Intestinal microbial marker for papillary thyroid carcinoma and application of intestinal microbial marker

Abstract

The invention relates to the technical field of biological medicine, in particular to an intestinal microbial marker for thyroid cancer and application of the intestinal microbial marker. According to the application, a metagenome sequencing method and a metabonomic sequencing method are used for finding out three types of specific bacteria (sBacteroidella CAG.47, sBacteroidella sp.3113 and sBacteroidella sp.3140A), and a combination of specific metabolites Deoxycholic acid (deoxycholic acid) is used for diagnosing papillary thyroid carcinoma, so that the papillary thyroid carcinoma can be diagnosed, and the papillary thyroid carcinoma can be diagnosed by using the combination of the three types of specific bacteria (sBacteroidella CAG.47, sBacteroidella sp.3140A) and the specific metabolites (Deoxycholic acid) of the specific bacteria (sBacteroidella CAG.47, sBacteroidella sp.3140A). According to the intestinal microbial marker for papillary thyroid carcinoma, high-risk groups of papillary thyroid carcinoma can be effectively and early screened or early patients can be found, so that further expansion of papillary thyroid carcinoma can be prevented as early as possible, the rupture risk can be reduced, and the treatment effect of papillary thyroid carcinoma can be monitored. Meanwhile, the intestinal microbial marker can be used for preparing diagnostic kits and therapeutic drugs. The invention overcomes the defects that the existing papillary thyroid carcinoma diagnosis cannot realize early screening, cannot predict the attack and development trend of papillary thyroid carcinoma and the like.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and specifically to intestinal microbial markers for papillary thyroid carcinoma and their applications. Background Art

[0002] Thyroid carcinoma (TC) is the most common malignant tumor of the endocrine system and head and neck. Patients with thyroid cancer often have no obvious symptoms or signs in the early stages. Traditional noninvasive diagnosis relies on regular and long-term imaging follow-up and blood test indicators, while invasive diagnosis relies on ultrasound-guided puncture biopsy and pathological results. Ultrasound only determines the morphological characteristics of the nodule, and its diagnostic accuracy is related to factors such as the experience and technical level of the ultrasound physician and the resolution of the ultrasound instrument. Blood test indicators are more often used to determine postoperative prognosis and lack diagnostic value for early screening. As an invasive procedure, puncture biopsy carries certain risks and pain. Therefore, based on traditional technologies, it is worth exploring the establishment of a simpler noninvasive auxiliary screening and identification model for thyroid cancer.

[0003] Current research has found a correlation between various thyroid diseases, including Graves' disease (GD), Hashimoto's thyroiditis (HT), and hypothyroidism, and gut microbial imbalances. Currently, there are no reliable metagenomic-based gut microbial biomarkers for thyroid cancer. Furthermore, existing diagnostic criteria cannot predict the onset of thyroid cancer early. Summary of the Invention

[0004] The present invention aims to address the deficiencies of the existing technology and provide intestinal microbial markers for thyroid cancer and their applications, which can achieve early screening of thyroid cancer, assist in the pathological typing of the disease, and conduct research on drug targets and pathogenesis.

[0005] The present invention provides an intestinal microbial marker for papillary thyroid carcinoma, which includes bacteria s__Bacteroides_fragilis_CAG.47, s__Bacteroides_sp._3_1_13, s__Bacteroides_sp._3_1_40A and the metabolite Deoxycholic.acid.

[0006] Based on the above markers, the present invention provides an application of an intestinal microbial marker in the preparation of an early screening tool for papillary thyroid carcinoma.

[0007] The early screening tool for papillary thyroid cancer is a papillary thyroid cancer early screening kit, which contains detection reagents for detecting the above-mentioned intestinal microbial markers.

[0008] The present invention provides an application of an intestinal microbial marker in the preparation of a drug for treating papillary thyroid carcinoma, specifically the preparation or screening of a drug for treating papillary thyroid carcinoma using the intestinal microbial marker as a target.

[0009] The present invention provides an application of an intestinal microbial marker in predicting the individual treatment effect of patients with papillary thyroid carcinoma.

