An in vitro diagnostic method for thyroid function abnormalities based on novel microRNA markers

Abstract

The application discloses an in-vitro diagnosis method for thyroid function abnormality based on a novel microRNA marker. The application collects peripheral blood of a to-be-tested individual, extracts total microRNA, adopts a real-time fluorescent quantitative polymerase chain reaction technology to jointly detect expression levels of thyroid tissue-specific microRNA-1 (miR-7-5p), thyroid tissue-specific microRNA-2 (miR-375) and immune-regulating microRNA (miR-146a-5p), constructs a comprehensive diagnosis index, and detects and calculates a dynamic change rate at a first time point and a second time point respectively, and carries out diagnosis and evaluation according to the comprehensive diagnosis index and the dynamic change rate. The application jointly detects the tissue-specific microRNA and the immune-regulating microRNA, and combines dynamic monitoring, thereby significantly improving the sensitivity and specificity of the diagnosis of the thyroid function abnormality, and realizing quantitative evaluation on an evolution trend of the disease.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and in particular to an in vitro diagnostic method for thyroid dysfunction based on novel microRNA biomarkers. Background Technology

[0002] Thyroid dysfunction, including hypothyroidism and hyperthyroidism, is one of the most common endocrine disorders in clinical practice. Its diagnosis primarily relies on serological testing, specifically measuring the levels of thyroid-stimulating hormone (TSH), total triiodothyronine (TTI), free triiodothyronine (FTTH), total thyroxine (TTH), and free thyroxine (FTTH). However, this traditional diagnostic system has significant limitations. First, these hormone levels are finely regulated by the hypothalamic-pituitary-thyroid axis, exhibiting feedback lag. For example, in the early stages of hyperthyroidism, TSH levels may not be fully suppressed while FTTH levels have already increased, leading to an ambiguous diagnostic window. Second, non-thyroid syndromes (such as severe infections, tumors, and liver and kidney diseases) can interfere with the hypothalamic-pituitary-thyroid axis, causing false changes in serum hormone levels that resemble thyroid dysfunction, easily leading to misdiagnosis. Third, current hormone testing cannot effectively differentiate the etiology of thyroid dysfunction; for example, it cannot clearly distinguish between Graves' disease (autoimmune hyperthyroidism) and toxic nodular goiter, directly impacting the choice of subsequent treatment strategies.

[0003] In recent years, microRNAs have attracted widespread attention as a novel class of biomarkers due to their crucial role in tissue-specific expression and regulation. Previous studies have reported that certain microRNAs are highly expressed in thyroid tissue and released into the bloodstream during thyroid dysfunction; however, their diagnostic efficacy is limited, with insufficient sensitivity and specificity. For example, the area under the receiver operating characteristic (AUC) curve for detecting miR-7-5p or miR-146a-5p alone is typically between 0.7 and 0.8, which is insufficient to meet the needs of high-precision clinical diagnosis. More importantly, most existing studies remain at the static detection level, only detecting microRNA expression levels at a single time point, ignoring the dynamic changes of microRNAs during disease development, which is crucial information reflecting the changing trends of thyroid function. Furthermore, current technologies lack strategies for effectively combining tissue-specific microRNAs with immunomodulatory microRNAs to construct dynamic models, resulting in an inability to effectively correlate the pathophysiological information of microRNAs with disease activity and evolution, thus failing to fully realize the potential of microRNAs as in vitro diagnostic biomarkers.

[0004] Therefore, developing an in vitro diagnostic method for thyroid dysfunction that can overcome the shortcomings of the existing technology, has higher sensitivity and specificity, and can provide dynamic information on the disease is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] To achieve the above objectives, the present invention provides an in vitro diagnostic method for thyroid dysfunction based on novel microRNA biomarkers, comprising the following steps:

[0006] Step 1: Collect peripheral venous blood samples from the individual to be tested and separate the plasma samples;

[0007] Step 2: Extract total microRNA from the plasma sample to obtain a plasma total microRNA solution;

[0008] Step 3: Using the total plasma microRNA solution as a template, reverse transcription reaction is performed using reverse transcription primers to synthesize complementary deoxyribonucleic acid. The reverse transcription primers contain specific stem-loop primers targeting thyroid tissue-specific microRNA-1, thyroid tissue-specific microRNA-2, and immunomodulatory microRNA.

[0009] Step 4: Using the complementary deoxyribonucleic acid as a template, the expression levels of the thyroid tissue-specific microRNA-1, the thyroid tissue-specific microRNA-2, and the immunomodulatory microRNA were detected by real-time quantitative polymerase chain reaction technology to obtain their respective cycle thresholds;

[0010] Step 5: Based on the cycle threshold, calculate the relative expression levels of the thyroid tissue-specific microRNA-1, the thyroid tissue-specific microRNA-2, and the immunomodulatory microRNA;

[0011] Step 6: Based on the relative expression levels, construct a comprehensive diagnostic index, wherein the comprehensive diagnostic index uses the relative expression levels of thyroid tissue-specific microRNA-1 and thyroid tissue-specific microRNA-2 as positive indicators, and the relative expression levels of immune-regulating microRNAs as negative indicators.

[0012] Step 7: For the individual to be tested, repeat steps 1 to 6 at the first time point and the second time point respectively to calculate the comprehensive diagnostic index at the first time point and the comprehensive diagnostic index at the second time point, and calculate the dynamic change rate based on the comprehensive diagnostic index at the first time point and the comprehensive diagnostic index at the second time point, and perform a diagnostic assessment based on the comprehensive diagnostic index and the dynamic change rate.

[0013] Preferably, the extraction of total microRNA from the plasma sample in step 2 is specifically performed as follows: Extraction is carried out using a magnetic bead extraction kit. 600 μL of lysis binding buffer is added to 200 μL of plasma sample. The lysis binding buffer contains 4 mol / L guanidine isothiocyanate, 25 mmol / L sodium citrate, and 0.5% sodium dodecyl sarcosinate. The mixture is vortexed for 15 seconds and incubated at room temperature for 5 minutes. Then, 20 μL of magnetic bead suspension is added. The magnetic beads in the suspension are nanoscale superparamagnetic beads with a silica-modified surface. After vortexing and mixing, the mixture is incubated at room temperature on a rotary mixer for 15 minutes. The incubated mixture is then placed on a magnetic rack and incubated for 2 minutes. After the magnetic beads are completely adsorbed, the supernatant is discarded. The magnetic beads are then washed sequentially with 700 μL of washing solution 1 and 700 μL of washing solution 2. Washing solution 1 is a phosphate buffer solution with a concentration of 1x containing 50% anhydrous ethanol, and washing solution 2 is a phosphate buffer solution with a concentration of 1x containing 75% anhydrous ethanol. After each wash, the magnetic beads are adsorbed using a magnetic rack and the supernatant is discarded. The magnetic beads are then left to dry at room temperature for 8 to 10 minutes. 30 μL of RNase-free water is added, and the mixture is shaken and incubated at 55 degrees Celsius for 5 minutes. The mixture is then placed back on the magnetic rack, and after the magnetic beads are completely adsorbed, the supernatant is collected to obtain the purified plasma total microRNA solution.

[0014] Preferably, in step 3, the thyroid tissue-specific microRNA-1 is miR-7-5p, the thyroid tissue-specific microRNA-2 is miR-375, and the immunomodulatory microRNA is miR-146a-5p; the total volume of the reverse transcription reaction system is 20 μL, comprising: 10 μL of the total plasma microRNA solution, 4 μL of 5-fold concentration reverse transcription buffer, 2 μL of deoxyribonucleic acid mixture, 1 μL of ribonuclease inhibitor, 1 μL of reverse transcriptase, and 2 μL of reverse transcription primer mixture, wherein the reverse transcription primer mixture contains specific stem-loop primers for miR-7-5p, specific stem-loop primers for miR-375, and specific stem-loop primers for miR-146a-5p; the reverse transcription reaction program is: incubation at 16°C for 30 minutes, incubation at 42°C for 30 minutes, incubation at 85°C for 5 minutes, and finally storage at 4°C to obtain complementary deoxyribonucleic acid products.

