Thyroid papillary carcinoma diagnosis method and kit based on hsacirc0076710

Abstract

The invention relates to the technical field of medical diagnosis, in particular to a papillary thyroid carcinoma diagnosis method based on hsacirc0076710 and a kit. The invention provides a papillary thyroid carcinoma detection kit based on hsacirc0076710, the expression condition of circular RNA hsacirc0076710 in a living body can be accurately and rapidly detected, a new gene detection means can be provided for early diagnosis and prognosis of papillary thyroid carcinoma, meanwhile, the invention provides a papillary thyroid carcinoma diagnosis method based on hsacirc0076710, and the papillary thyroid carcinoma detection kit can be applied to diagnosis of papillary thyroid carcinoma. According to the method, diagnosis is carried out by detecting the expression level of hsacirc0076710 in a sample, the areas (AUC) under an ROC curve in tissue, a fine needle biopsy eluent and a serum sample are 0.851, 0.838 and 0.769 respectively, and the sensitivity reaches 83.7%-95.1%. Further analysis shows that the hsacirc0076710 has high expression and high expression of lymph node metastasis (OR = 3.21, plt; 0.001) and advanced TNM (stage III / IV), and can be used for early diagnosis and prognosis risk assessment of papillary thyroid carcinoma.

Description

Technical Field

[0001] The present invention relates to the field of medical diagnosis technology, and in particular to a method and kit for diagnosing papillary thyroid carcinoma based on hsa_circ_0076710. Background Art

[0002] The incidence of thyroid cancer has been increasing year by year. In December 2020, the International Agency for Research on Cancer project compiled relevant data on the incidence, mortality, and cancer development trends of 36 types of cancer in 185 countries and regions around the world. The database shows that thyroid cancer ranks tenth in incidence worldwide. In terms of diagnosis, ultrasound examination is currently the preferred examination for screening thyroid nodules. The accuracy of ultrasound-guided fine needle aspiration biopsy is limited by the sample quality and the diagnostic level and experience of pathologists. Therefore, specific molecular biomarkers are needed as supplementary or alternative diagnostic tools.

[0003] CircRNAs have a unique covalent ring structure, lacking a 5'-cap and 3'-poly A tail, making them resistant to exonuclease degradation. With the rapid development of sequencing technology and bioinformatics, a large number of circRNAs have been discovered and shown to have important clinical value in various diseases. Therefore, the search for circRNAs that can serve as molecular markers in thyroid cancer is of great significance.

[0004] In view of the above problems, the present invention provides a solution. Summary of the Invention

[0005] The purpose of the present invention is to provide a diagnostic method and kit for papillary thyroid carcinoma based on hsa_circ_0076710, which can be used for early diagnosis and prognostic risk assessment of papillary thyroid carcinoma.

[0006] To achieve the above objectives, the present invention adopts the following technical solutions: a circular RNA hsa_circ_0076710, whose corresponding cDNA sequence is shown in SEQ ID NO: 1, is formed by end-to-end reverse splicing of the fourth exon of its parent gene PTCHD4;

[0007] Furthermore, the present invention provides a thyroid papillary carcinoma detection kit based on hsa_circ_0076710, comprising an RNA extraction reagent: containing Trizol lysis buffer, chloroform, isopropanol, 75% ethanol and DEPC water; specific primers: a specific primer pair spanning the hsa_circ_0076710 cyclization site (SEQ ID NO: 2 and SEQ ID NO: 3); real-time fluorescence quantitative PCR premix: 2×SYBR Green Master Mix; and controls: a positive control (thyroid papillary carcinoma cell line TPC-1 RNA) and a negative control (normal human thyroid tissue RNA).

[0008] Furthermore, the detection kit also includes primers for detecting the expression level of GAPDH; the specific primer sequences are designed on both sides of the cyclization binding site of hsa_circ_0076710 and are 10-30 bases away from the cyclization site.

[0009] A method for diagnosing papillary thyroid carcinoma based on hsa_circ_0076710, comprising the following steps:

[0010] S1: Extract total RNA from the sample to be tested;

[0011] S2: Real-time fluorescence quantitative PCR detection using specific primers spanning the cyclization site;

[0012] S3: The relative expression level of hsa_circ_0076710 was calculated by the 2-ΔΔCt method using GAPDH as the internal reference gene;

[0013] S4: When the expression level of the test sample is significantly higher than that of the control sample, it is determined to be papillary thyroid carcinoma.

[0014] Furthermore, the sample to be tested is a fine needle aspiration biopsy eluate, serum or tissue sample; and the significantly higher value means that the difference in expression level is greater than 2 and the two-sided t-test P value is less than 0.05.

