Detection product for auxiliary diagnosis of cancer and / or auxiliary judgment of prognosis of cancer patient

Abstract

The invention discloses a detection product for auxiliary diagnosis of cancers and / or auxiliary judgment of prognosis of cancer patients, the detection product comprises a reagent for detecting hascirc0082002 in a sample, and the types of the cancers comprise at least one of thyroid cancer, nasopharynx cancer, oral cancer and ovarian cancer. According to the invention, a detection product for auxiliary diagnosis of cancers and / or auxiliary judgment of prognosis of cancer patients can be provided.

Description

[0001] This application is filed on June 20, 2022 、Application No. 202210700049.2 、The name of the invention is biology Application of markers in the preparation of reagents for auxiliary diagnosis of cancer A divisional application for a patent application. Technical Field

[0002] The present invention relates to the field of biomedicine technology, and in particular to a detection product for assisting in diagnosing cancer and / or assisting in determining the prognosis of cancer patients. Background Art

[0003] Stable and effective early screening or diagnosis of cancers such as thyroid cancer, oral cancer, nasopharyngeal cancer, and ovarian cancer plays an important role in cancer control. Currently, there are many screening and diagnostic methods in clinical practice, such as puncture biopsy, imaging examination, serological marker detection, genetic testing, etc. However, various screening and diagnostic methods have their own advantages and disadvantages. The results of multiple screening and diagnostic methods can be combined to diagnose and screen cancer. For example, ultrasound-guided thyroid fine needle aspiration (FNAB) is one of the main means of diagnosing thyroid nodules in clinical practice, but there are still about 30% of patients who cannot be clearly diagnosed, and usually need to be assisted by other methods.

[0004] Circular RNAs (circRNAs) are a type of non-coding RNA. The configuration of circular RNAs is different from that of traditional linear RNAs. Circular RNA molecules are closed circular structures. Since there are no free 5' and 3' ends, circular RNAs are more conservative and stable than linear RNAs and are not easily degraded by nucleases. Circular RNAs are tissue-specific molecules produced by a special alternative splicing mechanism and play a key role in various biological processes, such as cancer growth regulation, as molecular sponges of miRNAs or regulatory factors of gene expression. In recent years, more and more people have realized that the differential expression of circular RNAs in cancer tissues can be used to predict and evaluate cancer.

[0005] The development of more markers or targets will be beneficial for auxiliary diagnosis, prognosis assessment and treatment of cancer. Summary of the invention

[0006] The present disclosure is made in view of the above-mentioned state of the prior art, and its purpose is to provide a biomarker and a biological target for use in auxiliary diagnosis and treatment of cancer.

[0007] To this end, the first aspect of the present disclosure provides an application of a biomarker in the preparation of a reagent for assisting in the diagnosis of cancer, wherein the biomarker is has_circ_0082002 in circular RNAs (the sequence of has_circ_0082002 can be obtained according to the authoritative circular RNA database CircBase (http: / / circbase.org / )). In the present disclosure, it is found through research that has_circ_0082002 in circular RNAs is related to various types of tumors, and has_circ_0082002 can be used as a biomarker to assist in the screening and diagnosis of cancer patients.

[0008] In the application involved in the first aspect of the present disclosure, optionally, the type of cancer includes at least one of thyroid cancer, nasopharyngeal cancer, oral cancer, and ovarian cancer. Therefore, has_circ_0082002 can be used as a biomarker for auxiliary diagnosis of thyroid cancer, nasopharyngeal cancer, oral cancer, and ovarian cancer.

[0009] In the application involved in the first aspect of the present disclosure, optionally, the type of cancer is papillary thyroid carcinoma. Therefore, has_circ_0082002 can be used as a biomarker for auxiliary diagnosis of papillary thyroid carcinoma.

[0010] In the application involved in the first aspect of the present disclosure, optionally, the reagent for assisting in the diagnosis of cancer is a reagent for detecting has_circ_0082002 in cancer tissue or blood. In this case, the has_circ_0082002 in cancer tissue or in the blood of the subject can be detected to assist in the screening and diagnosis of cancer patients.

[0011] In the application involved in the first aspect of the present disclosure, optionally, the reagent for assisting in the diagnosis of cancer includes an upstream primer and a downstream primer for real-time fluorescence quantitative detection of has_circ_0082002, wherein the sequence of the upstream primer is 5'-ACAATGTGAGATGTCTCCAGCAT-3', and the sequence of the downstream primer is 5'-GGTAAACAGGAGCACGAGGAT-3'. Thus, has_circ_0082002 can be quantified.

[0012] In the application involved in the first aspect of the present disclosure, optionally, the reagent for assisting in the diagnosis of cancer includes a probe for detecting has_circ_0082002. Thus, the probe can be used to detect has_circ_0082002.

[0013] The second aspect of the present disclosure provides an application of a biomarker in the preparation of a reagent for assisting in determining the prognosis of a cancer patient, wherein the biomarker is has_circ_0082002 in circular RNAs. In the present disclosure, research has found that has_circ_0082002 in circular RNAs is related to various types of tumors, and circular RNA has_circ_0082002, as a biomarker, can assist in determining the prognosis of cancer patients.

