Application of LINC01355, miR-27b-3p and POGZ in diagnosis and treatment of thyroid cancer

Abstract

The invention relates to an application of LINC01355, miR-27b-3p and POGZ in diagnosis and treatment of thyroid cancer. Specifically, the invention provides an application of LINC01355, miR-27b-3p and POGZ markers or a combination thereof in the occurrence risk of thyroid cancer, and also provides a detection reagent or a kit and risk assessment equipment for assessing the occurrence risk of thyroid cancer. The invention also provides a treatment method of combining the LINC01355 expression promoter, the miR-27b-3p inhibitor and the POGZ expression promoter. The treatment method disclosed by the invention has a remarkable treatment effect on the thyroid cancer.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and clinical medicine, and specifically, to the application of LINC01355, miR-27b-3p and POGZ in the diagnosis and treatment of thyroid cancer. Background Art

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

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

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

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

[0006] Therefore, there is an urgent need in this field to develop 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 more targeted drugs and methods for treating thyroid cancer. Summary of the Invention

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

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

[0009] Wherein, the detection reagent is used to detect the level of the risk marker, and the thyroid cancer risk marker includes:

[0010] (A1) LINC01355; and

[0011] (B) Any marker selected from the following group, or a combination thereof: (B1) miR-27b-3p; (B2) the gene, transcript, or protein of POGZ.

[0012] In another preferred example, the thyroid cancer risk marker is a marker selected from Table A below:

[0013] Table A

[0014] Code name Gene name Up-regulated / Down-regulated A1 LINC01355 Down-regulated B1 miR-27b-3p Up-regulated B2 POGZ Down-regulated.

[0015] In another preferred example, when the marker selected from Table A is an up-regulated marker, if the level or expression level C1 of the marker is higher than the control reference value C0, it indicates a high risk of thyroid cancer in the subject to be tested; when the marker selected from Table A is a down-regulated marker, if the level or expression level C1 of the marker is lower than the control reference value C0, it indicates a high risk of thyroid cancer in the subject to be tested.

[0016] In another preferred example, when the level C1 of the risk marker LINC01355 is significantly lower than the control reference value C0, the risk of thyroid cancer is high.

[0017] In another preferred example, when the level C1 of the risk marker miR-27b-3p is significantly higher than the control reference value C0, the risk of thyroid cancer is high.

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

[0019] In another preferred example, when the level C1a of the risk marker LINC01355 is significantly lower than the control reference value C0a, and the expression level C1b of POGZ is significantly lower than the control reference value C0b, it indicates a high risk of thyroid cancer in the test subject.

[0020] In another preferred example, the test subject is selected from the group consisting of: thyroid cancer patients, high-risk subjects for thyroid cancer, subjects without thyroid cancer, or combinations thereof.

[0021] In a second aspect of the present invention, a kit is provided, the kit contains a detection reagent for detecting a thyroid cancer risk marker,

[0022] wherein the thyroid cancer risk marker includes:

[0023] (A1) LINC01355; and

[0024] (B) Optionally, any marker selected from the following group, or a combination thereof: (B1) miR-27b-3p; (B2) the gene, transcript, or protein of POGZ.

[0025] In another preferred example, the detection reagent includes:

[0026] (a) Specific antibodies and specific binding molecules against the thyroid cancer risk markers; and / or

[0027] (b) Primers or primer pairs, probes or chips (such as nucleic acid chips or protein chips) that specifically amplify the mRNA or cDNA of the thyroid cancer risk markers.

[0028] In another preferred embodiment, the detection reagent comprises a primer pair or a probe, and the primer pair or the probe is a primer pair or a probe that specifically amplifies the mRNA or cDNA of the thyroid cancer risk marker.

[0029] In another preferred embodiment, the primer pairs are selected from the following group: primer pairs for amplifying LINC01355: SEQ ID NO.1, SEQ ID NO.2; primer pairs for amplifying POGZ: SEQ ID NO.3, SEQ ID NO.4; primer pairs for amplifying miR-27b-3p: SEQ ID NO.7, SEQ ID NO.8.

[0030] In another preferred embodiment, the thyroid cancer risk marker is of human origin.

[0031] In another preferred embodiment, the detection is for an in vitro sample.

[0032] In another preferred embodiment, the in vitro sample includes: tissue samples.

[0033] In another preferred embodiment, the detection reagent is conjugated with or carries a detectable label.

[0034] In another preferred embodiment, the detectable label is selected from the following group: chromophore, chemiluminescent group, fluorophore, isotope or enzyme.

[0035] In another preferred embodiment, the antibody is a monoclonal antibody or a polyclonal antibody.

[0036] In another preferred embodiment, the determination reagent comprises an antibody, a primer, a probe, a sequencing library, a nucleic acid chip (such as a DNA chip) or a protein chip.

[0037] In another preferred embodiment, the nucleic acid chip comprises a substrate and specific oligonucleotide probes spotted on the substrate, and the specific oligonucleotide probes comprise probes that specifically bind to the polynucleotide (mRNA or cDNA) of any of the thyroid cancer risk markers.

[0038] In another preferred embodiment, the protein chip comprises a substrate and specific antibodies spotted on the substrate, and the specific antibodies comprise specific antibodies against the thyroid cancer risk markers.

[0039] In another preferred embodiment, the antibody is a monoclonal antibody or a polyclonal antibody.

[0040] In another preferred embodiment, the kit contains genes, mRNAs, cDNAs, and / or proteins of thyroid cancer risk markers as reference substances or quality control substances.

[0041] In another preferred embodiment, the reagent includes primers, probes, gRNAs, or combinations thereof, more preferably primer pairs or probes for PCR, qPCR, or RT-PCR.

[0042] In another preferred embodiment, the detection of the thyroid cancer risk marker can be performed by the following methods: sequencing, PCR, or a combination thereof.