[0015] Preferably, during the real-time quantitative polymerase chain reaction (qPCR) detection in step 4, the expression level of an internal reference gene is simultaneously detected. The internal reference gene is U6 small nucleonucleotide. Each test microRNA's qPCR system is 20 μL, comprising: 2 μL of a 10-fold diluted complementary deoxyribonucleic acid product, 10 μL of a 2-fold concentration of qPCR premix, 0.4 μL of 10 μmol / L upstream and downstream specific primers, and 0.2 μL of 10 μmol / L upstream and downstream specific primers. The specific probe was added per liter, and the amount of RNase-free water was replenished to 20 μL. The real-time quantitative polymerase chain reaction program was as follows: pre-denaturation at 95 degrees Celsius for 10 minutes; then 40 cycles of denaturation at 95 degrees Celsius for 15 seconds and annealing extension at 60 degrees Celsius for 1 minute were performed; fluorescence signals were collected at the end of the annealing extension phase at 60 degrees Celsius in each cycle; after the reaction, the baseline threshold was set according to the amplification curve to obtain the cycle threshold of each microRNA to be tested and the cycle threshold of the U6 small nucleoribonucleic acid.

[0016] Preferably, the calculation of the relative expression levels of the thyroid tissue-specific microRNA-1, the thyroid tissue-specific microRNA-2, and the immunomodulatory microRNA in step 5 is specifically performed using the formula: Calculate the relative expression level of each microRNA to be tested, where The relative expression levels of thyroid tissue-specific microRNA-1 were obtained through the above calculations and denoted as follows: The relative expression level of the thyroid tissue-specific microRNA-2 is denoted as The relative expression level of the immune regulatory microRNA is denoted as .

[0017] Preferably, the comprehensive diagnostic index in step 6 is calculated using the following formula:

[0018]

[0019] in, , and These are the average relative expression levels of the thyroid tissue-specific microRNA-1, the thyroid tissue-specific microRNA-2, and the immunomodulatory microRNA, respectively, in a pre-constructed database of individuals with normal thyroid function.

[0020] Preferably, in step 7, the first time point is the initial medical visit, and the second time point is the 14th day after the initial medical visit; the dynamic change rate Calculated using the following formula:

[0021]

[0022] in, This refers to the comprehensive diagnostic index at the first time point. The comprehensive diagnostic index at the second time point; the judgment rule for the diagnostic assessment is: if Greater than the first preset threshold and If the value exceeds the second preset threshold, a diagnosis of hyperthyroidism is made; if... Less than the third preset threshold and If the value is less than the fourth preset threshold, a diagnosis of hypothyroidism is made; if... Between the third preset threshold and the first preset threshold and If the absolute value is less than the fifth preset threshold, the diagnosis is normal thyroid function; the first preset threshold, the second preset threshold, the third preset threshold, the fourth preset threshold and the fifth preset threshold are cutoff values ​​determined by receiver operating characteristic curve analysis based on the distribution of the comprehensive diagnostic index and its dynamic change rate of pre-constructed groups of individuals with normal thyroid function, patients with hypothyroidism and patients with hyperthyroidism.

[0023] Preferably, the step 1 of collecting peripheral venous blood samples from the individual to be tested and separating plasma samples specifically involves: placing the collected peripheral venous blood samples in an anticoagulant blood collection tube containing dipotassium EDTA; centrifuging at 4 degrees Celsius and 1600 times relative centrifugation for 15 minutes within 2 hours after collection to separate the upper plasma sample; aliquoting the plasma sample into RNase-free cryovials and storing them at -80 degrees Celsius for later use; before testing, slowly thawing the frozen plasma sample on ice, then centrifuging at 4 degrees Celsius and 12000 times relative centrifugation for 10 minutes, and using the supernatant for subsequent microRNA extraction.

[0024] Preferably, before step 1, a control sample preparation step is included: peripheral venous blood samples are collected from at least 30 clinically diagnosed individuals with normal thyroid function, at least 30 clinically diagnosed patients with hypothyroidism, and at least 30 clinically diagnosed patients with hyperthyroidism. These samples are processed according to the methods in steps 1 to 5 to obtain a database of the relative expression levels of thyroid tissue-specific microRNA-1, thyroid tissue-specific microRNA-2, and immunomodulatory microRNA in the individuals with normal thyroid function, and the mean and standard deviation of the relative expression levels of each microRNA are calculated. At the same time, a database of the relative expression levels of thyroid tissue-specific microRNA-1, thyroid tissue-specific microRNA-2, and immunomodulatory microRNA in the patients with hypothyroidism and hyperthyroidism is obtained. Based on the above database, the first preset threshold, the second preset threshold, the third preset threshold, the fourth preset threshold, and the fifth preset threshold are determined by receiver operating characteristic (ROC) curve analysis.

[0025] Preferably, the individual to be tested is an individual with suspected symptoms of thyroid dysfunction, which include at least one of palpitations, hand tremors, weight change exceeding 5% of original weight within one month, neck thickening, heat intolerance and excessive sweating or cold intolerance and reduced sweating; the first time point is any day from the 1st to the 3rd day after the appearance of the suspected symptoms of thyroid dysfunction, the second time point is any day from the 12th to the 16th day after the first time point, and the interval between the second time point and the first time point is 14 days.

[0026] The beneficial effects of this invention are:

[0027] 1. This invention creatively combines two thyroid tissue-specific microRNAs, miR-7-5p and miR-375, with one immunomodulatory microRNA, miR-146a-5p, and constructs a comprehensive diagnostic index. The synergistic upregulation of thyroid tissue-specific microRNAs is used as a positive indicator, while the negative regulatory effect of immunomodulatory microRNAs is used as a negative indicator, achieving a joint assessment of the functional status of thyroid follicular cells and the state of local immune inflammation. This comprehensive diagnostic index does not simply sum the information from the three markers; instead, it eliminates individual differences in basal microRNA expression levels through ratio normalization, amplifying characteristic expression profile changes under pathological conditions. This results in a diagnostic sensitivity and specificity exceeding 95%, significantly superior to single microRNA marker detection techniques.

[0028] 2. This invention innovatively introduces the monitoring of the dynamic change rate of the comprehensive diagnostic index within a 14-day time window, expanding static, point-like diagnostic information into dynamic, linear disease evolution trend information. For patients with hyperthyroidism, the dynamic change rate of the comprehensive diagnostic index shows a significant positive increase, reflecting disease activity; for patients with hypothyroidism, the dynamic change rate of the comprehensive diagnostic index shows a significant negative decrease, reflecting persistent hypothyroidism. This dynamic information provides an objective quantitative indicator for accurate diagnosis at initial consultation, while simultaneously enabling the assessment of disease evolution trends, overcoming the limitation of traditional hormone testing in providing dynamic disease information.

[0029] 3. This invention provides a complete, coherent, and self-consistent technical process, encompassing sample collection, plasma separation, total microRNA extraction using magnetic beads, reverse transcription to synthesize complementary deoxyribonucleic acid (DNA), real-time quantitative polymerase chain reaction (qPCR) detection to relative quantitative analysis, construction of a comprehensive diagnostic index, and calculation of dynamic change rate. The inputs and outputs of each step are closely interconnected, and all operational parameters, such as centrifugation force, temperature, time, reagent formulation, and primer / probe sequences, are clearly described. This ensures that those skilled in the art can fully reproduce the diagnostic method based on this solution, meeting the stringent disclosure requirements of patent law and demonstrating promising prospects for clinical translational applications. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0031] Figure 1 This is a flowchart of the steps of the method of the present invention. Detailed Implementation

[0032] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. It should also be noted that, to make the embodiments more comprehensive, the following embodiments are the best and preferred embodiments, and those skilled in the art can use other alternative methods to implement some well-known technologies; moreover, the accompanying drawings are only for more specific description of the embodiments and are not intended to specifically limit the present invention.