[0014] In the application involved in the second aspect of the present disclosure, optionally, the type of cancer is papillary thyroid carcinoma. Therefore, has_circ_0082002 can be used as a biomarker to assist in the prognosis of patients with papillary thyroid carcinoma.

[0015] The third aspect of the present disclosure provides an application of a biological target in the preparation of a product for treating cancer patients, wherein the biological target is has_circ_0082002 in circular RNAs. Through research, it is found that has_circ_0082002 in circular RNAs plays an important role in regulating cancer cell proliferation and circulation. In the present disclosure, by using circular RNA has_circ_0082002 as a therapeutic target, by targeting has_circ_0082002, cancer patients can be assisted in treatment.

[0016] In the application involved in the third aspect of the present disclosure, optionally, the type of cancer is papillary thyroid carcinoma. Thus, by targeting has_circ_0082002, patients with papillary thyroid carcinoma can be assisted in the treatment.

[0017] According to the present disclosure, a new biomarker and biological target can be provided for application in auxiliary diagnosis and treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. It can be understood that the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0019] Figure 1 Schematic diagram of the experimental workflow of the embodiment of the present disclosure.

[0020] Figure 2 Graphs showing the expression profiles of mRNAs and circular RNAs of the disclosed embodiments.

[0021] Figure 3Schematic diagram showing that 14 upregulated circular RNAs according to an embodiment of the present disclosure are used to construct a circular RNA-miRNA-mRNA network.

[0022] Figure 4 Schematic diagram of graphene oxide enrichment analysis of differential circular RNAs in the top 30 biological processes according to an embodiment of the present disclosure.

[0023] Figure 5 Schematic diagram showing the enrichment results of parental genes of differentially circRNAs in the top 30 pathways of the disclosed embodiments.

[0024] Figure 6 Schematic diagram showing the verification results of differential circRNA expression in papillary thyroid carcinoma and paired adjacent normal thyroid tissues according to the embodiment of the present disclosure.

[0025] Figure 7 Graphs show the expression levels of host genes in papillary thyroid carcinoma and adjacent normal tissues according to an embodiment of the present disclosure.

[0026] Figure 8 Schematic diagram of survival analysis showing the host genes of major differentially expressed circular RNAs in patients with papillary thyroid carcinoma according to an embodiment of the present disclosure.

[0027] Fig. 9 Schematic diagram showing the expression of circMET according to an embodiment of the present disclosure. DETAILED DESCRIPTION

[0028] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions. The embodiments described below by the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be interpreted as limiting the present invention.

[0029] Those skilled in the art will understand that unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.

[0030] It should also be understood that terms, such as those defined in commonly used dictionaries, should be understood to have a meaning consistent with that in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless as defined herein.

[0031] To facilitate understanding of the present invention, the present invention is further explained below with reference to specific embodiments in conjunction with the accompanying drawings, and the specific embodiments do not constitute a limitation on the embodiments of the present invention. Those skilled in the art should understand that the accompanying drawings are only schematic diagrams of the embodiments, and the components in the accompanying drawings are not necessarily necessary for implementing the present invention.

[0032] The nomenclatures and techniques used in connection with biotechnology, molecular oncology, and molecular and cellular biology described in this specification are those well known and commonly used in the art.

[0033] In the description of this specification, the "tumor" or "cancer" used are terms well known in the art and are used interchangeably throughout the disclosure. "Marker", "biomarker" or "biological target" means the entire gene / protein, or an EST (Expressed Sequence Tag, an expressed sequence tag, is a short cDNA partial sequence obtained by single sequencing of the 5' and 3' ends from a randomly selected cDNA clone, representing a small part of a complete gene), whose expression or expression level varies between certain conditions. When the expression of a gene is associated with a disease, the gene is a marker for the disease. The term "prognosis" or "prognostic marker" involves predicting disease progression without considering any reference to therapy / treatment / drug. Very qualitative prediction methods for, for example, age, tumor stage, and tumor grade are prognostic markers. This prognosis involves understanding the long-term outcome of the disease.

[0034] The term "predictive" or "predictive marker" in this specification relates to a marker that not only provides a reference to disease progression, but can also guide treatment based on the presence of markers such as circular RNAs hsa_circ_0082002.

[0035] In this embodiment, the first aspect provides an application of a biomarker in the preparation of a reagent for assisting in the diagnosis of cancer (hereinafter sometimes referred to as an application). In some examples, the biomarker is circular RNAs (circRNAs), and further, the biomarker is has_circ_0082002 in circular RNAs (the sequence of has_circ_0082002 can be obtained according to the authoritative circular RNA database CircBase (http: / / circbase.org / )). Thus, it is possible to assist in the diagnosis of whether the subject is a cancer patient by detecting the situation of has_circ_0082002 in the subject. In the present disclosure, another name for has_circ_0082002 is circMET, and circMET is sometimes used below to refer to has_circ_0082002 in the circular RNAs involved in this embodiment.