[0010] The present invention also provides an early screening method for papillary thyroid cancer. By performing gene sequencing on the intestinal microbiome of the test subject, the abundance of bacteria s__Bacteroides_fragilis_CAG.47, s__Bacteroides_sp._3_1_13, and s__Bacteroides_sp._3_1_40A in fecal samples is detected. When the total abundance of the three is lower than that of the healthy control group (p<0.01), and the area under the receiver operating characteristic curve of the combined detection of the three is ≥0.90, the patient is judged to be at high risk or in the early stage of papillary thyroid cancer.

[0011] This application uses metagenomic sequencing and metabolomics sequencing methods to find out three specific bacteria (s__Bacteroides_fragilis_CAG.47, s__Bacteroides_sp._3_1_13, s__Bacteroides_sp._3_1_40A) and a specific metabolite Deoxycholic.acid (deoxycholic acid) for the diagnosis of papillary thyroid carcinoma. According to the intestinal microbial markers of the above-mentioned papillary thyroid carcinoma, it is possible to effectively screen the high-risk population of papillary thyroid carcinoma or find early patients at an early stage, so as to prevent the further expansion of papillary thyroid carcinoma and reduce the risk of rupture as early as possible, and monitor the therapeutic effect of papillary thyroid carcinoma. At the same time, the intestinal microbial marker can be used to prepare diagnostic kits and therapeutic drugs. The present invention overcomes the shortcomings of existing papillary thyroid carcinoma diagnosis that cannot achieve early screening, cannot predict the incidence and development trend of papillary thyroid carcinoma, etc., can help disease pathology typing and drug target research, precision medication, pathogenesis research, etc., and provide new treatment options. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The specific implementation of this application is further described in detail below with reference to the accompanying drawings.

[0013] Figure 1 Correlation heatmap constructed for the relative abundance of differentially expressed bacterial species and fecal metabolites in this study.

[0014] Figure 2The areas under the ROC curves obtained based on specific bacteria used in the present invention were: s__Bacteroides_fragilis_CAG.47 (N1), s__Bacteroides_sp._3_1_13 (N2), and s__Bacteroides_sp._3_1_40A (N3), with AUCs of 0.933, 0.9, and 0.867, respectively.

[0015] Figure 3 The area under the ROC curve obtained for the specific metabolite used in the present invention was: Deoxycholic acid AUC was 0.9.

[0016] Figure 4 and Figure 5 The figure shows the comparison of tumor volumes of papillary thyroid carcinoma in nude mice before and after the intervention of deoxycholic acid of the present invention. DETAILED DESCRIPTION

[0017] The present invention aims to address the deficiencies of the existing technology and provide intestinal microbial markers for thyroid cancer and their applications, which can achieve early screening of thyroid cancer, assist in the pathological typing of the disease, and conduct research on drug targets and pathogenesis.

[0018] The present application is further described below with reference to specific embodiments, but the scope of protection of the present application is not limited thereto:

[0019] The present invention provides an intestinal microbial marker for papillary thyroid carcinoma, which includes bacteria s__Bacteroides_fragilis_CAG.47, s__Bacteroides_sp._3_1_13, s__Bacteroides_sp._3_1_40A and the metabolite Deoxycholic.acid.

[0020] Numerous experimental studies have demonstrated that the use of these specific bacteria and metabolites for diagnosis has high diagnostic and specificity. The areas under the receiver operating characteristic (ROC) curves (AUCs) for the bacteria s__Bacteroides_fragilis_CAG.47, s__Bacteroides_sp._3_1_13, and s__Bacteroides_sp._3_1_40A achieved discriminatory powers of 0.933, 0.9, and 0.867, respectively. The metabolite deoxycholic acid achieved an AUC of 0.9. Furthermore, oral administration of deoxycholic acid in a nude mouse thyroid cancer model significantly increased thyroid tumor size, further confirming the diagnostic value of this metabolite.

[0021] Based on the above markers, an application of an intestinal microbial marker in the preparation of an early screening tool for papillary thyroid cancer is provided.

[0022] Preferably, the early screening tool for papillary thyroid carcinoma is a papillary thyroid carcinoma early screening kit, which contains a detection reagent for detecting the aforementioned intestinal microbial markers. Using this kit to detect marker abundance and compare it with a predetermined value, early screening or prediction of papillary thyroid carcinoma can be performed on the subject.