[0033] Please see Figure 1This invention provides an in vitro diagnostic method for thyroid dysfunction based on novel microRNA biomarkers. This method involves jointly detecting the expression levels of thyroid tissue-specific microRNA-1, thyroid tissue-specific microRNA-2, and immunomodulatory microRNAs in the peripheral blood of the tested individual, and combining this with the dynamic change rates at first and second time points to construct a comprehensive diagnostic index, thereby achieving accurate diagnosis of thyroid dysfunction. The invention will be further described in detail below with reference to specific embodiments.

[0034] 1. Pre-construction of the control sample database

[0035] Before performing tests on individuals, a control sample database needs to be built in advance to determine the preset thresholds required for subsequent diagnostic assessments.

[0036] Peripheral venous blood samples were collected from at least 30 clinically diagnosed individuals with normal thyroid function. The diagnostic criteria for normal thyroid function were: thyroid-stimulating hormone (TSH) levels between 0.4 mIU / L and 4.0 mIU / L, free triiodothyronine (FTTH) levels between 3.1 pmol / L and 6.8 pmol / L, and free thyroxine (FTH) levels between 12 pmol / L and 22 pmol / L, with no history of thyroid disease or family history of thyroid disease.

[0037] Peripheral venous blood samples were collected from at least 30 patients with clinically diagnosed hypothyroidism. The diagnostic criteria for hypothyroidism were: thyroid-stimulating hormone (TSH) levels above 4.0 mIU / L and free thyroxine (FTTH) levels below 12 picomol / L.

[0038] Peripheral venous blood samples were collected from at least 30 patients with clinically diagnosed hyperthyroidism. The diagnostic criteria for hyperthyroidism were: thyroid-stimulating hormone (TSH) levels below 0.4 mIU / L, and free triiodothyronine (FTT) levels above 6.8 picomol / L or free thyroxine (FTTH) levels above 22 picomol / L.

[0039] Peripheral venous blood samples from all the individuals described above were processed and tested using the same methods described in steps 2 to 6 of this specific embodiment. Databases of relative expression levels of thyroid tissue-specific microRNA-1, thyroid tissue-specific microRNA-2, and immunomodulatory microRNAs were obtained for individuals with normal thyroid function, patients with hypothyroidism, and patients with hyperthyroidism, respectively. Based on these databases, the average relative expression level of thyroid tissue-specific microRNA-1 in the group of individuals with normal thyroid function was calculated and denoted as _____. ; Calculate the average relative expression level of thyroid tissue-specific microRNA-2 in a population of individuals with normal thyroid function, denoted as . ; Calculate the average relative expression level of immune regulatory microRNAs in a population of individuals with normal thyroid function, denoted as . .

[0040] Based on the aforementioned database, a first, second, third, fourth, and fifth preset threshold were determined through receiver operating characteristic (ROC) curve analysis. The specific method for ROC curve analysis is as follows: using the comprehensive diagnostic index as the test variable and the clinical diagnosis result as the state variable, an ROC curve was plotted. The comprehensive diagnostic index value corresponding to the maximum value of the Youden index was selected as the cutoff value distinguishing between hyperthyroidism and euthyroidism; this cutoff value is the first preset threshold. Similarly, a cutoff value distinguishing between hypothyroidism and euthyroidism was selected; this cutoff value is the third preset threshold. Using the rate of change as the test variable, the above analysis was repeated to obtain the cutoff value for the rate of change distinguishing between hyperthyroidism and euthyroidism as the second preset threshold, and the cutoff value for the rate of change distinguishing between hypothyroidism and euthyroidism as the fourth preset threshold. The fifth preset threshold was determined by analyzing the upper limit of the 95% confidence interval of the rate of change in the euthyroid individual population.

[0041] 2. Sample Collection and Processing

[0042] Step 1: Collect peripheral venous blood samples from the individual to be tested and separate the plasma samples.

[0043] Specifically, peripheral venous blood samples were collected from the individuals to be tested and placed in anticoagulant blood collection tubes containing dipotassium ethylenediaminetetraacetate (EDTA). Within 2 hours of collection, the anticoagulant blood collection tubes were placed in a centrifuge at 4°C, with a relative centrifugal force of 1600 times and a centrifugation time of 15 minutes. After centrifugation, the upper plasma layer was carefully aspirated using a pipette, avoiding the middle leukocyte layer and the lower erythrocyte layer, to obtain the upper plasma sample. The plasma sample was aliquoted into RNase-free cryovials, 200 μL per tube, and stored at -80°C for later use. Before testing, the frozen plasma samples were removed and slowly thawed on ice. After complete thawing, the plasma samples were transferred to RNase-free centrifuge tubes and centrifuged at 12000 times relative centrifugation force for 10 minutes in a pre-cooled centrifuge at 4°C. After centrifugation, carefully aspirate the supernatant with a pipette and transfer it to a new RNase-free centrifuge tube. This supernatant is the processed plasma sample, which will be used for subsequent microRNA extraction.

[0044] 3. Extraction of total microRNA from plasma

[0045] Step 2: Extract total microRNA from the plasma sample to obtain a plasma total microRNA solution.

[0046] Specifically, total microRNA was extracted from the plasma sample processed in step 1 using a magnetic bead extraction kit. 600 μL of lysis binding buffer, containing 4 mol / L guanidine isothiocyanate, 25 mmol / L sodium citrate, and 0.5% sodium dodecyl sarcosinate, was added to 200 μL of plasma sample. The mixture was vortexed for 15 seconds and then allowed to stand at room temperature for 5 minutes to allow the lysis binding buffer to fully lyse exosomes and protein complexes in the plasma, releasing the microRNA.

[0047] Subsequently, 20 μL of magnetic bead suspension was added to the mixture. The magnetic beads in the suspension were nanoscale superparamagnetic beads with silica-modified surfaces. After vortexing to mix again, the centrifuge tube was placed on a rotary mixer and incubated at room temperature at 20 rpm for 15 minutes to allow the released microRNA to fully bind to the surface of the magnetic beads.

[0048] Place the incubated mixture on a magnetic rack and let it stand for 2 minutes. Under the influence of the magnet, the magnetic beads will be attracted to the wall of the centrifuge tube near the magnetic rack. Carefully aspirate the supernatant using a pipette. Add 700 μL of washing buffer 1 (a phosphate buffer solution containing 50% anhydrous ethanol at a concentration of 1) to the centrifuge tube. Remove the centrifuge tube from the magnetic rack and vortex for 30 seconds to resuspend the magnetic beads. Place the centrifuge tube back on the magnetic rack and let it stand for 1 minute until the magnetic beads are completely attracted, then aspirate the supernatant. Add 700 μL of washing buffer 2 (a phosphate buffer solution containing 75% anhydrous ethanol at a concentration of 1) to the centrifuge tube. Remove the centrifuge tube from the magnetic rack and vortex for 30 seconds to resuspend the magnetic beads. Place the centrifuge tube back on the magnetic rack and let it stand for 1 minute until the magnetic beads are completely attracted, then aspirate the supernatant.

[0049] Leave the centrifuge tube uncovered at room temperature for 8 to 10 minutes to allow any residual ethanol to evaporate completely. Observe the surface of the magnetic beads to ensure there is no liquid sheen. Finally, add 30 μL of RNase-free water to the centrifuge tube and pipette repeatedly 10 times to fully resuspend the magnetic beads. Incubate the centrifuge tube in a 55°C metal bath for 5 minutes to promote the elution of microRNA bound to the magnetic beads into the water. After incubation, place the centrifuge tube back on the magnetic rack and let it stand for 2 minutes until the magnetic beads are completely adsorbed. Carefully collect the supernatant with a pipette and transfer it to a new RNase-free centrifuge tube. This supernatant is the purified plasma total microRNA solution.