[0036] In this embodiment, the second aspect provides an application of a biomarker in the preparation of a reagent for assisting in determining the prognosis of a cancer patient (hereinafter sometimes referred to as an application). In some examples, the biomarker is circular RNAs, and further, the biomarker is has_circ_0082002 in circular RNAs. Thus, the prognosis of a cancer patient can be determined by detecting the situation of has_circ_0082002 in a cancer patient. The prognosis may include the risk of recurrence of early cancer, and the early cancer patient can be classified as a high risk or low risk of cancer recurrence according to the prognosis result. In addition, after surgery or before chemotherapy for an early cancer patient, it can also be determined whether chemotherapy is required for the early cancer patient according to the prognosis result. Therefore, the content disclosed in the present invention helps to classify early cancer patients as low risk or high risk of cancer recurrence, thereby being able to intelligently divide the modules for treating low-risk and high-risk patients according to the optimal chemotherapy needs, thereby reducing the situation of undertreatment / overtreatment of patients, and also helps to design effective drugs or personalized treatments for cancer treatment. Prognosis can also include the degree of disease progression after treatment for patients with mid- to late-stage cancer. Therefore, the disease treatment plan or strategy can be adjusted based on the prognostic results.

[0037] In this embodiment, the third aspect provides an application of a biological target in preparing a product for treating cancer (hereinafter sometimes referred to as application). In some examples, the biological target is circular RNAs, and further, the biological target is has_circ_0082002 in circular RNAs. Thus, by targeting has_circ_0082002, cancer patients can be assisted in treatment.

[0038] In this embodiment, in the applications of the first aspect, the second aspect and the third aspect involved in this embodiment, the circular RNAs hsa_circ_0082002 highly expressed in patients with thyroid cancer, nasopharyngeal carcinoma, oral cancer and ovarian cancer were screened by whole transcriptome high-throughput sequencing, FISH (fluorescence in situ hybridization) and qRT-PCR (real-time quantitative PCR). In addition, it was found that (has_circ_0082002)circMET plays an important role in regulating cancer cell proliferation and circulation. Finally, it was confirmed that has_circ_0082002 can be used as a biomarker for diagnosis or prognosis prediction of thyroid cancer, nasopharyngeal carcinoma, oral cancer and ovarian cancer. In addition, targeting has_circ_0082002 is a promising treatment strategy for thyroid cancer, nasopharyngeal carcinoma, oral cancer and ovarian cancer. In other words, in the applications of the first aspect, the second aspect and the third aspect involved in this embodiment, the type of cancer includes at least one of thyroid cancer, nasopharyngeal carcinoma, oral cancer and ovarian cancer.

[0039] In this embodiment, in the applications of the first, second and third aspects of this embodiment, whole transcriptome high-throughput sequencing, FISH (fluorescence in situ hybridization) and qRT-PCR (real-time quantitative PCR) were used to screen out circular RNAs hsa_circ_0082002 that are highly expressed in patients with papillary thyroid cancer (PTC). In addition, it was found that (has_circ_0082002)circMET plays an important role in regulating the proliferation and circulation of papillary thyroid cancer cells. Finally, it was confirmed that has_circ_0082002 can be used as a biomarker for the diagnosis or prognosis prediction of papillary thyroid cancer. In addition, targeting has_circ_0082002 is a promising therapeutic strategy for papillary thyroid cancer. In other words, in the applications of the first, second and third aspects of this embodiment, the type of cancer is papillary thyroid cancer. Thus, it is possible to provide an application of a biomarker in the preparation of a reagent for assisting in the diagnosis of papillary thyroid cancer, an application of a biomarker in the preparation of a reagent for assisting in determining the prognosis of a patient with papillary thyroid cancer, and an application of a biological target in the preparation of a product for treating a patient with papillary thyroid cancer.

[0040] In this embodiment, in some examples, the has_circ_0082002 in the tissue or blood of the subject can be detected to assist in the diagnosis of cancer or to assist in the prognosis of a cancer patient. In some examples, the tissue of the subject may include cancer tissue, tissue at the lesion site, and malignant effusion (e.g., pleural effusion, ascites, cerebrospinal fluid, etc.). In some examples, the blood of the subject may include serum in peripheral blood. Serum is used as a test sample, and compared with tissue testing, the sample acquisition method and operation are convenient. In other words, the reagent for assisting the diagnosis of cancer can be a reagent for detecting has_circ_0082002 in cancer tissue or blood, and the reagent for assisting in the prognosis of a cancer patient can be a reagent for detecting has_circ_0082002 in cancer tissue or blood.

[0041] In some examples, the expression level of has_circ_0082002 in the subject can be detected to assist in the diagnosis of cancer or to assist in determining the prognosis of a cancer patient.