[0023] The present invention provides an application of an intestinal microbial marker in the preparation of a drug for treating papillary thyroid carcinoma. Specifically, the intestinal microbial marker is used as a target for the preparation or screening of a drug for treating papillary thyroid carcinoma. The effect of a candidate drug on the intestinal microbial marker of papillary thyroid carcinoma before and after use can also be used to determine whether the candidate drug can be used to treat or prevent papillary thyroid carcinoma.

[0024] The present invention provides an application of an intestinal marker in predicting the individual treatment effect of patients with papillary thyroid carcinoma.

[0025] The present invention also provides a method for early screening of papillary thyroid cancer, which involves performing genetic sequencing on the intestinal microbiome of the test subject, detecting the abundance of specific bacteria in fecal samples, and determining whether the patient is a high-risk or early-stage patient of papillary thyroid cancer based on the abundance difference and the area under the ROC curve of the combined detection.

[0026] In addition, the present invention provides a method for determining intestinal microbial markers for papillary thyroid carcinoma, by obtaining fecal samples for metagenomic sequencing, data processing, model training and screening, etc., to determine intestinal microbial markers for diagnosis.

[0027] Example

[0028] 1. Identifying gut microbial markers for papillary thyroid cancer

[0029] Sample Acquisition and Processing: Stool samples were obtained from 31 healthy individuals and 33 patients with papillary thyroid cancer. DNA extracted from the stool samples was sheared into approximately 400-bp fragments using a Covaris M220 sequencer (Covaris, USA). Polymerase chain reaction (PE) libraries were constructed and amplified using bridge PCR. Metagenomic sequencing was performed using the PE150 method. Raw data were quality-controlled and host-free using kneaddata. Reads with an average base weight of less than 20 were removed. Species annotation was performed using Kraken2, and the relative abundance of each species in each sample was calculated.

[0030] Model training and screening: The healthy people and thyroid papillary carcinoma patients were numbered, and 30 random numbers were generated by R language. The corresponding number of people was used as the training set, and the remaining 34 healthy people and thyroid papillary carcinoma patients were used as the test set. The training set was trained by machine learning random forest model, and the test set was classified and predicted to preliminarily evaluate the model. If the prediction accuracy is less than 75%, the random forest model parameters are optimized and retrained until the prediction accuracy is higher than 75%. Ten-fold cross-validation is performed to view the corresponding feature number when the minimum error rate is obtained. According to the ten-fold cross-validation results and the Mean Decrease Accuracy importance score, the intestinal microbial markers are selected. When the number of microorganisms is 32, the error rate is the lowest, which is the best model. The top 20 intestinal microorganisms ranked in descending order of Mean Decrease Accuracy are selected as the intestinal microbial markers of thyroid papillary carcinoma.

[0031] Specifically as follows:

[0032] s__Bacteroides_fragilis_CAG:47

[0033] s__Bacteroides_sp._3_1_13

[0034] s__Bacteroides_sp._3_1_40A

[0035] s__Bacteroides_vulgatus

[0036] s__Bacteroides_vulgatus_CAG:6

[0037] s__Bacteroides_xylanisolvens

[0038] s__Blautia_producta

[0039] s__Blautia_sp._CAG:37

[0040] s__Butyricicoccus_pullicaecorum

[0041] s__Clostridia_bacterium_UC5.1-1D10

[0042] s__Clostridium_sp._CAG:226

[0043] s__Clostridium_sp._CAG:269

[0044] s_Eubacterium desmolans

[0045] s_Firmicutes bacterium CAG:41

[0046] s_Fusicatenibacter saccharivorans

[0047] s_Marvinbryantia formatexigens

[0048] s_Roseburia hominis

[0049] s_Roseburia sp. CAG:47

[0050] s_unclassified g_Bacteroides

[0051] s_unclassified o_Bacteroidales

[0052] 2. Validation of the diagnostic value of the markers

[0053] Correlation analysis: Spearman correlation analysis was performed on the top 20 specific bacterial species and metabolites, and the results were shown in a heatmap as shown in Figure 1 Spearman correlation analysis results between the 20 most abundant bacteria species specific to the thyroid cancer group and the 41 fecal non-target metabolites specific to the thyroid cancer group. Red indicates positive correlation, blue indicates negative correlation. The depth of color indicates the size of the correlation, where darker colors indicate stronger correlation than lighter colors, *p<0.05, **p<0.01.