[0050] 4. Reverse transcription to synthesize complementary deoxyribonucleic acid

[0051] Step 3: Using the total plasma microRNA solution as a template, reverse transcription reaction is performed using reverse transcription primers to synthesize complementary deoxyribonucleic acid. The reverse transcription primers include specific stem-loop primers targeting thyroid tissue-specific microRNA-1, thyroid tissue-specific microRNA-2, and immunomodulatory microRNA.

[0052] Specifically, the thyroid tissue-specific microRNA-1 is miR-7-5p, the thyroid tissue-specific microRNA-2 is miR-375, and the immunomodulatory microRNA is miR-146a-5p.

[0053] The total volume of the reverse transcription reaction system is 20 μL, comprising: 10 μL of the plasma total microRNA solution, 4 μL of a 5-fold concentration reverse transcription buffer, 2 μL of deoxyribonucleotide mixture, 1 μL of ribonuclease inhibitor, 1 μL of reverse transcriptase, and 2 μL of reverse transcription primer mixture. The deoxyribonucleotide mixture contains 10 mmol / L of deoxyadenosine triphosphate, 10 mmol / L of deoxyguanosine triphosphate, 10 mmol / L of deoxycytidine triphosphate, and 10 mmol / L of deoxythymidine triphosphate. The concentration of the ribonuclease inhibitor is 40 units per μL. The concentration of the reverse transcriptase is 200 units per μL. The reverse transcription primer mixture contains specific stem-loop primers for miR-7-5p, miR-375, and miR-146a-5p, with a final concentration of 0.5 μmol / L for each primer in the mixture. The specific stem-loop primer sequences for miR-7-5p are shown in SEQ ID NO:1, the specific stem-loop primer sequences for miR-375 are shown in SEQ ID NO:2, and the specific stem-loop primer sequences for miR-146a-5p are shown in SEQ ID NO:3.

[0054] After mixing the above reaction mixture, it was placed in a polymerase chain reaction (PCR) instrument for reverse transcription. The reaction program was set as follows: incubation at 16°C for 30 minutes, 42°C for 30 minutes, 85°C for 5 minutes, and finally stored at 4°C. After the reaction, complementary deoxyribonucleic acid (DNA) products were obtained.

[0055] 5. Real-time quantitative polymerase chain reaction (qPCR) detection

[0056] Step 4: Using the complementary deoxyribonucleic acid as a template, the expression levels of the thyroid tissue-specific microRNA-1, the thyroid tissue-specific microRNA-2, and the immunomodulatory microRNA were detected by real-time quantitative polymerase chain reaction (qPCR) to obtain their respective cycle thresholds.

[0057] Specifically, while detecting the expression levels of the thyroid tissue-specific microRNA-1, the thyroid tissue-specific microRNA-2, and the immunomodulatory microRNA, the expression level of the internal reference gene, which is U6 small nucleotide RNA, is also detected.

[0058] First, the complementary deoxyribonucleic acid (DNA) product obtained in step 3 was diluted 10-fold with RNase-free water, and 2 μL of the diluted DNA product was used as a template. Each real-time quantitative polymerase chain reaction (RT-PCR) system for the microRNA to be tested consisted of 20 μL of the following: 2 μL of 10-fold diluted DNA product, 10 μL of 2-fold concentration R-PCR premix, 0.4 μL of 10 μmol / L upstream and downstream specific primers, 0.2 μL of 10 μmol / L specific probe, and RNase-free water to a final volume of 20 μL. The 2-fold concentration R-PCR premix contained hot-start DNA polymerase, a DNA nucleotide mixture, magnesium ions, and a reaction buffer.

[0059] The upstream primer sequence for miR-7-5p is 5'-ACACTCCAGCTGGGTGGTAGTTAGTGT-3', the downstream primer sequence is 5'-CTCAACTGGTGTCGTGGA-3', and the specific probe sequence is 5'-FAM-TGGTAGTTAGTGTGTCAG-BHQ1-3'. The upstream primer sequence for miR-375 is 5'-ACACTCCAGCTGGGTTTGTTCGTTCGG-3', the downstream primer sequence is 5'-CTCAACTGGTGTCGTGGA-3', and the specific probe sequence is 5'-FAM-TTTGTTCGTTCGGCTCG-BHQ1-3'. The upstream primer sequence for miR-146a-5p is 5'-ACACTCCAGCTGGGTGAGAACTGAATTC-3', the downstream primer sequence is 5'-CTCAACTGGTGTCGTGGA-3', and the specific probe sequence is 5'-FAM-TGAGAACTGAATTCCATG-BHQ1-3'. The upstream primer sequence for U6 small nucleonucleotide is 5'-CTCGCTTCGGCAGCACA-3', the downstream primer sequence is 5'-AACGCTTCACGAATTTGCGT-3', and the specific probe sequence is 5'-HEX-CACCATCAAGTTGCCC-BHQ1-3'.

[0060] The prepared reaction system was placed in a real-time quantitative polymerase chain reaction (qPCR) instrument for amplification. The reaction program was as follows: pre-denaturation at 95°C for 10 minutes; followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing extension at 60°C for 1 minute; at the end of the 60°C annealing extension phase of each cycle, the instrument automatically acquired the fluorescence signal. After the reaction, a baseline threshold was set according to the amplification curve, which was set to the value at the start of the exponential growth phase of the fluorescence signal, and was set to 10 times the standard deviation of the background fluorescence signal. The instrument automatically obtained the cycling threshold for each target microRNA and internal reference gene, which were denoted as the cycling threshold for the target microRNA and the cycling threshold for U6 small nucleonucleotide, respectively.

[0061] 6. Relative quantitative analysis

[0062] Step 5: Based on the cycle threshold, calculate the relative expression levels of the thyroid tissue-specific microRNA-1, the thyroid tissue-specific microRNA-2, and the immunomodulatory microRNA.

[0063] Specifically, using the formula Calculate the relative expression level of each microRNA to be tested, where Through the above calculations, the relative expression levels of the thyroid tissue-specific microRNA-1 were obtained, denoted as [missing information]. The relative expression level of the thyroid tissue-specific microRNA-2 is denoted as... The relative expression levels of the immune regulatory microRNAs are denoted as... .

[0064] 7. Construct a comprehensive diagnostic index

[0065] Step 6: Based on the relative expression levels, construct a comprehensive diagnostic index. The comprehensive diagnostic index uses the relative expression levels of thyroid tissue-specific microRNA-1 and thyroid tissue-specific microRNA-2 as positive indicators, and the relative expression levels of immune-regulating microRNAs as negative indicators.

[0066] Specifically, the comprehensive diagnostic index is calculated using the following formula:

[0067]

[0068] in, , and These are the average relative expression levels of the thyroid tissue-specific microRNA-1, the thyroid tissue-specific microRNA-2, and the immunomodulatory microRNA, respectively, in the pre-constructed database of individuals with normal thyroid function established in step 1.

[0069] 8. Dynamic monitoring and diagnostic assessment

[0070] Step 7: For the individual to be tested, repeat steps 1 to 6 at the first time point and the second time point respectively to calculate the comprehensive diagnostic index at the first time point and the comprehensive diagnostic index at the second time point, and calculate the dynamic change rate based on the comprehensive diagnostic index at the first time point and the comprehensive diagnostic index at the second time point, and perform a diagnostic assessment based on the comprehensive diagnostic index and the dynamic change rate.

[0071] Specifically, the first time point is the initial visit, and the second time point is 14 days after the initial visit. The dynamic change rate... Calculated using the following formula:

[0072]

[0073] in, This refers to the comprehensive diagnostic index at the first time point. This is the comprehensive diagnostic index at the second time point.

[0074] The diagnostic assessment criteria are as follows: if Greater than the first preset threshold and If the value exceeds the second preset threshold, a diagnosis of hyperthyroidism is made; if... Less than the third preset threshold and If the value is less than the fourth preset threshold, a diagnosis of hypothyroidism is made; if... Between the third preset threshold and the first preset threshold and If the absolute value is less than the fifth preset threshold, the diagnosis is normal thyroid function.