[0042] In this embodiment, tissues and / or serum are used as samples, and molecules related to tumor diagnosis in tissues and / or serum are searched and verified through a large number of experiments. It is found that circular RNAs has_circ_0082002 present in patients are positively correlated with various types of tumors, and has_circ_0082002 can be used as a molecular marker for auxiliary or preliminary diagnosis of malignant tumors.

[0043] In this embodiment, the qRT-PCR method was used to verify the expression of circular RNAs has_circ_0082002 in papillary thyroid carcinoma, nasopharyngeal carcinoma, oral cancer, and ovarian cancer. It was shown that the expression of has_circ_0082002 in the tissues and / or serum of patients with papillary thyroid carcinoma, nasopharyngeal carcinoma, oral cancer, and ovarian cancer was significantly upregulated, and the expression trend was consistent. has_circ_0082002 can be used as a molecular marker for the diagnosis of the above tumors.

[0044] In this embodiment, the reagent for assisting in the diagnosis of cancer is a reagent for detecting has_circ_0082002 in cancer tissue or blood. In some examples, the reagent for detecting has_circ_0082002 in the subject can be used to prepare a detection kit, thereby providing a systematic detection product for tumor auxiliary diagnosis or prognosis.

[0045] In some examples, the reagent for assisting in the diagnosis of cancer may include a real-time fluorescence quantitative detection reagent. The real-time fluorescence quantitative detection reagent includes an upstream primer and a downstream primer for real-time fluorescence quantitative detection of has_circ_0082002. In some examples, the sequence of the upstream primer may be: 5'-ACAATGTGAGATGTCTCCAGCAT-3', and the sequence of the downstream primer may be: 5'-GGTAAACAGGAGCACGAGGAT-3'.

[0046] In some examples, reagents for assisting in the diagnosis of cancer may include a probe for detecting has_circ_0082002.

[0047] In some examples, the sequence of the probe for detecting has_circ_0082002 may be: 5'-GGTTTATCCATTAAAGCAGTGCTCAGAGGTTTATCCTATTAAAGCAGTCATTAGAGGTTTATCTATTAA-3'.

[0048] In this embodiment, the primers or probes used can be designed by a dedicated primer design website Primer 3.0. In order to ensure the specificity of the primers and the accuracy of qPCR, the following primer design principles and probe design principles can be followed: 1. Primer design principles: Primer length: 18-23 bp, so that the extension temperature is not too high; PCR amplification product length: 100-200 bp; Primer GC content: 50-60%, the GC content of upstream and downstream primers should be as close as possible; Primer TM value: within the range of 57℃~62℃, and as close to 60℃ as possible; Primer self-complementarity: no more than 4 consecutive bases are complementary; Complementarity between two primers: no more than 4 consecutive bases are complementary; Avoid T / C / A / G continuous structures: Try to avoid more than 3 consecutive bases at the 3'UTR end; Avoid primers from forming secondary structures: free energy is less than 58.61KJ / mol; Avoid nonspecific amplification of the genome: design primers across exons; Blast test primer specificity: homology less than 70%.

[0049] 2. Probe design principles: Probe length: between 18 and 32 bp. If it is too long, the quenching effect will be poor. Probe GC content: 30-80%, avoid multiple repeated bases in the probe, and avoid ≥4 G bases; Probe TM value: between 66-72℃, preferably 70℃. Make sure the probe TM value is 5-10℃ higher than the primer TM value to ensure that the probe binds to the target fragment before the primer during annealing. Probe and primer position: The probe should be close to the upstream primer, that is, the probe should be close to the upstream primer on the same chain, and the 5' end of the probe should be at least 4 bp away from the 5' end of the upstream primer; In this embodiment, high-throughput sequencing of the whole transcriptome was used to screen out highly expressed circular RNAs (hsa_circ_0082002), and according to the authoritative circular RNAs database CircBase (http: / / circbase.org / ), it was analyzed that the high expression of this circular RNA can regulate the biological function of papillary thyroid carcinoma through the hsa-miR-6796-5p / ALDH1A3 signaling pathway.

[0050] The following is a detailed description of the application of the biomarkers provided by the present disclosure in the preparation of reagents for assisting the diagnosis of cancer and the application of biological targets in the preparation of products for treating cancer, mainly taking thyroid papillary carcinoma as an example. It is understandable that the embodiments should not be construed as limiting the scope of protection of the present disclosure. In addition, unless otherwise specified, the reagents or instruments used in this embodiment are all commercially available.

[0051] [Example] Experimental workflow: (1) Sample information: like Figure 1 As shown, Figure 1is a schematic diagram showing the experimental workflow of the embodiment of the present disclosure. A total of 68 patients from the Department of Endocrinology of Qilu Hospital were included and fine needle aspiration biopsy (FNAB) sampling was performed for the study. This study was approved by the Ethics Committee of Qilu Hospital and Shandong University, and all patients participating in this study obtained written informed consent before puncture.

[0052] The inclusion criteria for the sample were: 1) All samples were confirmed as papillary thyroid carcinoma (PTC) by surgery and pathology; 2) No infection at the FNAB biopsy site; 3) No other coagulation abnormalities.

