncRNA HCG11. The promoter activity of the 2,000-base-pair region upstream of the transcription start site (TSS) and six deletion constructs was evaluated. Figure 1 C).

[0200] Compared to the pGL 3-Basic vector, the pGL 3-2000 / 0 plasmid exhibited robust luciferase activity in 293T cells, with no significant difference observed between the full-length and deletion constructs. Figure 1 D).

[0201] Further truncation analysis identified two key fragments at -568 nt and -140 nt, containing regulatory elements essential for LncRNAHCG 11 transcription. Figure 1 F).

[0202] Chromatin immunoprecipitation (ChIP) assays confirmed that STAT3 can directly bind to these regions within the promoter of TPC-1 and LncRNA HCG 11 in WRO cells. Figure 1 G) indicates that STAT3 mediates the transcriptional regulation of LncRNA HCG 11 in thyroid cancer.

[0203] Subsequent experiments revealed that intervention in STAT3 expression (i.e., decreased STAT3 expression leading to decreased LncRNA HCG 11 expression) resulted in increased miR-450b-5p levels and decreased TAS2R14 expression. Figure 1 HI).

[0204] These findings confirm that STAT3 exerts regulatory control over both miR-450b-5p and TAS2R14 through its effect on LncRNA HCG11.

[0205] Therefore, the study of this invention reveals a new regulatory axis in thyroid cancer, in which STAT3 acts as a transcriptional regulator of LncRNAHCG11.

[0206] Example 3: LncRNA HCG11 downregulates miR-450b-5p expression in thyroid cancer cells.

[0207] Given the known interactions between lncRNAs and microRNAs, this invention hypothesizes that specific lncRNAs may coordinate the expression of miR-450b-5p in thyroid cancer tissues. To identify potential lncRNA regulators, this invention uses the ENCORI database for bioinformatics analysis to predict lncRNAs with complementary binding sites to miR-450b-5p.

[0208] Subsequent PCR analysis comparing normal thyroid cells (NTHY-ORI 3-1) with thyroid cancer cell lines (TPC-1 and WRO) showed that, among several candidates, LncRNAs NO P14-AS1, PURPL, and HCG 11 were significantly underexpressed in TPC-1 cells. Figure 2 A).

[0209] Quantitative PCR further confirmed the downregulation of these LncRNAs in TPC-1 and WRO cells compared to normal cell lines. Figure 2 B).

[0210] To identify the specific functions of these lncRNAs, this invention utilizes siRNAs to individually knock down their expression in cell lines. Notably, only the knockdown of lncRNA HCG 11 led to a significant upregulation of miR-450b-5p, such as... Figure 2 As shown in CE, this demonstrates the direct regulatory effect of LncRNA HCG 11 on miR-450b-5p expression.

[0211] To verify the direct interaction between LncRNA HCG11 and miR-450b-5p, this invention designed a luciferase reporter gene assay to incorporate the 3'UTR of LncRNA HCG11 with wild-type (WT) and mutant (MUT) sequences in 293T, TPC-1, and WRO cells. Figure 2 F).

[0212] This assay showed that, in response to miR-450b-5p levels, luciferase activity was specifically regulated by WT LncRNA HCG11, while the MUT form did not show this effect. Figure 2 The study confirmed the presence of the miR-450b-5p recognition sequence within LncRNA HCG 11.

[0213] Example 4: The role of lncRNA HCG11 in regulating the biological behavior of thyroid cancer cells

[0214] This embodiment analyzes the role of LncRNA HCG11 in regulating the biological behavior of thyroid cancer cells.

[0215] The results are as follows Figure 3 As shown, overexpression of LncRNA HCG11 significantly inhibited cell viability and invasion, while promoting apoptosis in thyroid cancer cells. Figure 3 AD). Furthermore, upregulation of LncRNA HCG11 is associated with reduced cell migration (AD). Figure 3 Conversely, knockdown of LncRNA HCG 11 led to increased cell viability, migration, and invasion, as well as reduced apoptosis in vitro.

[0216] To elucidate the molecular basis, this invention treated TPC-1 cells and WRO cells overexpressing LncRNA HCG11 with miR-450b-5p. Figure 3 (AJ). The results showed that miR-450b-5p treatment effectively reversed the inhibitory effect of LncRNA HCG11 overexpression on cell biological behavior.

[0217] Similarly, anti-miR-450b-5p treatment rescued phenotypes induced by LncRNA HCG11 knockdown, including in vitro cell proliferation, invasion, migration, and apoptosis. Figure 3 (AJ). These findings depict a new regulatory axis in thyroid cancer, in which the LncRNA HCG11 directly targets miR-450b-5p, providing new insights into the complex regulatory network controlling gene expression in this disease.

[0218] Example 5: Expression and clinical value of lncRNA HCG11 in thyroid tumors

[0219] To elucidate the expression and clinical value of lncRNA HCG11 in thyroid tumors, a comprehensive study was conducted in this embodiment, which found that the expression of long non-coding RNA (lncRNA) HCG11 in thyroid cancer tissues was significantly lower than that in normal tissues. Figure 4 AG).

[0220] The study also showed that downregulation of LncRNA HCG11 is common in different stages of thyroid cancer, regardless of age, race, or subtype. Figure 4 BE).

