CN108929909A - Screening kit for metastatic screening of thyroid papillary carcinoma - Google Patents

Abstract

The invention discloses a metastatic screening kit for papillary thyroid microcarcinoma and its application. The present invention effectively determines whether the test subject suffers from metastatic papillary thyroid microcarcinoma, and recommends early surgery for those who are positive, and continues to observe if the test is negative, avoiding over-treatment and under-treatment of thyroid microcarcinoma, and has excellent clinical application prospects.

Description

A screening kit for metastatic screening of papillary thyroid microcarcinoma

technical field

The invention relates to a detection method for thyroid micropapillary carcinoma.

Background technique

The incidence of thyroid cancer is increasing rapidly around the world, and it is the solid malignant tumor with the fastest annual growth rate. It is estimated that by 2019, thyroid cancer will become the third most common malignant tumor in women. Most of these new cases are papillary thyroid microcarcinoma (PTMC), that is, papillary thyroid cancer with tumor diameter ≤1cm. Depending on the country, PTMCs account for between 40% and 50% of new thyroid cancers. At present, whether there is overdiagnosis and treatment of papillary thyroid microcarcinoma patients at home and abroad is one of the hottest and most controversial topics in thyroid surgery: Should PTMC patients choose early surgery or continue to observe? However, at present, there is no method at home and abroad to accurately judge the aggressiveness and prognosis of PTMCs before surgery, and it is also impossible to predict in advance which PTMCs will cause greater harm to patients and require timely treatment.

Transcriptomics (RNA-seq) is to comprehensively and accurately identify transcript regulation, alternative splicing, gene fusion, etc. in tumors, and analyze the important role of frequent gene fusions in tumorigenesis and development. Transcriptomics, a molecular signature based on gene expression profiles, can not only identify the phenotype of cells, but also be used for disease diagnosis. So far, there are no transcriptomic studies and literature reports related to papillary thyroid carcinoma (PTC) and papillary thyroid microcarcinoma (PTMC) (tumor diameter ≤ 1 cm). Transcriptomics requires relatively low sample size, and a small amount of tissue can be detected, which is especially suitable for PTMC research.

Therefore, if an indicator can be found to accurately predict the high metastasis and poor prognosis of PTMC in advance, timely surgical treatment for high metastatic PTMC can achieve the best prognosis, and for relatively indolent PTMC, just choose observation. In this way, excessive treatment of PTMC can be avoided, which can not only reduce the psychological burden of patients, but also reduce the economic burden of the country and society, and can make more reasonable use of expensive and limited medical resources.

Contents of the invention

In order to solve the above problems, the present invention provides a method for determining whether a detection object (papillary thyroid microcarcinoma) is in an abnormal state (highly metastatic or poor prognosis).

The present invention discovers a biomarker for detecting the development process and prognosis judgment of papillary thyroid microcarcinoma markers.

The invention provides a metastatic screening kit of papillary thyroid microcarcinoma, which includes optional reagents for detecting the expression level of TESC in papillary thyroid microcarcinoma tissue.

TESC: tescalcin [Homo sapiens (human)],

Ensembl: ENSG00000088992MIM: 611585; Vega: OTTHUMG00000144168.

Preferably, the reagent for detecting the expression level of TESC in papillary thyroid microcarcinoma tissue is a reagent for PCR detection.

Preferably, the reagent for PCR detection comprises a pair of primers shown in SEQ ID NO.1-2.

Preferably, the reagent for detecting the expression level of TESC in papillary thyroid microcarcinoma tissue is a reagent for Western-blot detection method.

The present invention also provides the use of the reagent for detecting the expression level of TESC in papillary thyroid microcarcinoma tissue in the preparation of metastatic screening reagents for papillary thyroid microcarcinoma.

Preferably, the reagent for detecting the expression level of TESC in papillary thyroid microcarcinoma tissue is a reagent for PCR detection.