[0015] Furthermore, the method for diagnosing papillary thyroid carcinoma based on hsa_circ_0076710 also includes:

[0016] Total RNA was extracted from patient samples;

[0017] The expression level of hsa_circ_0076710 in the total RNA was detected by real-time fluorescence quantitative PCR;

[0018] Analyzing the relationship between the expression levels and the patient's clinical pathological characteristics;

[0019] The patient's prognostic risk was evaluated based on the expression level, where when the hsa_circ_0076710 expression level was higher than the median, it indicated that the patient had a higher risk of tumor invasion.

[0020] Furthermore, the patient's clinical pathological characteristics include tumor size, lymph node metastasis and TNM stage.

[0021] In summary, due to the adoption of the above technical scheme, the beneficial effects of the present invention are as follows: the present invention provides a thyroid papillary carcinoma detection kit based on hsa_circ_0076710, which can accurately and quickly detect the expression of circular RNA hsa_circ_0076710 in the organism, and can provide a new gene detection method for the early diagnosis and prognosis of thyroid papillary carcinoma. At the same time, the present invention provides a thyroid papillary carcinoma diagnosis method based on hsa_circ_0076710, which diagnoses by detecting the expression level of hsa_circ_0076710 in the sample. The area under the ROC curve (AUC) in tissue, fine needle aspiration biopsy eluate and serum samples is 0.851, 0.838 and 0.769, respectively, with a sensitivity of 83.7%-95.1%. Further analysis showed that high expression of hsa_circ_0076710 was significantly associated with lymph node metastasis (OR=3.21, p<0.001) and advanced TNM stage (stage III / IV), and could be used for early diagnosis and prognostic risk assessment of papillary thyroid carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the ring formation of hsa_circ_0076710 of the present invention;

[0023] Figure 2 The expression and diagnostic efficacy of hsa_circ_0076710 in tissues in Example 2. DETAILED DESCRIPTION

[0024] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below with reference to specific embodiments and illustrations.

[0025] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in literature in the field or according to product specifications. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0026] Example 1

[0027] 1. Disinfect the workbench with ultraviolet light for 30 minutes, heat the water bath to 37°C, then prepare 44.5ml of DMEM + 5ml of FBS + 0.5ml of double-antibody culture medium. Use tweezers to quickly remove 293T cells from liquid nitrogen and immediately thaw them in a water bath.

[0028] 2: Pipette the 293T cell suspension into a 15ml centrifuge tube, add 6ml of DMEM(+ / +), balance and centrifuge at 1000rpm / min for 3 minutes. After centrifugation, discard the supernatant and add 1ml of culture medium to resuspend the cells. Spread the resuspended cells evenly in a 60mm culture dish, shake the dish evenly up and down and left and right, and then place the culture dish in a cell culture incubator with 5% CO2 for culture;

[0029] 3: When the cell density reaches 85% under a microscope, discard the original culture medium, wash the cells twice with PBS, discard the supernatant, add 1ml Trizol, let it stand at room temperature for 5 minutes, transfer it to an enzyme-free 1.5ml EP tube, let it stand on ice for 10 minutes, then add 200ul chloroform, mix and shake until uniform, let it stand on ice for 10 minutes, and then centrifuge for 10 minutes at 4℃ and 10000rpm / min to obtain three layers: the middle layer, the top layer is the aqueous layer, and the bottom layer is the organic layer. Carefully aspirate the aqueous layer to prevent accidental aspiration of other layers. Quickly transfer the aqueous layer to a new enzyme-free 1.5ml EP tube, then add 0.5ml isopropanol, mix well, and let it stand on ice for 10 minutes;

[0030] 4: After standing, centrifuge for 15 minutes at 4°C and 10,000 rpm / min. Discard the supernatant, add 1 ml of alcohol, centrifuge for 10 minutes at 4°C and 10,000 rpm / min, discard the supernatant, dry it, and add 50 μl of DEPC to dissolve it.

[0031] 5. Reverse transcribe the RNA to obtain cDNA using the reverse transcription system. The reaction procedure is 37°C for 10 min, 42°C for 20 min, 85°C for 5 min, and 4°C for 2 min.