[0043] In another preferred embodiment, the detection of the thyroid cancer risk marker can be a quantitative detection.

[0044] In the third aspect of the present invention, a detection method is provided, including the steps of:

[0045] (a) Providing a detection sample;

[0046] (b) Detecting the level of the thyroid cancer risk marker in the detection sample and recording it as C1; and

[0047] (c) Comparing the level of the thyroid cancer risk marker with a control reference value C0;

[0048] Wherein, the thyroid cancer risk markers include:

[0049] (A1) LINC01355; and

[0050] (B) Optionally, any marker selected from the following group, or a combination thereof: (B1) miR-27b-3p; (B2) genes, transcripts, or proteins of POGZ;

[0051] If the level of the thyroid cancer risk marker of the detection subject meets the following conditions, it indicates that the risk of thyroid cancer in the subject to be tested is high:

[0052] (1) When a certain marker is an up-regulated marker in Table A, if its expression level C1 is significantly higher than the control reference value C0, it indicates that the risk of thyroid cancer in the subject is high; otherwise, it indicates that the risk of thyroid cancer is not high;

[0053] (2) When a certain marker is a down-regulated marker in Table A, if its expression level C1 is significantly lower than the control reference value C0, it indicates that the risk of thyroid cancer in the subject is high; otherwise, it indicates that the risk of thyroid cancer is not high.

[0054] In another preferred embodiment, the detection method is non-diagnostic and non-therapeutic.

[0055] In another preferred embodiment, the detection method is an in vitro method.

[0056] In another preferred embodiment, the test sample is selected from: thyroid cancer tissue, non-thyroid cancer tissue, or a combination thereof.

[0057] In a fourth aspect of the present invention, there is provided a device for assessing the risk of thyroid cancer development, the device comprising:

[0058] (a) An input module for inputting data of thyroid cancer risk markers of a certain test subject;

[0059] Wherein, the risk markers include:

[0060] (A1) LINC01355; and

[0061] (B) Optionally any marker selected from the following group, or a combination thereof: (B1) miR-27b-3p; (B2) the gene, transcript, or protein of POGZ;

[0062] (b) A processing module for comparing the expression level C1 of the input adenocarcinoma risk marker with a control reference value C0 to obtain an evaluation result; wherein, the evaluation includes:

[0063] (1) When a certain marker is an up-regulated marker, when its expression level C1 is higher than the control reference value C0, it indicates a high risk of thyroid cancer development in the subject; otherwise, it indicates a low risk of thyroid cancer development;

[0064] (2) When a certain marker is a down-regulated marker, when its expression level C1 is lower than the control reference value C0, it indicates a high risk of thyroid cancer development in the subject; otherwise, it indicates a low risk of thyroid cancer development; and

[0065] (c) An output module for outputting the evaluation result.

[0066] In another preferred embodiment, the device further comprises (d) a storage module for storing data including: comparison result values, control reference values.

[0067] In another preferred embodiment, the device further comprises (e) a control module for controlling the operation of each module.

[0068] In a fifth aspect of the present invention, there is provided a combination of active ingredients, the combination of active ingredients comprising:

[0069] (Z1) A first active ingredient, wherein the first active ingredient is a promoter for LINC01355 expression; and

[0070] Any active ingredient selected from the following group, or a combination thereof:

[0071] (Z2) A second active ingredient, wherein the second active ingredient is an inhibitor of miR-146a-3p;

[0072] (Z3) A third active ingredient, wherein the third active ingredient is a promoter for POGZ expression.

[0073] In a sixth aspect of the present invention, there is provided the use of an active ingredient combination in the preparation of a pharmaceutical composition for treating thyroid cancer, wherein the active ingredient combination comprises:

[0074] (Z1) A first active ingredient, wherein the first active ingredient is a promoter for LINC01355 expression; and

[0075] Any active ingredient selected from the following group, or a combination thereof:

[0076] (Z2) A second active ingredient, wherein the second active ingredient is an inhibitor of miR-146a-3p;

[0077] (Z3) A third active ingredient, wherein the third active ingredient is a promoter for POGZ expression.

[0078] In a seventh aspect of the present invention, there is provided a pharmaceutical composition, which contains:

[0079] (Z1) A first active ingredient, wherein the first active ingredient is a promoter for LINC01355 expression; and

[0080] Any active ingredient selected from the following group, or a combination thereof:

[0081] (Z2) A second active ingredient, wherein the second active ingredient is an inhibitor of miR-146a-3p;

[0082] (Z3) A third active ingredient, wherein the third active ingredient is a promoter for POGZ expression; and

[0083] (Z4) A pharmaceutically acceptable carrier.

[0084] It should be understood that within the scope of the present invention, the above-mentioned various technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1Shown is that LINC01355 targets miR-27b-3p which targets POGZ: (A) Expression characteristics of LINC01355 in 33 types of tumors; (B) LINC01355 levels in THCA and normal tissues of GEPIA patients; (C-D) Pearson test for the correlation between LINC01355 and POGZ in THCA tissues and whole blood; (E-F) Expression characteristics of LINC01355 and miR-27b-3p in normal HTori-3 cells and THCA cell lines; (G) Binding sites of LINC01355 with miR-27b-3p, POGZ and miR-27b-3p; (H) Dual-luciferase reporter gene to verify the targeted binding of LINC01355 with miR-27b-3p; (I) Dual-luciferase reporter gene to verify the targeted binding of POGZ with miR-27b-3p; *P<0.05 vs. normal. ***P<0.001 vs. HTori-3 or NC; P<0.001 vs. Mut-LINC01355 or Mut-POGZ.