Preferably, the reagent for PCR detection comprises a pair of primers shown in SEQ ID NO.1-2.

Preferably, the reagent for detecting the expression level of TESC in papillary thyroid microcarcinoma tissue is a reagent for Western-blot detection method.

The kit of the present invention detects the expression level of TESC in the tumor tissues of patients with papillary thyroid microcarcinoma to be detected, screens out highly metastatic patients, and timely surgically treats highly metastatic PTMCs to achieve the best prognosis. PTMC, just select Observation. In this way, excessive treatment of PTMC can be avoided, which can not only reduce the psychological burden of patients, but also reduce the economic burden of the country and society, and can make more reasonable use of expensive and limited medical resources.

Apparently, according to the above content of the present invention, according to common technical knowledge and conventional means in this field, without departing from the above basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above-mentioned content of the present invention will be further described in detail below through specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Description of drawings

Figure 1 TESC expression level detection results, a: real-time quantitative PCR detection of TESC RNA expression level in tissues; b: detection of TESC protein expression level in 2 cases of highly metastatic tumor tissues and 3 cases of non-metastatic tumor tissues by Western method; c: Immunohistochemical detection of protein expression levels of TESC in high-metastatic and non-metastatic tumor tissues Differentially expressed genes in high-metastatic and non-metastatic groups.

Detailed ways

Example 1 The relationship between the expression level of TESC and the status of papillary thyroid microcarcinoma

1. Pathology Collection

Collected PTMC cases from December 2016 to May 2018 in West China Hospital of Sichuan University, and signed informed consent for specimen collection before operation. Those who met the above conditions were routinely collected surgically resected pathological tissue samples. Within 15 minutes after excising the glandular lobe where the cancer focus is located, the cancer focus and normal tissue are collected and filled with liquid nitrogen, and the patient’s information is registered in detail to make a record. After a few months, a pathological test is performed to determine whether there is high lymph node metastasis or no metastasis. Strictly select and include The expression level of TESC was detected in the tumor tissues of the non-metastasis and high-metastasis groups, 35 cases without metastases and 26 cases with high metastases.

The criteria for sample inclusion are as follows:

①Papillary microcarcinoma in the left or right lobe of the thyroid gland (because the isthmus is thin, TMC can easily invade the capsule, so isthmus cancer is ruled out), and color Doppler ultrasound shows that the maximum diameter of the nodules is between 5-10mm;

② Non-pregnant or breastfeeding women (because the incidence rate of women is significantly higher than that of men, and in order to avoid the interference of sex hormones and other factors, transcriptome sequencing only includes female patients, and male patients are used for later functional verification);

③The cases were all operated for the first time.

2. Detection of expression level of TESC

2.1 Fluorescent real-time quantitative PCR detection of RNA expression level of target gene in tissue

Take out the tissue stored in liquid nitrogen, place it on ice for 5 minutes to soften it, pulverize it with a tissue homogenizer, weigh it, and add 1ml TRIZOL per 100mg tissue. Add 0.2ml of chloroform to every 1ml of TRIZOL reagent lysed sample, and cap the tube tightly. Shake the tube vigorously by hand for 15 seconds, and incubate at 15 to 30°C for 2 to 3 minutes. Centrifuge at 12000 rpm for 15 minutes at 4°C. After centrifugation, the mixed liquid will be divided into the lower red phenol chloroform phase, the middle layer and the upper layer of the colorless aqueous phase. The RNA was all partitioned into the aqueous phase. The volume of the upper layer of the aqueous phase is about 60% of the TRIZOL reagent added during homogenization. Transfer the upper aqueous phase to a clean RNase-free centrifuge tube. Add an equal volume of isopropanol and mix to precipitate the RNA. After mixing, incubate at 15 to 30°C for 10 minutes, then centrifuge at 12000rpm at 4°C for 10 minutes. At this point the RNA pellet that was not visible prior to centrifugation will form a gelatinous pellet on the bottom and sides of the tube. Remove the supernatant, and add at least 1 ml of 75% ethanol (75% ethanol prepared with DEPCH2O) to every 1 ml of the sample lysed by TRIZOL reagent to wash the RNA precipitate. After mixing, centrifuge at 7000 rpm for 5 minutes at 4°C. Carefully aspirate most of the ethanol solution and allow the RNA pellet to air dry for 5-10 min at room temperature. When dissolving RNA, first add 40 μl of RNase-free water and blow it several times with a gun repeatedly to dissolve it completely, and store the obtained RNA solution at -80°C until use. Agilent 2100Bioanalyzer (Agilent RNA 6000 Nano Kit) was used to detect the concentration of total RNA.