[0053] The exclusion criteria for the samples were: 1) Infection at the puncture site; 2) Severe bleeding disorders; 3) Non-PTC tissue types.

[0054] Among the 68 patients, 33 patients who did not undergo surgery in our hospital and whose samples were not suitable for analysis were excluded. The remaining 35 patients had pathological results that met the requirements (all samples were independently examined by two pathologists and identified as papillary thyroid carcinoma). Subsequently, 4 samples that failed analysis were excluded. Finally, 31 pairs of papillary thyroid carcinoma tissues and adjacent normal tissues were obtained for RNA isolation and analysis.

[0055] Among them, 4 pairs of papillary thyroid carcinoma tissues and adjacent normal tissues were subjected to high-throughput sequencing, and bioinformatics analysis was used to analyze differentially expressed circRNAs (also known as circular RNAs), and the host genes of 14 upregulated circRNAs were analyzed. qRT-PCR was performed on 27 pairs of papillary thyroid carcinoma tissues and adjacent normal tissues to verify the highly expressed circRNAs in high-throughput sequencing. Finally, circMET (also known as hsa_circ_0082002) was functionally verified.

[0056] (2) Sample collection and testing: Sample collection 1) Collect fine needle aspiration samples from the patients identified above, and the number of cells obtained should be greater than 10 for operability 4. 2) Remove non-researched tissue types (such as connective tissue, adipose tissue, etc.), determine the lesions and normal tissues, and remove the normal tissues around the lesions. 3) Quickly clean the stains on the surface of the tissue with 1XPBS or physiological saline prepared with pre-cooled RNase-free water, and absorb the liquid on the surface. 4) The treated tissue is quickly placed in a pre-cooled RNase-free threaded cryotube with a number, quickly cooled in liquid nitrogen for 1 hour, and stored at -80℃ for a long time to avoid repeated freezing and thawing before RNA extraction.

[0057] Total RNA extraction and quality control Total RNA was purified from all samples using TRIzol (Thermo Fisher Scientific, Pudong, Shanghai, China). The concentration and quality of total RNA for each sample were assessed. RNA quality was measured by OD260 / 280 ratio and OD260 / 230 ratio using a Nano Drop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). OD260 / 280 between 1.8 and 2.1 or OD260 / 230 greater than 1.8 was considered acceptable. RNA integrity and DNA contamination were then assessed using denaturing agarose gel electrophoresis.

[0058] Specifically: 1) Add chloroform 1 / 5 of the volume of RNAex to the lysate and mix thoroughly. Let stand at room temperature for 5 minutes. 2) Centrifuge at 12000g at 4℃ for 15 minutes. Carefully remove the centrifuge tube. At this time, the homogenate is divided into three layers, namely: supernatant (containing RNA), middle protein layer and lower organic phase. 3) Transfer the supernatant to another new centrifuge tube (do not aspirate the middle protein layer). 4) Add isopropanol 1 / 2 times the volume of RNAex to the supernatant, mix thoroughly, and let stand at room temperature for 10 minutes. 5) Centrifuge at 12000g at 4℃ for 10 minutes. After centrifugation, discard the supernatant, being careful not to touch the RNA precipitate. 6) Add 80% ethanol (pre-cooled at -20℃) of the same volume as RNAex to the centrifuge tube, wash the RNA precipitate and the wall of the centrifuge tube, centrifuge at 7500g at 4℃ for 5 minutes, carefully discard the supernatant, and do not touch the precipitate (Note: When discarding the supernatant, remove as much supernatant as possible, and use a pipette to absorb the droplets remaining on the wall and mouth of the tube). 7) Open the centrifuge tube cap, vacuum or dry the precipitate at room temperature for about 5 minutes (Note: If there is a lot of ethanol residue, the drying time can be extended. Remember not to use centrifugal drying or heating drying, otherwise the RNA will be difficult to dissolve). 8) Add an appropriate amount of RNA-free water to the centrifuge tube to dissolve the RNA. Store the dissolved RNA at -80℃.

[0059] High-throughput sequencing analysis High-throughput sequencing was performed on 4 pairs of PTCs and paired tissues to investigate the differentially expressed circular RNAs between tumor tissues and adjacent tissues. Sequencing was performed by Shanghai JingGene Technology Co., Ltd. RNA was sequenced on an Illumina Nova seq 6000 sequencer. The sequencing process was completely controlled by the data acquisition software provided by Illumina (SanDiego, CA, USA), and the sequencing results were analyzed in real time. The edgeR software was used to perform differential expression analysis of circular RNAs. To obtain differentially expressed circular RNAs, the screening conditions were set to a fold change of expression level in tumor tissue relative to normal tissue ≥ 2 and P < 0.05. The statistical power of this experimental design was calculated to be 0.533478 by power analysis.