[0086] Figure 2 Shown is that LINC01355 promotes POGZ expression via miR-27b-3p: (A) Construction of overexpression of LINC01355 in KTC-1 and CAL-62 cells; (B) Construction of overexpression of miR-27b-3p in KTC-1 and CAL-62 cells; (C-D) Effects of overexpressing LINC01355 and / or miR-27b-3p on POGZ protein expression; ***P<0.001 vs. NC + mimic NC; P<0.001 vs. LINC01355 + mimic NC; ^^^P<0.001 vs. NC + mimic.

[0087] Figure 3 Shown is that LINC01355 inhibits THCA cell proliferation by targeting miR-27b-3p: (A-B) Effects of overexpressing LINC01355 and / or miR-27b-3p on cell viability by CCK-8; (C-D) Effects of overexpressing LINC01355 and / or miR-27b-3p on cell proliferation by colony formation assay; ***P<0.001 vs. NC + mimic NC; P<0.001 vs. LINC01355 + mimic NC; ^^P<0.01, ^^^P<0.001 vs. NC + mimic.

[0088] Figure 4Shown is that LINC01355 inhibits THCA cell invasion by targeting miR-27b-3p: (A-C) Transwell assay was used to detect the effect of overexpression of LINC01355 and / or miR-27b-3p on invasion; ***P<0.001 vs. NC+mimic NC; P<0.001 vs. LINC01355+mimic NC; ^^P<0.01, ^^^P<0.001 vs. NC+mimic.

[0089] Figure 5 Shown are the effects of LINC01355 and miR-27b-3p on THCA-bearing nude mice: (A) A THCA xenograft model was constructed with THCA cells overexpressing LINC01355 and / or miR-27b-3p. (B-C) Effects of overexpression of LINC01355 and / or miR-27b-3p on tumor volume and weight; (D) Effects of overexpression of LINC01355 and / or miR-27b-3p on POGZ mRNA in tumor tissues; ***P<0.001 vs. NC+mimic NC; P<0.001 vs. LINC01355+mimic NC; ^^P<0.01, ^^^P<0.001 vs. NC+mimic. Detailed implementation manners

[0090] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time the thyroid cancer risk marker LINC01355 and its interaction relationship with miR-27b-3p and POGZ, and accordingly developed a kit and device for detecting (or diagnosing) the risk of thyroid cancer occurrence. Using LINC01355, miR-27b-3p and POGZ as detection markers, the risk of thyroid cancer occurrence can be detected with high sensitivity and high specificity.

[0091] The inventors found that elevated miR-27b-3p inhibits the expression of POGZ, while elevated LINC01355 can promote POGZ and counteract the inhibitory effect of miR-27b-3p, thereby inhibiting the development of THCA. Therefore, a combination of active ingredients containing a LINC01355 expression promoter, a miR-146a-3p inhibitor and a POGZ expression promoter can be used for the treatment of thyroid cancer. The present invention was completed on this basis.

[0092] Terms

[0093] As used herein, the term "sample" or "specimen" refers to a material specifically associated with a subject from which specific information related to the subject can be determined, calculated, or inferred. A sample can be composed, in whole or in part, of biological material from the subject.

[0094] As used herein, the term "expression" includes the production of mRNA from a gene or gene segment, and includes the production of a protein encoded by the RNA or gene segment, and also includes the appearance of a detectable substance related to the expression. For example, cDNA, the binding of a binding ligand (such as an antibody) to a gene or other oligonucleotide, protein, or protein fragment, and the chromogenic portion of the binding ligand are all included within the scope of the term "expression". Thus, an increase in the half-band density on an immunoblot such as a Western blot is also within the scope of the term "expression" based on biological molecules.

[0095] As used herein, the term "reference value" or "control reference value" refers to a value that is statistically related to a specific result when compared with the analysis result. In a preferred embodiment, the reference value is determined by comparing the mRNA expression and / or protein expression of risk markers for papillary thyroid carcinoma and performing statistical analysis. Some such studies are shown in the Examples section herein. However, studies from the literature and the user experience of the methods disclosed herein can also be used to produce or adjust the reference value. The reference value can also be determined by considering circumstances and results specifically related to the patient's ethnicity, medical history, genetics, age, and other factors.

[0096] LINC01355

[0097] Long non-coding RNA (LncRNA) can competitively sponge microRNA (miRNA), thereby reversing the inhibitory effect of miRNA on gene expression.

[0098] LINC01355 is a newly discovered tumor-related LncRNA that regulates proliferation, invasion, and CD8+ T cell-related immunosuppression in oral cancer. LINC01355 has also been reported to promote the proliferation of gastric cancer cells by targeting miR-431-5p to promote the Wnt pathway. In breast cancer, LINC01355 inhibits tumor growth by regulating the transcriptional function of FOXO3.

[0099] This indicates that LINC01355 may have different roles in different cancers, but its role in THCA is unclear.

[0100] miR-27b-3p

[0101] miRNA is a short (comprising about 22 nucleotides), conserved endogenous RNA. They effectively bind to the 3'-UTR region of mRNA. This complementary base pairing can prevent translation or induce mRNA degradation.

[0102] miR-27b-3p is a newly discovered tumor-related miRNA. In colorectal cancer and breast cancer, miR-27b-3p has a tumor-promoting effect. While in gastric cancer and cervical cancer, miR-27b-3p inhibits tumors and is targeted by NEAT1 or AFAP1-AS1.

[0103] POGZ

[0104] POGZ (Pogo transposable element with ZNF domain) is a component of the centromere and is involved in chromosome condensation, mitosis, and DNA repair. Its neurobiological function as an autism-related protein is well-known.

[0105] However, recent studies have revealed its cancer regulatory role. For example, POGZ is upregulated in osteosarcoma tissues and is associated with poor prognosis. Elevated POGZ can promote the proliferation of docetaxel-resistant prostate cancer cells and inhibit apoptosis. However, the role of POGZ in THCA remains to be determined.