Synthesis of sample cDNA: Synthesize using kit with product number Femantas K1631, reverse transcription buffer 4 μl, random primer 0.2 μl, reverse transcriptase 0.5 μl, DEPC water 13.3 μl, RNA template 2 μl, total volume 20 μl. Mix the solution by flicking the bottom of the tube and centrifuge briefly at 6000 rpm. The mixture was firstly dried in a 70°C dry bath for 3 minutes, then in a 37°C water bath for 60 minutes. Immediately after taking it out, put it in a dry bath at 95°C for 3 minutes to obtain the final reverse transcription solution, which is the cDNA solution, and store it at -80°C until use.

Fluorescent real-time quantitative PCR: Reaction system: 2×PCR enzyme Mix 10ul, upstream primer F 0.5ul, downstream primer R 0.5ul, add water to a total volume of 20ul. Reaction steps: 45 PCR cycles (94° C. for 20 seconds; 60° C. for 5 seconds). After completing the reaction, record the Ct value and calculate the expression level. The kit used is bio-rad ssofast evagreen, product number 172-5204AP.

The primer sequences are as follows:

TESC: Forward: TCAACCCCATCCGATCCAAA

Reverse: ATCTCAGCTTCTCCTTCCGG

2.2 Western method (biorad 1620177, tesc antibody purchased from Novus, product number nbp2-13426) was used to detect the protein expression level of TESC in 2 cases of highly metastatic tissues and 3 cases of non-metastatic tumor tissues.

3 The relationship between the expression level of TESC and the status of papillary thyroid microcarcinoma

3.1 Fluorescent real-time quantitative PCR detection of RNA expression level of target gene in tissue

The experimental results are shown in Figure 1a and Table 1:

Table 1 Correlation between TESC in tissue and papillary thyroid microcarcinoma status

Average expression level of TESC Non-metastatic papillary thyroid microcarcinoma samples 1.83 copy/ng cDNA Highly metastatic samples of papillary thyroid microcarcinoma 8.55 copy/ng cDNA

As shown in Table 1 and Figure 1a, among patients with papillary thyroid microcarcinoma, the expression level of TESC in non-metastatic patients was low, while the expression level of TESC in high-metastatic patients was significantly higher, and the difference between the two was significant.

In actual testing, if the expression level of TESC in the sample to be tested is found to be greater than 8.55 copy/ng cDNA, surgery is recommended; if it is 1.83 copy/ng cDNA to 8.55 copy/ng cDNA, close follow-up and reexamination are recommended to monitor the expression level of TESC , surgery if necessary, if it is less than 1.83 copy/ng cDNA, it is recommended to observe.

2.2 Western method was used to detect the protein expression level of TESC in 2 high-metastatic tissues and 3 non-metastatic tumor tissues.

As shown in Figure 1b and c, in patients with papillary thyroid microcarcinoma, the average protein expression level of TESC in non-metastatic patients was low, while the protein expression level of TESC in high metastatic patients was significantly higher, and the difference between the two was significant.