[0060] Construction of CeRNA network and functional enrichment analysis Regression model analysis and sequence matching based on gene expression values ​​were used to establish a miRNA sponge adsorption regulatory network and identify core endogenous competing RNAs (ceRNAs). Cytoscape3.4.0 was used to construct a biological interaction network. Using this network, we predicted which microRNAs could be bound by differential circular RNAs by analyzing gene regulatory interactions and identifying core regulatory genes. According to the location information of circular RNAs on the genome, the corresponding gene information can be obtained. The coding genes of differential circular RNAs were annotated using the GeneOntology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes, http: / / www.genome.ad.jp / kegg) databases.

[0061] Reverse transcription and real-time quantitative PCR (qRT-PCR) validation RNA was reverse transcribed into cDNA using the EvoM ML Reverse Transcription Kit II (AG11711, Precision Biotechnology (Hunan) Co., Ltd.) to obtain a reaction solution with a volume of 20 µL. The reaction conditions were as follows: 37°C for 15 min, 85°C for 5 s, and then stored at 4°C.

[0062] qRT-PCR was performed using the SYBR Green ProTaq HS Premix qPCR Kit (Takara, Japan) in a LightCycler 480 system (Roche Diagnostics, Germany) with 10 µL of PCR reaction mixture including cDNA (1 µL). The reaction mixture was incubated at 95°C for 5 min in a Micro-Amp TM Fast Optical 96-well reaction plate, followed by 35 cycles at 95°C (10 s), 60°C (30 s), and 72°C (30 s). Each experiment was repeated three times. −∆∆Ct The expression levels of related RNAs were calculated by the method. The expression levels of circRNAs were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Among them, the primers for real-time fluorescence quantitative detection of circular RNA circ_0082002 expression were: upstream primer: 5'-ACAATGTGAGATGTCTCCAGCAT-3', downstream primer: 5'-GGTAAACAGGAGCACGAGGAT-3'.

[0063] Host gene expression and survival analysis The relevant papillary thyroid carcinoma data in the GEPIA Cancer Genome Atlas (TCGA) database platform (http: / / gepia.cancer-pku.cn) were analyzed to analyze the significant differential expression of host genes and survival lines between the tumor group and the control group.

[0064] Fluorescence in situ hybridization (FISH) CircMET probe was synthesized by Suzhou Gemma Gene Co., Ltd. and used for qualitative detection of CircMET in PTC tissues and cell lines. The CircMET probe sequence is: 5' to 3'GGT+TTATCCAT+TAAAGCAGTGCTCAGAGGT+TTA+TCC+TAT+TAAAGCAGTCAT+TAGAGGT+TTA+TCTAT+TAA.

[0065] Cell transfection Three PTC cancer cell lines, TPC-1, SW-579, and B-CPAP, and the benign human thyroid follicular cell line Nthy ori3-1, were purchased from the American Type Culture Collection (ATCC) and maintained in Roswell Park Memorial Institute (RPMI) modified medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. Cell culture was performed at 37°C in a humidified CO2 (5%) environment. Cell identity was verified by short tandem repeat analysis, and potential mycoplasma contamination was excluded by conventional PCR. Index cells were used for all analyses. siRNA targeting circMET junction sites was purchased from Suzhou Gemma Gene Co., Ltd. All oligonucleotides and vectors were transfected into the TPC-1 cell line using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA). After 24 h, the transfection efficiency was detected by qRT-PCR.

[0066] Cell proliferation and cell cycle analysis The proliferation curve of PTC cell lines was determined by cell counting kit-8 (CCK-8; Beyotime, Haimen, China). Specifically, cells were cultured in 96-well plates at 2 × 103 cells per well in triplicate. After culturing for 0, 24, 48, and 72 h, the OD values ​​were measured at 450 nm after incubation of cells with CCK-8 reagent at 37 °C for 2 h. Flow cytometric analysis was detected by cell cycle and apoptosis detection kit (Meilune, Cat: MA0334, China), followed by detection according to the manufacturer's protocol (BD Biosciences, San Jose, CA).

[0067] Statistical analysis Statistical analysis was performed using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA) and SPSS version 21.0 software (SPSS Inc., Chicago, IL, USA). Continuous variables were expressed as mean ± standard deviation, and t-test was performed to evaluate the differential expression of circRNAs between PTC and normal tissues. The average expression of circRNAs was used to define low or high expression. P < 0.05 was considered statistically significant.