[0106] Thyroid cancer risk marker

[0107] 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 "LINC01355, miR-27b-3p, POGZ, or a combination thereof".

[0108] In the present invention, the terms "thyroid cancer risk marker gene" and "polynucleotide of thyroid cancer risk marker" are used interchangeably and both refer to the nucleotide sequence of any thyroid cancer risk marker shown in LINC01355, miR-27b-3p, POGZ, or a combination thereof.

[0109] It should be understood that nucleotide substitutions in codons are acceptable when they encode the same amino acid. Additionally, it should be understood that nucleotide changes are also acceptable when they result in conservative amino acid substitutions due to nucleotide substitutions.

[0110] In the case of obtaining information on thyroid cancer risk markers, nucleic acid sequences encoding them can be constructed based on this information, and specific probes can be designed according to the nucleotide sequences. The full-length nucleotide sequence or its fragments can generally be obtained by PCR amplification, recombination, or artificial synthesis methods. For the PCR amplification method, primers can be designed according to the nucleotide sequences of thyroid cancer risk markers disclosed in the present invention, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art can be used as templates for amplification to obtain the relevant sequences. When the sequence is relatively long, it is often necessary to perform PCR amplification two or more times, and then splice the fragments amplified each time together in the correct order.

[0111] Once the relevant sequences are obtained, the relevant sequences can be obtained in large quantities by recombination methods. This is usually to clone them into vectors, then transfer them into cells, and then isolate the relevant sequences from the proliferated host cells by conventional methods.

[0112] In addition, artificial synthesis methods can also be used to synthesize the relevant sequences, especially when the fragment length is relatively short. Usually, very long fragments can be obtained by first synthesizing multiple small fragments and then ligating them.

[0113] Currently, it is already possible to completely obtain the DNA sequence encoding the protein (or its fragments, derivatives) of the present invention by chemical synthesis. Then this DNA sequence can be introduced into various existing DNA molecules (such as vectors) and cells known in the art.

[0114] By conventional recombinant DNA techniques, the polynucleotide sequences of the present invention can be used to express or produce recombinant thyroid cancer risk markers.

[0115] Detection methods

[0116] Based on the fact that the expression level of the thyroid cancer risk marker LINC01355 in tissues of thyroid cancer patients decreases, the expression level of POGZ in tissues also decreases, while the level of miR-27b-3p increases in tissues. The present invention also provides corresponding methods for diagnosing the risk of thyroid cancer onset.

[0117] The present invention relates to diagnostic test methods for quantitatively and locally detecting the levels of human thyroid cancer risk markers. These tests are well-known in the art. The levels of thyroid cancer risk markers detected in the tests can be used for diagnosing (including auxiliary diagnosis) the risk of thyroid cancer occurrence and / or the prognosis evaluation of thyroid cancer.

[0118] A preferred method is to quantitatively detect thyroid cancer risk markers.

[0119] Preferably, a method for detecting whether there is a thyroid cancer risk marker in a sample is to use a specific antigen for detection, which includes: contacting the sample with a specific antibody of the antigen protein; observing whether an antibody complex is formed, and the formation of an antibody complex indicates the presence of a thyroid cancer risk marker in the sample.

[0120] The thyroid cancer risk marker of the present invention can be used for the diagnosis of thyroid cancer. The antigen protein of the thyroid cancer risk marker can be immobilized on a protein chip for detecting the thyroid cancer risk marker in a sample.

[0121] Based on the research of the present invention, the level of the thyroid cancer risk marker of the present invention is significantly increased in thyroid cancer patients. Therefore, the thyroid cancer 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 cancer occurrence. During detection, when the thyroid cancer risk marker is an up-regulated marker and the ratio (C1 / C0) of the marker level C1 to the corresponding level C0 in the normal population is ≥ 1.5, preferably ≥ 2, more preferably ≥ 3; when the thyroid cancer risk marker is a down-regulated marker and the ratio (C1 / C0) of the corresponding level C0 in the normal population to the marker level C1 is ≥ 1.5, preferably ≥ 2, more preferably ≥ 3; then the risk of thyroid cancer occurrence can be regarded as increased.

[0122] Detection kit

[0123] Based on the correlation between the thyroid cancer risk marker of the present invention and the risk of thyroid cancer occurrence and prognosis, therefore, the thyroid cancer risk marker of the present invention can be used as a diagnostic marker for thyroid cancer occurrence and / or a marker for evaluating thyroid cancer prognosis.

[0124] The thyroid cancer risk marker provided by the present invention includes the following combinations:

[0125] (A1) LINC01355; and

[0126] (B) Any marker selected from the following group, or a combination thereof: (B1) miR-27b-3p; (B2) the gene, transcript, or protein of POGZ.

[0127] The present invention also provides a kit for diagnosing the occurrence of thyroid cancer, the kit contains a detection reagent, and the detection reagent is used for detecting the thyroid cancer risk marker of the present invention. Preferably, the kit contains the antigen of the thyroid cancer risk marker of the present invention, or an active fragment thereof.

[0128] In another preferred example, the kit further includes a label or an instruction manual, and the label or the instruction manual indicates that the kit is used for diagnosing the risk of thyroid cancer occurrence and / or evaluating the prognosis of thyroid cancer.

[0129] Drug Composition and Administration Method

[0130] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity in humans and / or animals and is acceptable to humans and / or animals.

[0131] As used herein, a "pharmaceutically acceptable" component is a substance that is suitable for humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, a substance having a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier used for administering a therapeutic agent, including various excipients and diluents.