[0221] Furthermore, this invention found a positive correlation between low expression of lncRNA HCG11 and poor prognosis in thyroid cancer patients, suggesting that lncRNA HCG11 may play a role in inhibiting thyroid cancer growth. Figure 4 F).

[0222] Furthermore, using a nuclear fragmentation method, this invention investigated the distribution of lncRNA HCG11 in the nucleus and cytoplasm. The results showed that lncRNA HCG11 co-localized with Tubulin in TPC-1 and WRO cells, indicating that it is primarily localized in the cytoplasm. Figure 4 HI).

[0223] In summary, the findings of this invention collectively highlight the downregulation of LncRNA HCG11 in thyroid cancer tissues and its potential role as a diagnostic and prognostic biomarker and therapeutic target for thyroid cancer.

[0224] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of a thyroid cancer risk marker or its detection reagent, characterized in that, This is used to prepare diagnostic reagents or kits for (a) determining the risk of developing thyroid cancer; and / or (b) assessing the prognosis of thyroid cancer. The diagnostic reagent is used to detect the level of the thyroid cancer risk marker in the sample to be tested, and the thyroid cancer risk marker includes: (A1) LncRNA HCG11.

2. The use as described in claim 1, characterized in that, The risk biomarkers also include any biomarker selected from the group consisting of: (B1) the gene, mRNA or protein of STAT3; (B2) miR-450b-5p; or a combination thereof.

3. A diagnostic reagent combination, characterized in that, The diagnostic reagent combination includes: (a) LncRNA HCG11 detection reagent; and (b) Detection reagents selected from the following group: STAT3 detection reagent; miR-450b-5p detection reagent; or combinations thereof; The STAT3 detection reagent is used to detect the level of STAT3 genes, mRNA, or proteins.

4. A reagent kit, characterized in that, The kit contains a detection reagent for detecting the levels of thyroid cancer risk markers, wherein the thyroid cancer risk markers include: (A1)LncRNA HCG11; and (B) Any biomarker selected from the following groups, or a combination thereof: (B1) The gene, mRNA, or protein of STAT3; (B2) miR-450b-5p; or a combination thereof; or, The kit contains the diagnostic reagent combination as described in claim 3.

5. The kit according to claim 4, characterized in that, The detection reagent includes primer pairs or probes that specifically amplify the thyroid cancer risk markers; and the primer pairs are selected from the group consisting of: primer pairs for amplifying LncRNA HCG11: SEQ ID NO: 1 and 2; primer pairs for amplifying STAT3: SEQ ID NO: 5 and 6; primer pairs for amplifying miR-450b-5p: SEQ ID NO: 3 and 4; primer pairs for amplifying the internal reference GAPDH: SEQ ID NO: 7 and 8; or combinations thereof.

6. A risk assessment device for thyroid cancer, characterized in that, The evaluation equipment includes: (a) An input module configured to input risk biomarker data from a tissue of a test subject; wherein the risk biomarkers include: (A1) LncRNA HCG11; (b) An evaluation module configured to compare the expression level C1 of an input adenocarcinoma risk marker with a control reference value C0 to obtain an evaluation result; wherein the evaluation includes: (1) When a certain marker is upregulated, if its expression level C1 is higher than the control reference value C0, it indicates that the subject has a high risk of thyroid cancer; otherwise, it indicates that the risk of thyroid cancer is not high. (2) When a biomarker is downregulated, a lower expression level (C1) below the control reference value (C0) indicates a high risk of thyroid cancer in the subject; conversely, a higher expression level (C1) indicates a lower risk of thyroid cancer. (c) Output module, which is used to output the evaluation results.

7. The device as described in claim 6, characterized in that, The risk biomarkers also include any biomarker selected from the following group, or a combination thereof: (B1) the gene, mRNA or protein of STAT3; (B2) miR-450b-5p.

8. A combination of active ingredients, characterized in that, The combination of active ingredients includes: (Z1) The first active ingredient, wherein the first active ingredient is a LncRNA HCG11 expression promoter; and Selected from any active ingredient from the following group, or a combination thereof: (Z2) Second active ingredient, wherein the second active ingredient is a STAT3 expression promoter; (Z3) The third active ingredient is a miR-450b-5p inhibitor.

9. Use of an active ingredient combination in the preparation of a pharmaceutical composition for treating thyroid cancer, characterized in that, The combination of active ingredients includes: (Z1) The first active ingredient, wherein the first active ingredient is a LncRNA HCG11 expression promoter; and Selected from any active ingredient from the following group, or a combination thereof: (Z2) Second active ingredient, wherein the second active ingredient is a STAT3 expression promoter; (Z3) The third active ingredient is a miR-450b-5p inhibitor.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: (Z1) The first active ingredient, wherein the first active ingredient is a LncRNA HCG11 expression promoter; and Selected from any active ingredient from the following group, or a combination thereof: (Z2) Second active ingredient, wherein the second active ingredient is a STAT3 expression promoter; (Z3) The third active ingredient, wherein the third active ingredient is a miR-450b-5p inhibitor; and (Z4) Pharmaceutically acceptable carrier.

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