[0075] The first preset threshold, the second preset threshold, the third preset threshold, the fourth preset threshold, and the fifth preset threshold are cutoff values ​​determined by receiver operating characteristic curve analysis based on the distribution of the comprehensive diagnostic index and its dynamic change rate of individuals with normal thyroid function, patients with hypothyroidism, and patients with hyperthyroidism pre-constructed in step 1.

[0076] 9. Explanation regarding the individuals to be tested

[0077] The individuals to be tested are those with suspected symptoms of thyroid dysfunction. These suspected symptoms include at least one of the following: palpitations, hand tremors, a weight change of more than 5% of the original body weight within one month, neck thickening, heat intolerance with excessive sweating, or cold intolerance with reduced sweating.

[0078] The first time point is any day from the 1st to the 3rd day after the appearance of the suspected symptoms of thyroid dysfunction. The second time point is any day from the 12th to the 16th day after the first time point, and the interval between the second time point and the first time point is 14 days.

[0079] 10. Description of existing technical features

[0080] The conventional operations involved in this specific embodiment, such as centrifugation, vortexing, magnetic rack separation, metal bath incubation, conventional setup and use of the polymerase chain reaction (PCR) instrument, and data acquisition and analysis methods for the real-time quantitative PCR instrument, are all prior art known to those skilled in the art. The reagents used, such as lysis binding buffer, magnetic bead suspension, washing buffer 1, washing buffer 2, RNase-free water, reverse transcription buffer, deoxyribonucleic acid mixture, ribonuclease inhibitor, reverse transcriptase, and real-time quantitative PCR premix, are all conventional reagents in the art, obtainable commercially or prepared according to conventional methods. The primer and probe sequences involved are sequences that can be conventionally designed by those skilled in the art based on the target microRNA sequence. The receiver operating characteristic (ROC) curve analysis involved is a conventional statistical analysis method in the art. The specific operational details and conventional parameter settings of the above-mentioned prior art features can be fully implemented by those skilled in the art based on the guidance of this specific embodiment and their general technical knowledge.

[0081] Example

[0082] This embodiment provides an in vitro diagnostic method for thyroid dysfunction based on novel microRNA biomarkers, applied to a test subject with typical suspected symptoms of thyroid dysfunction.

[0083] 1. Individuals to be tested and control sample population

[0084] The subject of this example was a 35-year-old woman who complained of palpitations and hand tremors without any obvious cause over the past three weeks, a weight loss of 6 kg within one month (more than 5% of her original weight), and also experienced heat intolerance, excessive sweating, and neck thickening. This subject met the characteristics of suspected thyroid dysfunction.

[0085] Before testing the individuals to be tested, this embodiment pre-constructs a control sample database. Specifically:

[0086] Peripheral venous blood samples were collected from 30 clinically diagnosed individuals with euthyroid function. The diagnostic criteria for euthyroidism were: thyroid-stimulating hormone (TSH) levels between 0.4 mIU / L and 4.0 mIU / L, free triiodothyronine (FTT) levels between 3.1 pmol / L and 6.8 pmol / L, and FTT levels between 12 pmol / L and 22 pmol / L, with no history of thyroid disease or family history. Peripheral venous blood samples were also collected from 30 clinically diagnosed patients with hypothyroidism. The diagnostic criteria for hypothyroidism were: TSH levels above 4.0 mIU / L and FTT levels below 12 pmol / L. Peripheral venous blood samples were also collected from 30 clinically diagnosed patients with hyperthyroidism. The diagnostic criteria for hyperthyroidism were: TSH levels below 0.4 mIU / L and FTT levels above 6.8 pmol / L or FTT levels above 22 pmol / L.

[0087] Peripheral venous blood samples from all the individuals described above were processed and tested using the same methods described later in this embodiment, resulting in databases of the relative expression levels of thyroid tissue-specific microRNA-1, thyroid tissue-specific microRNA-2, and immunomodulatory microRNAs in individuals with normal thyroid function, patients with hypothyroidism, and patients with hyperthyroidism. Based on these databases, receiver operating characteristic (ROC) curve analysis was used to determine the first, second, third, fourth, and fifth preset thresholds required for subsequent diagnostic assessment. The specific method for ROC curve analysis was as follows: using the comprehensive diagnostic index as the test variable and the clinical diagnosis result as the state variable, a ROC curve was plotted. The comprehensive diagnostic index value corresponding to the maximum value of the Youden index was selected as the cutoff value distinguishing between hyperthyroidism and normal thyroid function, i.e., the first preset threshold; similarly, the cutoff value distinguishing between hypothyroidism and normal thyroid function was selected, i.e., the third preset threshold. The dynamic change rate was used as the test variable, and the above analysis was repeated to obtain the second and fourth preset thresholds. The fifth preset threshold was determined by analyzing the upper limit of the 95% confidence interval of the dynamic change rate in the normal thyroid function group.

[0088] 2. Sample Collection and Processing

[0089] On the second day after the onset of suspected thyroid dysfunction symptoms (i.e., the first time point), a 5 ml peripheral venous blood sample was collected from the individual and placed in an anticoagulant blood collection tube containing dipotassium EDTA. Within 1.5 hours of collection, the blood collection tube was placed in a centrifuge at 4 degrees Celsius, with a relative centrifugal force of 1600 times, for 15 minutes. After centrifugation, the upper plasma layer was carefully aspirated using a pipette, avoiding the middle leukocyte layer. The separated plasma sample was aliquoted into RNase-free cryovials, 200 μL per tube, and immediately stored at -80 degrees Celsius for later use.

[0090] Before microRNA extraction, the frozen plasma sample was removed and thawed slowly on ice for approximately 30 minutes. Once completely thawed, the plasma sample was transferred to a 1.5 mL RNase-free centrifuge tube and centrifuged at 12000x relative centrifugation for 10 minutes in a pre-chilled 4°C centrifuge. After centrifugation, a small amount of precipitate was visible at the bottom of the tube. The supernatant was carefully aspirated using a pipette and transferred to a new RNase-free centrifuge tube. This supernatant is the processed plasma sample, used for subsequent microRNA extraction.

[0091] 3. Extraction of total microRNA from plasma

[0092] Total microRNA was extracted from the treated plasma samples using a magnetic bead extraction kit. 600 μL of lysis binding buffer was added to 200 μL of plasma sample. The lysis binding buffer consisted of 4 mol / L guanidine isothiocyanate, 25 mmol / L sodium citrate, and 0.5% sodium dodecyl sarcosinate. The mixture was vortexed for 15 seconds to ensure sufficient contact between the plasma and the lysis binding buffer. The mixture was then allowed to stand at room temperature for 5 minutes to allow the lysis binding buffer to fully lyse exosomes, protein-microRNA complexes, and other components in the plasma, releasing the microRNA.

[0093] Subsequently, 20 μL of magnetic bead suspension was added to the mixture. The magnetic beads in the suspension were nanoscale superparamagnetic beads with a surface modified with silica, a particle size of 300 nm, and a concentration of 50 mg / mL. After vortexing to mix again, the centrifuge tube was placed on a rotary mixer and incubated at room temperature at a speed of 20 rpm for 15 minutes to allow the released microRNA to fully bind to the surface of the magnetic beads.

[0094] Place the incubated mixture on a magnetic rack and let it stand for 2 minutes. Under the influence of magnetism, the magnetic beads are attracted to the wall of the centrifuge tube near the magnetic rack, forming a clear precipitate. Carefully aspirate the supernatant with a pipette, being careful not to disturb the magnetic bead precipitate. Add 700 μL of washing buffer 1, which is a phosphate buffer solution with a concentration of 50% anhydrous ethanol, to the centrifuge tube. Remove the centrifuge tube from the magnetic rack and vortex for 30 seconds to resuspend the magnetic beads and wash thoroughly. Place the centrifuge tube back on the magnetic rack and let it stand for 1 minute until the magnetic beads are completely attracted, then aspirate the supernatant. Repeat the above washing steps, but use 700 μL of washing buffer 2, which is a phosphate buffer solution with a concentration of 75% anhydrous ethanol, for washing. After both washings are complete, leave the centrifuge tube open at room temperature for 8 minutes to allow any residual ethanol to evaporate completely. Observe the surface of the magnetic beads; ensure there is no liquid shine, indicating that the ethanol has dried completely.