[0132] The pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and combinations thereof. Usually, the pharmaceutical preparation should be matched with the administration method. The dosage forms of the pharmaceutical composition of the present invention are injection, oral preparation (tablet, capsule, oral liquid), transdermal agent, sustained-release agent. For example, it is prepared by a conventional method with physiological saline or an aqueous solution containing glucose and other adjuvants. The said pharmaceutical composition should be manufactured under aseptic conditions.

[0133] The effective amount of the active ingredient described in the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by those of ordinary skill in the art according to various factors (such as through clinical trials). Such factors include but are not limited to: the pharmacokinetic parameters of the said active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the patient's body weight, the patient's immune status, the route of administration, etc. Usually, when the active ingredient of the present invention is administered at a dose of about 0.00001 mg - 50 mg / kg of animal body weight per day (preferably 0.0001 mg - 10 mg / kg of animal body weight), satisfactory effects can be obtained. For example, due to the urgent requirements of the treatment condition, several separate doses can be administered per day, or the dose can be proportionally reduced.

[0134] The pharmaceutically acceptable carriers described in the present invention include (but are not limited to): water, saline, liposome, lipid, protein, protein-antibody conjugate, peptide substance, cellulose, nanogel, or combinations thereof. The selection of the carrier should be matched with the administration method, which is well known to those of ordinary skill in the art.

[0135] In the present invention, the said expression vector can be directly administered to the subject, or the said expression vector can be prepared into a pharmaceutical combination with a pharmaceutically acceptable carrier and then administered. The said administration includes intravenous injection.

[0136] Treatment Method

[0137] The present invention also provides a method for treating thyroid cancer with low expression of LINC01355 and / or POGZ, and / or high expression of miR-27b-3p, that is, administering a safe and effective amount of the pharmaceutical composition of the present invention to a subject in need, so as to treat thyroid cancer with low expression of LINC01355 and / or POGZ, and / or high expression of miR-27b-3p.

[0138] Generally, "thyroid cancer with low expression of LINC01355" means that in the tumor described, the expression level E1 of LINC01355 has a significant difference compared with the amount E0 of LINC01355 in the adjacent cancer tissue or normal tissue. Preferably, the "low expression" means E0≥1.5E1, more preferably E0≥2E1; "thyroid cancer with low expression of POGZ" means that in the tumor described, the expression level E1 of POGZ has a significant difference compared with the amount E0 of POGZ in the adjacent cancer tissue or normal tissue. Preferably, the "low expression" means E0≥1.5E1, more preferably E0≥2E1; "thyroid cancer with high expression of miR-27b-3p" means that in the tumor described, the expression level E1 of miR-27b-3p has a significant difference compared with the amount E0 of miR-27b-3p2 in the adjacent cancer tissue or normal tissue. Preferably, the "high expression" means E1≥1.5E0, more preferably E1≥2E0.

[0139] Whether LINC01355 and / or POGZ are lowly expressed, and / or whether miR-27b-3p is highly expressed in tumor tissues can be detected by conventional methods.

[0140] The main advantages of the present invention:

[0141] 1. The risk markers of the present invention can efficiently and accurately predict the occurrence risk of thyroid cancer.

[0142] 2. The present invention provides gene targets for the development of drugs for treating thyroid cancer.

[0143] 3. The microRNA (miR-27b-3p) of the present invention can be chemically synthesized and modified to directly become a nucleic acid drug for treatment.

[0144] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0145] Materials and Methods

[0146] Bioinformatics Analysis

[0147] The GEPIA tool was used to analyze THCA cases in the TCGA database. A total of 512 tumor and 337 normal tissue samples were included for differential analysis.

[0148] Pearson test was used to examine the correlation between LINC01355 and POGZ in THCA tissues. The String tool was used to dissect the protein-protein interaction (PPI) of POGZ.

[0149] Cell Culture

[0150] Human normal thyroid cells HTori-3 (YS4003C, Yoji, Shanghai, China) and THCA cells ACT-1 (YB-H1078, Science, China), KTC-1 (AC340144, ATCC), TTA1 (YB-H1077, Science, China) and CAL-62 (CL-0618, Pricilla, China) were preserved in DMEM medium (Gibco, USA) supplemented with 10% fetal bovine serum. The cells were cultured in an incubator at 37 °C, 95% humidity, and 5% CO₂.

[0151] Cell Transfection

[0152] KTC-1 and CAL-62 cells with relatively low expression of LINC01355 were transfected to overexpress LINC01355 and / or miR-27b-3p. KTC-1 and CAL-62 cells were used to construct cell models overexpressing LINC01355 and / or miR-27b-3p.

[0153] On this basis, KTC-1 and CAL-62 groups were formed as 2:NC + mimic NC, LINC01355 + mimic NC, NC + mimic, and LINC01355 + mimic (GenePharma Co., Ltd., China).

[0154] 50 pmol (0.67 μg) of plasmid was diluted in 25 μL of serum-free DMEM as reagent A, and 1 μL of Entranter TM -R4000 (Engreen) was mixed with 24 μL of serum-free DMEM for 25 min as reagent B. 25 μL of reagent A and 25 μL of reagent B were thoroughly mixed (aspirated 10 times with a pipette), and after standing for 15 min, it was used as the transfection complex. 0.45 mL of cells in complete medium were transfected with 50 μL of the transfection complex.

[0155] LINC01355, miR-27b-3p mimic, and the corresponding NC / mimic NC were transfected using the above method.

[0156] RT-qPCR

[0157] 400 μL of Trizol (Thermo Fisher, USA) was added to the cells or tissues in a 1.5 mL sterile RNase-free EP tube. After grinding the cells or tissues into a homogenate, 600 μL of Trizol was added, mixed for 5 min, centrifuged for 5 min (4 °C, 12,000×g), and the pellet was discarded. Trizol and chloroform were mixed at a ratio of 1:2 and added to the centrifugation product of the previous step, and shaken up and down for 15 s. Then, isopropanol was added to precipitate the RNA in the aqueous phase. The collected RNA pellets were carefully washed with 75% ethanol to remove impurities. The RNA was dissolved in DEPC water and stored at -80 °C.