[0054] At the same time, intestinal microbial metabolites were detected in both groups, a total of 8169 metabolites were detected, of which 41 metabolites were different between the two groups. Using Spearman correlation analysis to evaluate the correlation between the 41 different metabolites in the sample and the 20 most different and abundant bacterial species, it was determined that deoxycholic acid, the most specific, was positively and strongly correlated with the following 8 bacterial species, most of which were Bacteroides:

[0055] s_Bacteroides fragilis CAG.47

[0056] s_Bacteroides sp. 3_1_13

[0057] s__Bacteroides_sp._3_1_40A

[0058] s__Bacteroides_vulgatus

[0059] s__Bacteroides_vulgatus_CAG.6

[0060] s__Bacteroides_xylanisolvens

[0061] s__unclassified_g__Bacteroides

[0062] s__unclassified_o__Bacteroidales

[0063] Among them, such as Figure 2 and 3 As shown, the discriminatory power of the areas under the receiver operating characteristic (ROC) curve (AUC) for bacteria s__Bacteroides_fragilis_CAG.47, s__Bacteroides_sp._3_1_13, and s__Bacteroides_sp._3_1_40A were 0.933, 0.9, and 0.867, respectively. The discriminatory power of the area under the ROC curve for the metabolite deoxycholic acid was 0.9, indicating that the specific intestinal marker has diagnostic value.

[0064] Animal experiment verification: Figure 4 and 5 As shown in the figure, the metabolite Deoxycholic acid was gavaged into nude mice to induce thyroid papillary carcinoma. The results showed that the tumor volume was significantly increased compared with the control group, proving its diagnostic value.

[0065] 3. Early screening for papillary thyroid cancer: Gene sequencing of the intestinal microbiota isolated from the test subjects was performed to detect the total abundance of bacteria s__Bacteroides_fragilis_CAG.47, s__Bacteroides_sp._3_1_13, and s__Bacteroides_sp._3_1_40A in fecal samples of patients with papillary thyroid cancer. The total abundance of bacteria s__Bacteroides_fragilis_CAG.47, s__Bacteroides_sp._3_1_13, and s__Bacteroides_sp._3_1_40A was significantly lower in fecal samples of patients with papillary thyroid cancer than in the healthy control group (p<0.01). When the area under the receiver operating characteristic (ROC) curve (AUC) of the combined detection of the three was ≥0.90, the patients were identified as high-risk or early-stage patients for papillary thyroid cancer, and early screening for papillary thyroid cancer was performed.

[0066] 4. Preparation of an early screening kit for papillary thyroid carcinoma: Combining detection reagents capable of detecting the above-mentioned intestinal microbial markers into an early screening kit for papillary thyroid carcinoma, using the kit to detect the abundance of intestinal microbial markers of papillary thyroid carcinoma, and comparing the obtained abundance value with the predetermined value to perform early screening or prediction of papillary thyroid carcinoma in the test subject.

Claims

1. A gut microbial marker for papillary thyroid carcinoma, characterized in that: The markers include bacteria s__Bacteroides_fragilis_CAG.47, s__Bacteroides_sp._3_1_13, s__Bacteroides_sp._3_1_40A and the metabolite deoxycholic acid.

2. Application of an intestinal microbial marker in the preparation of an early screening tool for papillary thyroid cancer.

3. The use according to claim 2, characterized in that The early screening tool for papillary thyroid carcinoma is a papillary thyroid carcinoma early screening kit, which contains a detection reagent for detecting the intestinal microbial marker according to claim 1.

4. Application of an intestinal microbial marker in the preparation of a drug for the treatment of papillary thyroid carcinoma.

5. The use according to claim 4, characterized in that The intestinal microbial markers are used as targets for the preparation or screening of drugs for treating papillary thyroid cancer.

6. Application of a gut microbial marker in predicting individual treatment outcomes in patients with papillary thyroid carcinoma.

7. A method for early screening of papillary thyroid carcinoma, characterized in that: The intestinal microbiota of the test subjects was sequenced, and the abundance of bacteria s__Bacteroides_fragilis_CAG.47, s__Bacteroides_sp._3_1_13, and s__Bacteroides_sp._3_1_40A in fecal samples was detected. When the total abundance of the three was lower than that of the healthy control group (p<0.01) and the area under the receiver operating characteristic curve of the combined detection of the three was ≥0.90, the patients were judged to be high-risk or early-stage patients with papillary thyroid cancer.

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