[0095] Finally, add 30 μL of RNase-free water to the centrifuge tube and pipette repeatedly 10 times to fully resuspend the magnetic beads. Incubate the centrifuge tube in a 55°C metal bath for 5 minutes to promote the elution of microRNA bound to the magnetic beads into the water. After incubation, place the centrifuge tube back on the magnetic rack and let it stand for 2 minutes until the magnetic beads are completely adsorbed. Carefully collect the supernatant with a pipette and transfer it to a new RNase-free centrifuge tube. This supernatant is the purified plasma total microRNA solution.

[0096] 4. Reverse transcription to synthesize complementary deoxyribonucleic acid

[0097] Using the total plasma microRNA solution obtained in step 3 as a template, reverse transcription was performed using stem-loop reverse transcription primers. The total volume of the reverse transcription reaction system was 20 μL, and its specific composition was as follows: 10 μL total plasma microRNA solution, 4 μL reverse transcription buffer at a concentration of 5x, 2 μL deoxyribonucleotide mixture (containing 10 mmol / L deoxyadenosine triphosphate, 10 mmol / L deoxyguanosine triphosphate, 10 mmol / L deoxycytidine triphosphate and 10 mmol / L deoxythymidine triphosphate), 1 μL ribonuclease inhibitor (concentration of 40 units per μL), 1 μL reverse transcriptase (concentration of 200 units per μL), and 2 μL reverse transcription primer mixture.

[0098] The reverse transcription primer mixture contains specific stem-loop primers for three target microRNAs. The three target microRNAs are: thyroid tissue-specific microRNA-1, miR-7-5p, with its specific stem-loop primer sequence shown in SEQ ID NO:1; thyroid tissue-specific microRNA-2, miR-375, with its specific stem-loop primer sequence shown in SEQ ID NO:2; and immunomodulatory microRNA, miR-146a-5p, with its specific stem-loop primer sequence shown in SEQ ID NO:3. The final concentration of each stem-loop primer in the mixture is 0.5 μmol / L.

[0099] After mixing the above reaction mixture, it was placed in a polymerase chain reaction (PCR) instrument for reverse transcription. The reaction program was set as follows: incubation at 16°C for 30 minutes, 42°C for 30 minutes, and 85°C for 5 minutes to inactivate the enzyme, followed by storage at 4°C. After the reaction, complementary deoxyribonucleic acid (DNA) products were obtained.

[0100] 5. Real-time quantitative polymerase chain reaction (qPCR) detection

[0101] Using the complementary deoxyribonucleic acid product obtained in step 4 as a template, the expression levels of miR-7-5p, miR-375, and miR-146a-5p were detected by real-time quantitative polymerase chain reaction (qPCR). Simultaneously, the expression level of the internal reference gene U6 small ribonucleic acid was also detected.

[0102] First, the complementary deoxyribonucleic acid (CDNA) product obtained in step 4 was diluted 10-fold with RNase-free water, and 2 μL of the diluted CDNA product was used as a template. The real-time quantitative polymerase chain reaction (qPCR) system for each microRNA to be tested was 20 μL, specifically composed of: 2 μL of diluted CDNA product, 10 μL of 2-fold concentration qPCR premix (the premix contains hot-start CDNA polymerase, a CDNA mixture, magnesium ions, and reaction buffer), 0.4 μL of 10 μmol / L upstream and downstream specific primers, 0.2 μL of 10 μmol / L specific probe, and 20 μL of RNase-free water.

[0103] The upstream primer sequence for miR-7-5p is 5'-ACACTCCAGCTGGGTGGTAGTTAGTGT-3', the downstream primer sequence is 5'-CTCAACTGGTGTCGTGGA-3', and the specific probe sequence is 5'-FAM-TGGTAGTTAGTGTGTCAG-BHQ1-3'.

[0104] The upstream primer sequence for miR-375 is 5'-ACACTCCAGCTGGGTTTGTTCGTTCGG-3', the downstream primer sequence is 5'-CTCAACTGGTGTCGTGGA-3', and the specific probe sequence is 5'-FAM-TTTGTTCGTTCGGCTCG-BHQ1-3'.

[0105] The upstream primer sequence for miR-146a-5p is 5'-ACACTCCAGCTGGGTGAGAACTGAATTC-3', the downstream primer sequence is 5'-CTCAACTGGTGTCGTGGA-3', and the specific probe sequence is 5'-FAM-TGAGAACTGAATTCCATG-BHQ1-3'.

[0106] The upstream primer sequence for U6 small nucleotide is 5'-CTCGCTTCGGCAGCACA-3', the downstream primer sequence is 5'-AACGCTTCACGAATTTGCGT-3', and the specific probe sequence is 5'-HEX-CACCATCAAGTTGCCC-BHQ1-3'.

[0107] The prepared reaction system was placed in a real-time quantitative polymerase chain reaction (qPCR) instrument for amplification. The reaction program was as follows: pre-denaturation at 95°C for 10 minutes; followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing extension at 60°C for 1 minute; at the end of the 60°C annealing extension phase of each cycle, the instrument automatically acquired the fluorescence signal. After the reaction, a baseline threshold was set according to the amplification curve, and the threshold was set to the value at the start of the exponential growth phase of the fluorescence signal, typically set to 10 times the standard deviation of the background fluorescence signal. The instrument automatically obtained the cycle threshold for each microRNA to be tested and the internal reference gene U6 small nucleotide RNA. In this example, the detection results of the tested individuals at the first time point were: the cycle threshold for miR-7-5p was 28.3, the cycle threshold for miR-375 was 27.9, the cycle threshold for miR-146a-5p was 25.1, and the cycle threshold for U6 small nucleotide RNA was 23.5.

[0108] 6. Relative quantitative analysis

[0109] Calculate the relative expression level of each microRNA to be tested. The relative expression level is calculated using the formula... Calculation, where .

[0110] For miR-7-5p, Its relative expression level .

[0111] For miR-375 Its relative expression level .

[0112] For miR-146a-5p, Its relative expression level .

[0113] 7. Construct a comprehensive diagnostic index

[0114] Based on the relative expression levels of the three microRNAs obtained in step 6, a comprehensive diagnostic index, denoted as DI, is constructed. The comprehensive diagnostic index DI is calculated using the following formula:

[0115]

[0116] in, , and These are the average relative expression levels of miR-7-5p, miR-375, and miR-146a-5p, respectively, in the pre-constructed database of individuals with normal thyroid function established in step 1. In this embodiment, the average relative expression level of miR-7-5p in the pre-constructed database of individuals with normal thyroid function is 0.0521, the average relative expression level of miR-375 is 0.0438, and the average relative expression level of miR-146a-5p is 0.0785.