[0068] (3) Experimental results Circular RNAs expression profiling Based on pathology, 4 pairs of samples were selected for high-throughput sequencing. Figure 2 As shown, Figure 2 is an expression profile of mRNAs and circRNAs of the disclosed embodiments. Figure 2 A) and volcano plots (see Figure 2 Figure 2 (B) shows the expression profiles of mRNAs in PTC and adjacent normal tissues. A total of 2339 differentially expressed mRNAs were identified, of which 248 were downregulated and 2091 were upregulated compared with normal tissues. Figure 2 C) and scatter plots (see Figure 2 Part D of Figure 3 shows the expression profiles of circRNAs in PTC and adjacent normal tissues. Above and below the dotted line represent fold changes of circRNAs >2 in PTC tissues and adjacent normal tissues, respectively. Above the dotted line is up-regulated, and below it is down-regulated. The x-axis and y-axis are the SRPBM-normalized signal values ​​for each sample. A total of 5,835 circRNAs were identified in PTC and adjacent thyroid tissues. Among them, 103 circRNAs were differentially expressed, of which 14 were significantly down-regulated in PTC and 89 were significantly up-regulated in PTC. circRNAs genes are distributed on almost all human chromosomes, including 22 autosomes and sex chromosomes (see Figure 2 The differentially expressed circular RNAs were mainly present in exons, accounting for about 94% of the total RNA. 14 significantly upregulated circRNAs were further screened.

[0069] Core endogenous competing RNAs (ceRNAs) network Potential miRNAs with differentially expressed circRNAs were predicted by sequence alignment. Then, a circRNA and mRNA network was constructed to further study the interaction between circRNA and mRNA in PTC. Then, a circRNA-miRNA-mRNA network containing the top 14 most significantly upregulated circRNAs was constructed (see Figure 3 , Figure 3 is a schematic diagram showing that 14 upregulated circular RNAs of an embodiment of the present disclosure are used to construct a circular RNA-miRNA-mRNA network). Figure 3 In the study, 14 upregulated circular RNAs were used to construct a circular RNAs-miRNA-mRNA network, which included circular RNAs (represented by rectangles), downstream miRNAs (represented by triangles), and target mRNAs (represented by ovals).

[0070] According to the locations of putative binding sites in circRNA sequences, the predicted miRNAs for circMET were hsa-miR-6796-5p and hsa-miR-1197. Several potential axes were also found in these ceRNA networks, namely circMET / hsa-miR-1197 / B3GNT3, hsa-miR-6796-5p / CDH3, and hsa\U circ\U 0000660 / hsa-miR-1200 / MMP7.

[0071] Enrichment analysis GO and KEGG pathway analysis were performed on circRNA genes. GO annotated them according to their biological processes, cellular components, and molecular functions. Genes were annotated to cell (GO:0005623, P=5.523E-01), protein binding (GO:0005515, P=3.005E-03), stimulus response (GO:0050896, P=5.881E-05), and cellular immune regulation process (GO:0050794, P=6.073E-02) (see Figure 4 , Figure 4 is a schematic diagram of differential circRNAs graphene oxide enrichment analysis of the first 30 biological processes of the disclosed embodiment). The host gene MET related to gene expression, metabolic process and cellular process regulation was subsequently enriched. The genomic pathways of the 30 closely related circRNA genes were analyzed using KEGG. Some key pathways, such as cell adhesion molecules, focal adhesion and ECM receptor interactions, were enriched. These pathways are related to tumor occurrence, development, invasion and metastasis (see Figure 5 , Figure 5 is a schematic diagram showing the enrichment results of parental genes of differentially circRNAs in the top 30 pathways of the embodiment of the present disclosure).

[0072] qRT-PCR validation of differentially expressed circular RNAs To validate these findings, seven most upregulated circRNAs were selected in PTC and adjacent tissues based on fold change values: hsa_circ_0031584 (P = 0.0004), circMET (has_circ_0082002) (P = 0.0011), hsa_circ_0000660 (P = 0.0121), hsa_circ_0067938 (P = 0.0373), hsa_circ_0002483 (P = 0.0990), hsa_circ_0003692 (P = 0.0034), and hsa_circ_0006509 (P = 0.0005). P values ​​were < 0.05, which was consistent with the trend of sequencing results (see Figure 6 , Figure 6 is a schematic diagram showing the verification results of differential circRNA expression in papillary thyroid carcinoma and paired adjacent normal thyroid tissues according to an embodiment of the present disclosure).

[0073] Host gene expression analysis Using the GEPIA platform analysis of the TCGA database, the most critical genes were examined based on the results of 7 circRNAs, including FNDC3B, SKIL, MCTP2, MET, PTK2, MYO9A, and ARHGAP5. Among the host genes of differentially expressed circRNAs, only MET was found to be significantly differentially expressed ( Figure 7 , Figure 7 Graph showing the expression levels of host genes in papillary thyroid carcinoma and adjacent normal tissues according to an embodiment of the present disclosure).

[0074] The overall survival (OS) was calculated according to the GEPIA software (see Figure 8 , Figure 8 is a schematic diagram of survival analysis of the host genes of the main differentially expressed circular RNAs in patients with papillary thyroid carcinoma according to an embodiment of the present disclosure). In the TCGA database, PTC patients with higher expression of FNDC3B, MCTP2, and MET had a higher OS rate (overall survival rate) (HR were 0.52, 0.45, and 0.54, respectively; Figure 8 A, 8C, and 8D). PTC patients with higher MYO9A expression had a relatively lower OS rate (hazard ratio HR = 2.2, Figure 8 Part F). Other genes did not change OS rates ( Figure 8 B, 8E and 8G).