[0158] RNA was reverse transcribed into cDNA using the PrimeScript RT Reagent kit (RR047A, Takara) under the conditions of: 37 °C / 15 min; qPCR amplification was performed using SYBR Green reagent (Takara, Japan) under the following conditions: 94 °C, 3 min; 94 °C, 15 s; 58 °C, 20 s; 72 °C, 30 s; 40 cycles. The expression of LINC01355 and POGZ was normalized to GAPDH, and miR-27b-3p was normalized to U6 by the 2- ΔΔCt method. The primers were designed (from 5' end to 3' end) as shown in Table 1:

[0159] Table 1

[0160]

[0161]

[0162] Western blot

[0163] Use 100 μL of lysis buffer + 1 μL of protease inhibitor + 1 μL of PMSF as the lysis buffer. Remove the culture medium, wash, and then lyse the cells or tissues. After centrifugation (12,000×g, 4 °C, 15 min), collect the supernatant into another 1.5 mL tube. Take 2.5 μL of the sample and 22.5 μL of triple distilled water, and measure the protein concentration using BCA. Separation is performed using 8% - 12% SDS-PAGE.

[0164] Cut the gel according to the label instructions and the position of the target band. The eluted gel is soaked in transfer buffer for 15 minutes. After washing, add 5 mL of milk powder blocking solution and gently shake overnight at 4 °C. Add rabbit anti-primary antibodies POGZ (1:1000, ab167408, Abcam, USA) and MAD2L2 (1:1000, ab180579) (4 °C, overnight) respectively. Then incubate with the secondary antibody (1:2000, ab6721) for 2 h at 37 °C. Use an ECL kit (Amersham Biote) and IPP6.0 for blot visualization, and obtain the relative expression level with the internal reference GAPDH.

[0165] CCK-8 assay

[0166] Add 100 μL of cell suspension (5×10 4 / mL) to the wells of a 96-well plate. After 24 h, 48 h, and 72 h, add 10 μL of CCK-8 solution respectively. Gently mix on an orbital shaker at 37 °C for 1 minute to ensure uniform mixing. Then perform a 2-hour dehydrogenation reaction. Use an Elx808 microplate reader (25-315S, Lonza, USA) to detect the optical density (OD) value at a wavelength of 450 nm.

[0167] Colony formation assay

[0168] Suspend 200 cells evenly in the culture medium by digestion and resuspension. Then culture for 14 days under the above conditions. Depending on the actual situation, the culture medium needs to be changed every 2 - 3 days.

[0169] Macroscopic colonies appear at this time. Discard the supernatant and carefully wash twice with PBS. Remove 4% PFA after fixation for 15 minutes. Add Giemsa and keep for 20 minutes, then slowly wash off the staining solution and air dry.

[0170] Invert the plate, cover it with a grid transparent film, and count the number of colonies with more than 20 cells.

[0171] Transwell experiment

[0172] Matrigel (1:8 dilution, Corning, USA) was added to the upper chamber and incubated at 37°C for 30 min, and 600 μL of complete culture medium (20% FBS) was added to the lower chamber of the 24-well plate-transwell device.

[0173] The cells (5×10 / mL) were cultured in serum-free medium at 37°C for 24 h for starvation treatment. After digestion, 100 μL of cell solution (5×10 4 / mL) to hydrate the Transwell chamber. After 24 h, the uninvaded cells were washed away. The cells in the infiltrated lower chamber were stained with 0.1% crystal violet for 20 minutes at room temperature and then fixed with 95% ethanol.

[0174] Five fields were randomly selected within the ×400-fold field of view, and the number of invading cells was counted.

[0175] Dual luciferase reporter assay

[0176] For dual luciferase reporter gene assay, 1 μg of wild-type (wt)-LINC01355 / POGZ-pGL4 (Promega, USA) or mutant (mut)-LINC01355 / POGZ-pGL4 (QuickMutation TM Kit induction, Beyotime, China), 50 nmol miR-27b-3p mimic / mimic NC and 150 ng Renilla (RG062M, Beyotime, China) were used 2000 transfected to 3×10 4 The cells were transfected at 37°C for 36 hours. The luciferase activity was detected using a dual-luciferase reporter gene assay kit (Promega, USA). All data were normalized to Renilla luciferase activity.

[0177] Statistical analysis

[0178] All experiments were performed three times independently. Data were expressed as mean ± SD, and statistical analysis was performed using one-way analysis of variance (ANOVA) and Tukey's multiple comparison test (GraphPad Prism version 7.0). T-test was used to analyze the differences between the two groups. P < 0.05 was considered statistically significant.

[0179] Example 1. Bioinformatics analysis results

[0180] 1.1 LINC01355 is reduced in THCA and associated with POGZ

[0181] Through database analysis, the present invention found that the expression of LINC01355 is different in different tumors, and its expression is relatively low in some tumors including THCA ( Figure 1 A).

[0182] The level of LINC01355 in THCA tissues is lower than that in normal thyroid tissues ( Figure 1 B).

[0183] In addition, as shown in Figure 1 C-1D, in THCA tissues and peripheral blood, LINC01355 is positively correlated with POGZ (r = 0.67, P < 0.001; r = 0.59, P < 0.001). This indicates that POGZ in THCA may be regulated by LINC01355.

[0184] 1.2 miR-27b-3p targeted by LINC01355 targets POGZ

[0185] As shown in Figure 1 E-1F, in four THCA cell lines (ACT-1, KTC-1, TTA1, CAL-62), the level of LINC01355 is lower than that in normal cells HTori-3, that is, LINC01355 is down-regulated, while miR-27b-3p is up-regulated (P < 0.001).