[0117] Substitute the relative expression level of the individual at the first time point into the formula:

[0118]

[0119] The calculation yields:

[0120]

[0121]

[0122] 8. Dynamic monitoring and diagnostic assessment

[0123] For this individual, a peripheral venous blood sample was collected again on the 16th day after the onset of suspected thyroid dysfunction symptoms, i.e., the 14th day after the first time point, as the second time point. Steps 2 through 7 were repeated to obtain the comprehensive diagnostic index at the second time point. Using the same detection and calculation methods, the detection results of the tested individuals at the second time point were as follows: the cycle threshold for miR-7-5p was 27.1, the cycle threshold for miR-375 was 26.8, the cycle threshold for miR-146a-5p was 23.9, and the cycle threshold for U6 small nucleonucleotide was 23.2. The relative expression levels were calculated as follows: , , Substituting into the comprehensive diagnostic index formula:

[0124]

[0125] The calculation yields:

[0126]

[0127]

[0128] Calculate the dynamic rate of change It can be calculated using the following formula:

[0129]

[0130] Substitute the values:

[0131]

[0132] Based on the preset thresholds determined through receiver operating characteristic (ROC) curve analysis in step 1, in this embodiment: the first preset threshold is -1.20, the second preset threshold is 30.0%, the third preset threshold is -3.50, the fourth preset threshold is -25.0%, and the fifth preset threshold is 15.0%. The comprehensive diagnostic index DI_{T0} of the individual under test at the first time point is -2.4319, which falls between the third preset threshold of -3.50 and the first preset threshold of -1.20; its dynamic change rate... The absolute value of this value is greater than the fifth preset threshold of 15.0%. According to the discrimination rules, the individual does not meet the typical discrimination criteria of "hyperthyroidism", "hypothyroidism" or "normal thyroid function". However, combined with the significant positive increase in its clinical symptoms and dynamic change rate, it is suggested that the individual is in the active period of thyroid function development towards hyperthyroidism.

[0133] To further verify the diagnostic efficacy of this embodiment, the individual was simultaneously subjected to conventional serological hormone testing. The results at the first time point showed: thyroid-stimulating hormone (TSH) level was 0.02 mIU / L, free triiodothyronine (FTT) level was 10.2 pmol / L, and free thyroxine (FTH) level was 28.5 pmol / L. These results met the diagnostic criteria for hyperthyroidism. The results at the second time point showed: TSH level was 0.01 mIU / L, FTT level was 12.5 pmol / L, and FTH level was 31.8 pmol / L. The conventional hormone testing results confirmed the correctness of the diagnostic conclusion of this embodiment and further verified that the dynamic change rate provided in this embodiment can reflect the activity and progression trend of the disease. While conventional hormone testing provided a clear diagnosis at the first time point, it could not provide quantitative indicators of disease activity.

[0134] 9. Comparative Experiment

[0135] To demonstrate the significant advancement of the present invention compared to existing technologies, comparative examples are provided in this embodiment. Comparative Example 1 employs a traditional serological hormone detection method, specifically detecting the levels of thyroid-stimulating hormone (TSH), free triiodothyronine (FTTH), and free thyroxine (FTTH) using an electrochemiluminescence immunoassay analyzer. Comparative Example 2 employs a single microRNA biomarker detection method, specifically detecting only the relative expression level of miR-146a-5p, and diagnosing based on static detection values. The diagnostic threshold is a cutoff value of 0.2500, pre-determined through receiver operating characteristic (ROC) curve analysis. Hyperthyroidism is diagnosed when the relative expression level of miR-146a-5p is greater than 0.2500.

[0136] Thirty patients clinically diagnosed with hyperthyroidism, 30 patients clinically diagnosed with hypothyroidism, and 30 individuals with normal thyroid function were selected. The methods described in this embodiment, Comparative Example 1, and Comparative Example 2 were used for testing, respectively. The diagnostic results of the methods described in this embodiment were determined based on a comprehensive diagnostic index and dynamic change rate. Comparative Example 1 used the levels of thyroid-stimulating hormone (TSH), free triiodothyronine (FT3), and free thyroxine (FT4) as the diagnostic criteria, strictly adhering to clinical diagnostic standards. Comparative Example 2 used only the static relative expression level of miR-146a-5p as the diagnostic criteria. The final clinical diagnosis was used as the gold standard, and the diagnostic sensitivity, specificity, and area under the receiver operating characteristic (AUC) curve for each method were calculated. The results are shown in Table 1.

[0137] Table 1. Comparison of the efficacy of different diagnostic methods

[0138] Method of this embodiment 98.3 96.7 0.992 Comparative Example 1 (Traditional Hormone Testing) 95.0 93.3 0.965 Comparative Example 2 (Static Detection of a Single MicroRNA Marker) 81.7 78.3 0.862

[0139] As shown in Table 1, the method of this embodiment is significantly superior to Comparative Example 1 and Comparative Example 2 in terms of sensitivity, specificity, and area under the receiver operating characteristic (AUC). In particular, compared to Comparative Example 2, this embodiment, by jointly detecting three microRNAs and introducing dynamic monitoring, increased the AUC from 0.862 to 0.992, fully demonstrating the synergistic effect of multi-indicator combination and dynamic monitoring in the technical solution of this invention. Furthermore, although Comparative Example 1 also has high diagnostic efficacy, it cannot provide dynamic disease information and is prone to false positives in patients with non-thyroid disease syndromes. In contrast, the method of this embodiment, by introducing a combination of thyroid tissue-specific microRNAs and immunomodulatory microRNAs, effectively eliminates interference from non-thyroid diseases, further improving specificity.

[0140] In summary, the in vitro diagnostic method for thyroid dysfunction based on novel microRNA biomarkers provided by this invention, by jointly detecting miR-7-5p, miR-375, and miR-146a-5p and introducing dynamic change rates over time, constructs a comprehensive diagnostic index and a dynamic diagnostic model, achieving accurate and dynamic diagnosis of thyroid dysfunction. This method has significant clinical application value and promising prospects for wider application.

[0141] This invention encompasses any substitutions, modifications, equivalent methods, and solutions made within the spirit and scope of this invention. To provide the public with a thorough understanding of this invention, specific details are described in detail in the following preferred embodiments; however, those skilled in the art will fully understand the invention even without these details. Furthermore, to avoid unnecessary misunderstanding of the essence of this invention, well-known methods, processes, procedures, components, and circuits are not described in detail.

[0142] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An in vitro diagnostic method for thyroid dysfunction based on novel microRNA biomarkers, characterized in that, Includes the following steps: Step 1: Collect peripheral venous blood samples from the individual to be tested and separate the plasma samples; Step 2: Extract total microRNA from the plasma sample to obtain a plasma total microRNA solution; Step 3: Using the total plasma microRNA solution as a template, reverse transcription reaction is performed using reverse transcription primers to synthesize complementary deoxyribonucleic acid. The reverse transcription primers contain specific stem-loop primers targeting thyroid tissue-specific microRNA-1, thyroid tissue-specific microRNA-2, and immunomodulatory microRNA. Step 4: Using the complementary deoxyribonucleic acid as a template, the expression levels of the thyroid tissue-specific microRNA-1, the thyroid tissue-specific microRNA-2, and the immunomodulatory microRNA were detected by real-time quantitative polymerase chain reaction technology to obtain their respective cycle thresholds; Step 5: Based on the cycle threshold, calculate the relative expression levels of the thyroid tissue-specific microRNA-1, the thyroid tissue-specific microRNA-2, and the immunomodulatory microRNA; Step 6: Based on the relative expression levels, construct a comprehensive diagnostic index, wherein the comprehensive diagnostic index uses the relative expression levels of thyroid tissue-specific microRNA-1 and thyroid tissue-specific microRNA-2 as positive indicators, and the relative expression levels of immune-regulating microRNAs as negative indicators. Step 7: For the individual to be tested, repeat steps 1 to 6 at the first time point and the second time point respectively to calculate the comprehensive diagnostic index at the first time point and the comprehensive diagnostic index at the second time point, and calculate the dynamic change rate based on the comprehensive diagnostic index at the first time point and the comprehensive diagnostic index at the second time point, and perform a diagnostic assessment based on the comprehensive diagnostic index and the dynamic change rate.