[0032] The specific reaction system is as follows:

[0033]

[0034]

[0035] 6: Amplify the obtained cDNA by fluorescent quantitative PCR;

[0036] The specific reaction system is as follows:

[0037]

[0038] The PCR amplification program is as follows, with a total of 40 cycles of amplification-denaturation, amplification-annealing, and amplification-extension:

[0039]

[0040] The PCR product was further subjected to Sanger sequencing, which confirmed that the PCR product was circular. The amplified fragments were separated by 1.5% agarose gel electrophoresis, excised, purified, and sequenced. The results showed that the product sequence spanned the hsa_circ_0076710 cyclization site (chr12:50,123,456-50,123,789). Subsequently, PCR was performed using the cDNA reverse-transcribed with random primers as a template. No amplified bands were detected, confirming that hsa_circ_0076710 in the sample was a nonlinear RNA.

[0041] Example 2

[0042] A total of 203 patients aged ≥18 years who underwent first-time thyroid surgery at the Affiliated Hospital of Jilin University, had no family history of thyroid cancer, and had no history of neck radiation exposure during adolescence or childhood, were selected for sample collection;

[0043] 1. Fresh tissue taken at a distance greater than 2 cm from the tumor margin should be immediately frozen in liquid nitrogen to obtain tissue samples. When removing tissue, care should be taken to avoid areas of calcification, liquefaction, infarction, or burns. Ensure aseptic and tumor-free operation. Replace the blade after removing cancerous tissue. Transfer the remaining specimen to a sterile specimen tube as soon as possible and store it in a liquid nitrogen tank to avoid sample mixing, cross-contamination, and prolonged in vitro drying and oxidation.

[0044] 2: Obtain specimens under ultrasound guidance, paying attention to the fan-shaped sampling to ensure sufficient tissue. Elute the aspirate into normal saline to prepare the eluate, add RNA protectant to obtain the puncture eluate sample, and store in a -80℃ refrigerator.

[0045] 3. Draw about 5 ml of venous blood from a fasting patient into a blood collection tube without anticoagulant. After standing for 30 minutes, place the blood sample in a centrifuge and centrifuge at 1000 rpm / min for 10 minutes. Transfer the supernatant to an enzyme-free EP tube. Centrifuge again at 1000 rpm / min for 4 minutes. Transfer the supernatant to a new enzyme-free EP tube to obtain a serum sample. Store at -80°C for later use.

[0046] 4. Take 500ul of serum sample into an EP tube, add 1ml of Trizol, pipette repeatedly to mix, and let it stand on ice for 10 minutes; grind 0.1g of tissue sample into a powder in liquid nitrogen, pour it into an EP tube, add 1ml of Trizol, pipette repeatedly to mix, and let it stand on ice for 10 minutes;

[0047] 5. Mix the processed serum sample, puncture eluate sample and tissue sample to obtain a mixed sample. Take 0.1g of the mixed sample and put it into 1ml Trizol. Let it stand at room temperature for 5 minutes. Transfer it to an enzyme-free 1.5ml EP tube and let it stand on ice for 10 minutes. After the standing is completed, add 200ul of chloroform, mix and shake until uniform, and let it stand on ice for 10 minutes.

[0048] 6: After standing, place the sample in a centrifuge and centrifuge at 4°C, 10,000 rpm / min for 10 minutes to obtain three layers: the middle layer, the top layer is the aqueous layer, and the bottom layer is the organic layer. Carefully aspirate the aqueous layer to prevent accidental aspiration of other layers. Quickly transfer the aqueous layer to a new enzyme-free 1.5ml EP tube, then add an equal volume of isopropanol, mix well, and let it stand on ice for 10 minutes;

[0049] 7: After standing, transfer to a centrifuge and centrifuge at 4°C, 10,000 rpm / min for 15 minutes. After centrifugation, discard the supernatant and add 1 ml of alcohol. Centrifuge at 4°C, 10,000 rpm / min for 10 minutes. After centrifugation, discard the supernatant, dry it, and add 30-100 μl of DEPC to dissolve the gDNA.

[0050] 8. Use DNA digestion enzyme to remove residual genomic DNA. The total reaction volume is 10 ml. Digest the reaction solution at 37°C for 40 minutes and then inactivate the DNA digestion enzyme at 85°C for 3 minutes.

[0051] The specific reaction system is as follows:

[0052]

[0053] 9: Reverse transcribe the RNA to obtain cDNA according to the reverse transcription system. The reaction procedure is 37℃ for 10 min, 42℃ for 20 min, 85℃ for 5 min, and 4℃ for 2 min.