[0186] Among them, the level of LINC01355 in the CAL-62 cell line is the lowest, and the level of LINC01355 in the KTC-1 cell line is the second; the level of miR-27b-3p in the CAL-62 cell line is the highest, and the level of miR-27b-3p in the KTC-1 cell line is the second. Therefore, the CAL-62 cell line and the KTC-1 cell line can be used as preferred cell lines for cell experiments.

[0187] Through sequence matching and dual-luciferase reporter gene detection, it was found that as shown in Figure 1 G and Figure 1 H, after transfection with miR-27b-3p mimic and Wt-LINC01355, Luc / Ren decreased. As shown in Figure 1 G and Figure 1 I, after transfection with miR-27b-3p mimic and Wt-POGZ, Luc / Ren also decreased. This indicates that POGZ is targeted by miR-27b-3p, and miR-27b-3p is targeted by LINC01355.

[0188] Example 2. Cell experiments

[0189] 2.1 LINC01355 increases the expression of POGZ via miR-27b-3p

[0190] To clarify the targeted regulation of POGZ by the LINC01355 / miR-27b-3p axis, the present invention constructed THCA cells overexpressing LINC01355 and miR-27b-3p. Among them, CAL-62 cell line and KTC-1 cell line, which were preferred in Example 1, were selected as THCA cells. As shown in Figure 2 A-2B.

[0191] On this basis, CAL-62 and KTC-1 cells were divided into NC + mimic NC, LINC01355 + mimic NC, NC + mimic, and LINC01355 + mimic.

[0192] The results are shown in Figure 2 C-2D. The increase in LINC01355 led to the upregulation of POGZ protein. After miR-27b-3p was upregulated, the POGZ protein level was inhibited, and overexpression of LINC01355 could reverse the POGZ protein level. It shows that the mechanism by which LINC01355 promotes POGZ is inseparable from miR-27b-3p.

[0193] 2.2 LINC01355 inhibits THCA cells by targeting miR-27b-3p

[0194] To further clarify the effect of the LINC01355 / miR-27b-3p axis regulating POGZ on THCA cells, the present invention detected the proliferation and invasion of NC + mimic NC, LINC01355 + mimic NC, NC + mimic, and LINC01355 + mimic.

[0195] The results are shown in Figure 3 A-3E. Upregulation of LINC01355 inhibited the proliferation viability and colony formation level, while upregulation of miR-27b-3p promoted the proliferation viability and colony formation. Overexpression of miR-27b-3p blocked the inhibitory effect of LINC01355 on cell viability.

[0196] The invasion level of the LINC01355 + mimic NC group was lower than that of the NC + mimic NC group, and the invasion level of the NC + mimic NC group increased. The invasion ability of the LINC01355 + mimic group was higher than that of the LINC01355 + mimic NC group and lower than that of the NC + mimic group ( Figure 4 A-C).

[0197] Overexpression of miR-27b-3p blocked the inhibitory function of LINC01355, suggesting that the mechanism by which LINC01355 inhibits invasion is closely related to miR-27b-3p.

[0198] Example 3. Xenograft experiment

[0199] Effects of LINC01355 and miR-27b-3p on nude mice bearing THCA tumors

[0200] 3.1 Experimental method

[0201] BALB / C nude mice (4 weeks old, male, from Vital River (Charles River) Co., Ltd., China) were used to evaluate cell tumorigenesis. The breeding environment was 24 ± 1°C and the relative humidity was 60 ± 5%.

[0202] First, 5×10 6 KTC-1 cells transfected with LINC01355 and / or miR-27b-3p mimic were resuspended in 200 μL of PBS respectively. Then they were injected into the axillary region of nude mice at one time. After 4 weeks, euthanasia was performed, and the tumors were collected and weighed.

[0203] When the tumor diameter was greater than 2 cm (not involved in this experiment), the animals were euthanized. The experimental protocol involving animals was approved by the Ethics Committee of Gongli Hospital.

[0204] 3.2 Experimental results

[0205] Cells in the subcutaneous injection NC + mimic NC, LINC01355 + mimic NC, NC + mimic, and LINC01355 + mimic groups were used to obtain experimental models, as Figure 5 shown in A.

[0206] The experimental results were as Figure 5 shown in B-5C. The increase in LINC01355 led to a decrease in tumor volume and mass, while the increase in miR-27b-3p promoted an increase in tumor volume and mass. The tumor volume and mass in the LINC01355 + mimic group were higher than those in the LINC01355 + mimic NC group and lower than those in the NC + mimic group.

[0207] In addition, as Figure 5 shown in D, the increase in LINC01355 promoted the level of POGZ mRNA in tumor tissues, while overexpression of miR-27b-3p inhibited the level of POGZ mRNA. The POGZ mRNA in the LINC01355 + mimic group was lower than that in the LINC01355 + mimic NC group and higher than that in the NC + mimic group.

[0208] Overexpression of miR-27b-3p can weaken the inhibitory effect of LINC01355 on the tumorigenesis of THCA cells in vivo, indicating that the mechanism of LINC01355 inhibiting THCA in vivo is also inseparable from miR-27b-3p.

[0209] Discussion

[0210] The incidence rate of THCA is approximately 15 per 100,000 person-years, and the 5-year survival rate in China is approximately 84.3%. Although the malignancy of THCA is relatively low, there are still about 5% of patients with locally advanced THCA who have problems such as inoperability and a high local recurrence rate, which is the main reason for the increasing mortality of THCA. Due to the lack of understanding of the occurrence and development mechanisms of THCA, there are no effective treatment methods.