[0075] Functional analysis of hsa_circ_0082002 in PTC patient tissues and cell lines Based on the TCGA database analysis and the above results, circMET (i.e., hsa_circ_0082002) was selected as a promising circRNA, and the expression of circMET in PTC tissues and PTC cell lines was studied. The results are shown in Fig. 9 , Fig. 9 is a schematic diagram showing the expression of circMET in the embodiment of the present disclosure. FISH confirmed that the expression of circMET (i.e., has_circ_0082002) in PTC tissues was enhanced compared with that in non-cancerous tissues ( Fig. 9 Part A). qRT-PCR detected that the expression of circMET in SW-579 (P=0.0006), TPC-1 (P<0.0001) and B-CPAP (P=0.0028) cell lines was significantly higher than that in Nthy-ori3-1 cell line ( Fig. 9Part B), and the TPC-1 cell line was selected for further study. Three siRNAs targeting circMET junction sites were designed, of which si-circRNA2 (P<0.0001) and si-circRNA3 (P<0.0001) were selected for further study because their silencing efficiency in TPC-1 cells was higher than that of si-circRNA1 (P=0.0023). qRT PCR and FISH results showed that the expression of circMET was significantly downregulated in the TPC1 cell line transfected with siRNA fragments ( Fig. 9 CD part). In the proliferation experiment, the CCK-8 method was used to determine the growth curve. The CCK-8 results showed that circMET silencing significantly inhibited the proliferation of TPC-1 cells ( Fig. 9 Part E, 0h P=0.036, 24h P=0.0028, 48h P<0.0001, 72h P<0.0001). Cell cycle analysis showed that after circMET silencing, TPC-1 cells were more distributed in the G1 phase (P=0.4922) and less distributed in the S phase (P=0.2800) and G2 / M phase (P=0.0006), indicating that by silencing circMET, TPC-1 cells were arrested in the G1 phase ( Fig. 9 FG part).

[0076] (4) Conclusion This study found that 14 circRNAs were significantly upregulated in PTC compared with adjacent normal thyroid tissue. Among them, circMET may be involved in the pathogenesis and progression of PTC by affecting cell proliferation and cell cycle. circRNA-miRNA-mRNA interactions were predicted and potential regulatory pathways were discussed in this analysis.

[0077] In this study, 89 upregulated and 14 downregulated circRNAs were found in PTC patients. Among them, circMET (has_circ_0082002) was confirmed to be a diagnostic or predictive biomarker for papillary thyroid carcinoma (PTC). In addition, targeting circMET is a potential therapeutic strategy for PTC.

[0078] In summary, circular RNAs has_circ_0082002 can be used as a biomarker for auxiliary diagnosis of cancer. The above biomarkers can be used to prepare cancer auxiliary diagnosis reagents, to prepare reagents for auxiliary judgment of the prognosis of cancer patients, and can also be used to prepare drugs for treating cancer.

[0079] Although the present disclosure is specifically described above in conjunction with the accompanying drawings and embodiments, it is to be understood that the above description does not limit the present disclosure in any form. Those skilled in the art may modify and change the present disclosure as needed without departing from the essential spirit and scope of the present disclosure, and these modifications and changes all fall within the scope of the present disclosure.

Claims

1. A detection product for assisting in the diagnosis of cancer and / or assisting in determining the prognosis of a cancer patient, characterized in that: include: A reagent for detecting has_circ_0082002 in a sample, wherein the type of cancer comprises at least one of thyroid cancer, nasopharyngeal cancer, oral cancer, and ovarian cancer.

2. The detection product according to claim 1, characterized in that: The type of cancer is papillary thyroid carcinoma.

3. The detection product according to claim 2, characterized in that: The high expression of has_circ_0082002 regulates the biological function of papillary thyroid carcinoma through the hsa-miR-6796-5p / ALDH1A3 signaling pathway.

4. The detection product according to claim 1, characterized in that: The sample is tissue or serum of a subject.

5. The detection product according to claim 1, characterized in that: The reagent includes an upstream primer and a downstream primer for real-time fluorescence quantitative detection of has_circ_0082002, the sequence of the upstream primer is 5'-ACAATGTGAGATGTCTCCAGCAT-3', and the sequence of the downstream primer is 5'-GGTAAACAGGAGCACGAGGAT-3'.

6. The detection product according to claim 1 or 5, characterized in that: The reagents include a probe for detecting has_circ_0082002.

7. The detection product according to claim 6, characterized in that: The sequence of the probe is: 5'-GGTTTATCCATTAAAGCAGTGCTCAGAGGTTTATCCTATTAAAGCAGTCATTAGAGGTTTATCTATTAA-3'.

8. The detection product according to claim 1, characterized in that: has_circ_0082002 is positively correlated with the cancer, and the expression level of has_circ_0082002 in the subject can be detected to assist in the diagnosis of cancer or the prognosis of cancer patients.

9. The detection product according to claim 1, characterized in that: The detection product is a detection kit.

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