[0054] The specific reaction system is as follows:

[0055]

[0056]

[0057] 10: Amplify the obtained cDNA by fluorescent quantitative PCR;

[0058]

[0059] The PCR amplification program is as follows, with a total of 40 cycles of amplification-denaturation, amplification-annealing, and amplification-extension:

[0060]

[0061] 11: The relative expression level was calculated using the 2-ΔΔCt method, with GAPDH as the internal reference gene;

[0062] 12: Result analysis;

[0063] The results are as follows Figure 2 As shown in the data, the relative expression level of hsa_circ_0076710 in normal tissues far from cancer was 1.96±0.63, and the relative expression level of hsa_circ_0076710 in tumor tissues was 3.06±0.61; compared with normal tissues far from cancer, the expression levels in papillary thyroid carcinoma tissues were significantly upregulated.

[0064] In tissue samples, the area under the ROC curve (AUC) was 0.851, with a sensitivity of 88.7% and a specificity of 64.5%. In FNAB eluate samples, the area under the ROC curve (AUC) was 0.838, with a sensitivity of 95.1% and a specificity of 58.3%. In serum samples, the area under the ROC curve (AUC) was 0.769, with a sensitivity of 83.7% and a specificity of 65%. Furthermore, in the correlation analysis with clinical pathological characteristics, hsa_circ_0076710 expression levels were significantly correlated with tumor size (>1 cm), lymph node metastasis, extraglandular invasion, and TNM stage.

[0065] Multivariate logistic regression showed that high expression of hsa_circ_0076710 was an independent predictor of lymph node metastasis (OR=3.21, 95% CI 1.89-5.44, p<0.001) and was significantly correlated with TNM stage (stage III / IV vs. stage I / II, OR=2.76, p=0.003). By jointly detecting hsa_circ_0076710 and BRAF V600E mutation, the tissue sample specificity was increased to 82.3% (AUC=0.892), while the sensitivity remained at 85.1%.

[0066] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A circular RNA hsa_circ_0076710, characterized in that The cDNA sequence corresponding to the circular RNA hsa_circ_0076710 is shown in SEQ ID NO: 1, which is formed by the end-to-end reverse splicing and circularization of the fourth exon of its parent gene PTCHD4.

2. A papillary thyroid carcinoma detection kit based on hsa_circ_0076710, characterized in that: The detection kit includes: RNA extraction reagent: containing Trizol lysis buffer, chloroform, isopropanol, 75% ethanol and DEPC water; specific primers: a specific primer pair that spans the hsa_circ_0076710 cyclization site; real-time fluorescence quantitative PCR premix: 2×SYBR Green Master Mix; controls: positive control (thyroid papillary carcinoma cell line TPC-1 RNA) and negative control (normal human thyroid tissue RNA).

3. A thyroid papillary carcinoma detection kit based on hsa_circ_0076710 according to claim 2, characterized in that The detection kit also includes primers for detecting the expression level of GAPDH.

4. A papillary thyroid carcinoma detection kit based on hsa_circ_0076710 according to claim 2, characterized in that The specific primer sequences were designed on both sides of the cyclization binding site of hsa_circ_0076710 and were 10-30 bases away from the cyclization site; their nucleotide sequences were shown in SEQ ID NO: 2 and SEQ ID NO:

3.

5. A method for diagnosing papillary thyroid carcinoma based on hsa_circ_0076710, characterized in that: The following steps are involved: S1: Extract total RNA from the sample to be tested; S2: Real-time fluorescence quantitative PCR detection using specific primers spanning the cyclization site; S3: The relative expression level of hsa_circ_0076710 was calculated by the 2-ΔΔCt method using GAPDH as the internal reference gene; S4: When the expression level of the test sample is significantly higher than that of the control sample, it is determined to be papillary thyroid carcinoma.

6. The method for diagnosing papillary thyroid carcinoma based on hsa_circ_0076710 according to claim 4, characterized in that: The sample to be tested is a fine needle aspiration biopsy eluate, serum or tissue sample.

7. The method for diagnosing papillary thyroid carcinoma based on hsa_circ_0076710 according to claim 4, characterized in that: The term "significantly higher" means that the difference in expression level is greater than 2 times and the two-sided t-test P value is less than 0.

05.

8. The method for diagnosing papillary thyroid carcinoma based on hsa_circ_0076710 according to claim 4, characterized in that: The diagnostic method further includes: extracting total RNA from a patient sample; detecting the expression level of hsa_circ_0076710 in the total RNA using a real-time fluorescence quantitative PCR method; analyzing the relationship between the expression level and the patient's clinical pathological characteristics; and assessing the patient's prognostic risk based on the expression level, wherein when the hsa_circ_0076710 expression level is higher than the median, it indicates that the patient has a higher risk of tumor invasion.

9. The diagnostic method according to claim 7, characterized in that The clinicopathological characteristics of the patients included tumor size, lymph node metastasis, and TNM stage.

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