2. The in vitro diagnostic method for thyroid dysfunction based on novel microRNA biomarkers according to claim 1, characterized in that, Step 2, the extraction of total microRNA from the plasma sample, specifically involves: extraction using a magnetic bead extraction kit. 600 μL of lysis binding buffer, containing 4 mol / L guanidine isothiocyanate, 25 mmol / L sodium citrate, and 0.5% sodium dodecyl sarcosinate, is added to 200 μL of plasma sample. The mixture is vortexed for 15 seconds and incubated at room temperature for 5 minutes. Then, 20 μL of magnetic bead suspension (containing silica-modified nanoscale superparamagnetic beads) is added. After vortexing, the mixture is incubated at room temperature on a rotary mixer for 15 minutes. The incubated mixture is then placed on a magnetic rack and incubated for 2 minutes. After the magnetic beads are completely adsorbed, the supernatant is discarded. The magnetic beads are then washed sequentially with 700 μL of washing buffer 1 and 700 μL of washing buffer 2. Washing buffer 1 is a phosphate buffer solution with a concentration of 1:1 (50% anhydrous ethanol), and washing buffer 2 is a phosphate buffer solution with a concentration of 1:1 (75% anhydrous ethanol). After each wash, the magnetic beads are adsorbed using a magnetic rack and the supernatant is discarded. The magnetic beads are left uncapped and dried at room temperature for 8 to 10 minutes. 30 μL of RNase-free water is added, and the mixture is shaken and incubated at 55°C for 5 minutes. The mixture is then placed back on the magnetic rack. After the magnetic beads are completely adsorbed, the supernatant is collected to obtain the purified plasma total microRNA solution.

3. The in vitro diagnostic method for thyroid dysfunction based on novel microRNA biomarkers according to claim 1, characterized in that, In step 3, the thyroid tissue-specific microRNA-1 is miR-7-5p, the thyroid tissue-specific microRNA-2 is miR-375, and the immunomodulatory microRNA is miR-146a-5p. The total volume of the reverse transcription reaction system is 20 μL, containing: 10 μL of the total plasma microRNA solution, 4 μL of 5-fold concentration reverse transcription buffer, 2 μL of deoxyribonucleic acid mixture, 1 μL of ribonuclease inhibitor, 1 μL of reverse transcriptase, and 2 μL of reverse transcription primer mixture. The reverse transcription primer mixture contains specific stem-loop primers for miR-7-5p, specific stem-loop primers for miR-375, and specific stem-loop primers for miR-146a-5p. The reverse transcription reaction program is: incubation at 16°C for 30 minutes, incubation at 42°C for 30 minutes, incubation at 85°C for 5 minutes, and finally storage at 4°C to obtain complementary deoxyribonucleic acid products.

4. The in vitro diagnostic method for thyroid dysfunction based on novel microRNA biomarkers according to claim 1, characterized in that, In step 4, during the real-time quantitative polymerase chain reaction (qPCR) detection, the expression level of the internal reference gene, U6 small nucleotide, is simultaneously detected. Each test microRNA's qPCR system is 20 μL, containing: 2 μL of 10-fold diluted complementary deoxyribonucleic acid product, 10 μL of 2-fold concentrated qPCR premix, 0.4 μL of 10 μmol / L upstream and downstream specific primers, 0.2 μL of 10 μmol / L specific probe, and RNase-free water to a final volume of 20 μL. The qPCR program is as follows: 95°C pre-denaturation for 10 minutes; followed by 40 cycles of 95°C denaturation for 15 seconds and 60°C annealing extension for 1 minute; fluorescence signals are collected at the end of the 60°C annealing extension phase of each cycle; after the reaction, a baseline threshold is set based on the amplification curve to obtain the cycle threshold for each test microRNA and the cycle threshold for the U6 small nucleotide.

5. The in vitro diagnostic method for thyroid dysfunction based on novel microRNA biomarkers according to claim 4, characterized in that, Step 5, which involves calculating the relative expression levels of thyroid tissue-specific microRNA-1, thyroid tissue-specific microRNA-2, and the immunomodulatory microRNA, specifically employs the following formula: Calculate the relative expression level of each microRNA to be tested, where The relative expression levels of thyroid tissue-specific microRNA-1 were obtained through the above calculations and denoted as follows: The relative expression level of the thyroid tissue-specific microRNA-2 is denoted as The relative expression level of the immune regulatory microRNA is denoted as .

6. The in vitro diagnostic method for thyroid dysfunction based on novel microRNA biomarkers according to claim 5, characterized in that, The comprehensive diagnostic index mentioned in step 6 is calculated using the following formula: in, , and These are the average relative expression levels of the thyroid tissue-specific microRNA-1, the thyroid tissue-specific microRNA-2, and the immunomodulatory microRNA, respectively, in a pre-constructed database of individuals with normal thyroid function.

7. The in vitro diagnostic method for thyroid dysfunction based on novel microRNA biomarkers according to claim 6, characterized in that, In step 7, the first time point is the initial visit, and the second time point is 14 days after the initial visit; the dynamic change rate Calculated using the following formula: in, This refers to the comprehensive diagnostic index at the first time point. The comprehensive diagnostic index at the second time point; the judgment rule for the diagnostic assessment is: if Greater than the first preset threshold and If the value exceeds the second preset threshold, a diagnosis of hyperthyroidism is made; if... Less than the third preset threshold and If the value is less than the fourth preset threshold, a diagnosis of hypothyroidism is made; if... Between the third preset threshold and the first preset threshold and If the absolute value is less than the fifth preset threshold, the diagnosis is normal thyroid function; the first preset threshold, the second preset threshold, the third preset threshold, the fourth preset threshold and the fifth preset threshold are cutoff values ​​determined by receiver operating characteristic curve analysis based on the distribution of the comprehensive diagnostic index and its dynamic change rate of pre-constructed groups of individuals with normal thyroid function, patients with hypothyroidism and patients with hyperthyroidism.

8. The in vitro diagnostic method for thyroid dysfunction based on novel microRNA biomarkers according to claim 7, characterized in that, The step 1 involves collecting peripheral venous blood samples from the individuals to be tested and separating the plasma samples. Specifically, the collected peripheral venous blood samples are placed in anticoagulant blood collection tubes containing dipotassium EDTA. Within 2 hours of collection, the samples are centrifuged at 4 degrees Celsius and 1600 times relative centrifugation for 15 minutes to separate the upper plasma sample. The plasma sample is then aliquoted into RNase-free cryovials and stored at -80 degrees Celsius for later use. Before testing, the frozen plasma samples are slowly thawed on ice and then centrifuged at 4 degrees Celsius and 12000 times relative centrifugation for 10 minutes. The supernatant is then used for subsequent microRNA extraction.

9. The in vitro diagnostic method for thyroid dysfunction based on novel microRNA biomarkers according to claim 1, characterized in that, Before step 1, a control sample preparation step is included: peripheral venous blood samples are collected from at least 30 clinically diagnosed individuals with normal thyroid function, at least 30 clinically diagnosed patients with hypothyroidism, and at least 30 clinically diagnosed patients with hyperthyroidism. These samples are processed according to the methods in steps 1 to 5 to obtain a database of the relative expression levels of thyroid tissue-specific microRNA-1, thyroid tissue-specific microRNA-2, and immunomodulatory microRNAs in the individuals with normal thyroid function, and the mean and standard deviation of the relative expression levels of each microRNA are calculated. At the same time, a database of the relative expression levels of thyroid tissue-specific microRNA-1, thyroid tissue-specific microRNA-2, and immunomodulatory microRNAs in the patients with hypothyroidism and hyperthyroidism is obtained. Based on the above database, a first preset threshold, a second preset threshold, a third preset threshold, a fourth preset threshold, and a fifth preset threshold are determined through receiver operating characteristic (ROC) curve analysis.

10. The in vitro diagnostic method for thyroid dysfunction based on novel microRNA biomarkers according to claim 1, characterized in that, The individuals to be tested are those with suspected symptoms of thyroid dysfunction, including at least one of palpitations, hand tremors, weight change exceeding 5% of original weight within one month, neck thickening, heat intolerance and excessive sweating or cold intolerance and reduced sweating; the first time point is any day from the 1st to the 3rd day after the appearance of the suspected symptoms of thyroid dysfunction, the second time point is any day from the 12th to the 16th day after the first time point, and the interval between the second time point and the first time point is 14 days.

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