[0211] LncRNAs are newly discovered tumor diagnosis and treatment targets in recent years. For example, SLC26A4-AS1, LINC00671, and EGFEM1P are considered as clinical markers of THCA and are involved in regulating cell proliferation and invasion. LINC01355 (ENSG00000261326) is a newly discovered tumor-related LncRNA, which was first discovered to be involved in the inhibition of breast cancer in 2019. According to existing reports, LINC01355 acts as a tumor promoter in oral cancer and gastric cancer, and as a tumor suppressor in renal cancer and breast cancer. The GEPIA database shows that LINC01355 is lowly expressed in THCA and is positively correlated with POGZ. This indicates that POGZ in THCA may be regulated by LINC01355.

[0212] To further clarify the mechanism by which LINC01355 regulates POGZ and its effect on THCA, in this study, the target miRNAs of LINC01355 and POGZ were predicted respectively, and miR-27b-3p was obtained. This study confirmed the targeting relationship between miR-27b-3p and POGZ and LINC01355 ( Figure 1 G-1I and Figure 2 ). The elevation of miR-27b-3p inhibited the expression of POGZ, while LINC01355 not only promoted the expression of POGZ but also reversed the inhibitory effect of miR-27b-3p on POGZ. miR-27b-3p (the synthetic mimic in the figure) increased the proliferation and viability of THCA cells, and this effect was blocked by LINC01355 ( Figure 3 and 4 ).

[0213] ceRNA (competing endogenous RNA) is a new gene regulatory network mechanism proposed in 2011. Therefore, essentially, the regulatory mechanism of POGZ is the well-known ceRNA mechanism.

[0214] As far as the present invention is concerned, this is the first report on the ceRNA pathobiology regulated by POGZ.

[0215] At the animal level, LINC01355 attenuated the promoting effect of miR-27b-3p on THCA tumorigenesis and the inhibitory effect on POGZ ( Figure 2 ).

[0216] These indicate that LINC01355 is a target of POGZ / miR-27b-3p and thus participates in the progression of THCA. The clinical value of LINC01355, miR-27b-3p, and POGZ in THCA awaits further research with a larger sample size in the future.

[0217] In this study, the present invention focused on the tumor-suppressive effect of LINC01355. Through the combination of theoretical prediction and experimental verification using cell and nude mouse models, the present invention found that the expression of LINC01355 was coordinated by miR-27b-3p targeting POGZ. This study may provide inspiring insights for the research of LINC01355 and facilitate the diagnosis and treatment of THCA.

[0218] All the documents mentioned in the present invention are cited herein as references, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Use of a reagent for detecting thyroid cancer risk markers, characterized in that, For the preparation of a detection reagent or kit, which is used for (a) detecting the risk of thyroid cancer occurrence; and / or (b) prognostic evaluation of thyroid cancer; Wherein, the detection reagent is used to detect the level of the risk marker, and the thyroid cancer risk markers include: (A1) LINC01355; and (B) Optionally any one marker selected from the following group, or a combination thereof: (B1) miR-27b-3p; (B2) the gene, transcript, or protein of POGZ.

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

3. A kit, characterized in that, The kit contains a detection reagent, which is used to detect thyroid cancer risk markers, Wherein, the thyroid cancer risk markers include: (A1) LINC01355; and (B) Optionally any one marker selected from the following group, or a combination thereof: (B1) miR-27b-3p; (B2) the gene, transcript, or protein of POGZ.

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

5. The kit according to claim 4, wherein The primer pairs are selected from the following group: the primer pair for amplifying LINC01355: SEQ ID NO.1, SEQ ID NO.2; the primer pair for amplifying POGZ: SEQ ID NO.3, SEQ ID NO.4; the primer pair for amplifying miR-27b-3p: SEQ ID NO.7, SEQ ID NO.

8.

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

7. A device for assessing the risk of thyroid cancer, characterized in that, The device includes: (a) An input module, which is used to input the thyroid cancer risk marker data of a certain detection subject; Wherein, the risk markers include: (A1) LINC01355; and (B) Optionally any one marker selected from the following group, or a combination thereof: (B1) miR-27b-3p; (B2) the gene, transcript, or protein of POGZ; (b) A processing module, which is used to compare the expression level C1 of the input adenocarcinoma risk marker with the control reference value C0 to obtain an evaluation result; wherein, the evaluation includes: (1) When a certain marker is an up-regulated marker, when its expression level C1 is higher than the control reference value C0, it indicates that the risk of thyroid cancer occurrence of the subject is high; otherwise, it indicates that the risk of thyroid cancer occurrence is not high; (2) When a certain marker is a down-regulated marker, when its expression level C1 is lower than the control reference value C0, it indicates that the risk of thyroid cancer occurrence of the subject is high; otherwise, it indicates that the risk of thyroid occurrence is not high; and (c) An output module, which is used to output the evaluation result.

8. An active ingredient combination, characterized in that, The active ingredient combination includes: (Z1) A first active ingredient, which is a promoter for LINC01355 expression; and Any active ingredient selected from the following group, or a combination thereof: (Z2) A second active ingredient, which is an inhibitor of miR-146a-3p; (Z3) A third active ingredient, which is a promoter for POGZ expression.

9. Use of a combination of active ingredients in the preparation of a pharmaceutical composition for treating thyroid cancer, characterized in that, The combination of active ingredients includes: (Z1) A first active ingredient, which is a promoter for LINC01355 expression; and Any active ingredient selected from the following group, or a combination thereof: (Z2) A second active ingredient, which is an inhibitor of miR-146a-3p; (Z3) A third active ingredient, which is a promoter for POGZ expression.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: (Z1) A first active ingredient, which is a promoter for LINC01355 expression; and Any active ingredient selected from the following group, or a combination thereof: (Z2) A second active ingredient, which is an inhibitor of miR-146a-3p; (Z3) A third active ingredient, which is a promoter for POGZ expression; and (Z4) A pharmaceutically acceptable carrier.

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