From the above results, it can be seen that compared with non-metastatic papillary thyroid carcinoma patients, the RNA expression level and protein expression level of TESC in patients with high papillary thyroid carcinoma metastases were significantly increased, indicating that the metastatic nature of papillary thyroid microcarcinoma It is positively correlated with the expression level of TESC in tumor tissue, and the high expression of TESC will significantly increase the possibility of high metastatic nature of papillary thyroid microcarcinoma.

Therefore, the highly metastatic patients can be screened out by detecting the expression level of TESC in the tumor tissues of the papillary thyroid microcarcinoma patients.

Embodiment 2 The present invention detects the composition of the kit of TESC and using method thereof

1. PCR detection kit

1. The composition of the kit

Detection kit (50 copies):

components volume upstream primer 0.5ul (10μM) downstream primer 0.5ul (10μM) 2×PCR Enzyme Mix 10 μL dd water to a total volume of 20 μL

The primer sequences are as follows:

TESC: Forward: TCAACCCCATCCGATCCAAA

Reverse: ATCTCAGCTTCTCCTTCCGG

2. How to use the kit

Take out the tumor tissue of the sample to be tested, place it on ice for 5 minutes to soften it, pulverize it with a tissue homogenizer, weigh it, and add 1ml TRIZOL per 100mg tissue. Add 0.2ml of chloroform to every 1ml of TRIZOL reagent lysed sample, and cap the tube tightly. Shake the tube vigorously by hand for 15 seconds, and incubate at 15 to 30°C for 2 to 3 minutes. Centrifuge at 12000 rpm for 15 minutes at 4°C. After centrifugation, the mixed liquid will be divided into the lower red phenol chloroform phase, the middle layer and the upper layer of the colorless aqueous phase. The RNA was all partitioned into the aqueous phase. The volume of the upper layer of the aqueous phase is about 60% of the TRIZOL reagent added during homogenization. Transfer the upper aqueous phase to a clean RNase-free centrifuge tube. Add an equal volume of isopropanol and mix to precipitate the RNA. After mixing, incubate at 15 to 30°C for 10 minutes, then centrifuge at 12000rpm at 4°C for 10 minutes. At this point the RNA pellet that was not visible prior to centrifugation will form a gelatinous pellet on the bottom and sides of the tube. Remove the supernatant, and add at least 1 ml of 75% ethanol (75% ethanol prepared with DEPCH2O) to every 1 ml of the sample lysed by TRIZOL reagent to wash the RNA precipitate. After mixing, centrifuge at 7000 rpm for 5 minutes at 4°C. Carefully aspirate most of the ethanol solution and allow the RNA pellet to air dry for 5-10 min at room temperature. When dissolving RNA, first add 40 μl of RNase-free water and blow it several times with a gun repeatedly to dissolve it completely, and store the obtained RNA solution at -80°C until use. Agilent 2100Bioanalyzer (Agilent RNA 6000 Nano Kit) was used to detect the concentration of total RNA.

Synthesis of sample cDNA: Synthesize using kit with product number Femantas K1631, reverse transcription buffer 4 μl, random primer 0.2 μl, reverse transcriptase 0.5 μl, DEPC water 13.3 μl, RNA template 2 μl, total volume 20 μl. Mix the solution by flicking the bottom of the tube and centrifuge briefly at 6000 rpm. The mixture was firstly dried in a 70°C dry bath for 3 minutes, then in a 37°C water bath for 60 minutes. Immediately after taking it out, put it in a dry bath at 95°C for 3 minutes to obtain the final reverse transcription solution, which is the cDNA solution, and store it at -80°C until use.

Fluorescent real-time quantitative PCR: Reaction system: 2×PCR enzyme Mix 10ul, upstream primer F 0.5ul, downstream primer R 0.5ul, add water to a total volume of 20ul. Reaction steps: 45 PCR cycles (94° C. for 20 seconds; 60° C. for 5 seconds). After completing the reaction, record the Ct value and calculate the expression level.

To sum up, the kit of the present invention screens out highly metastatic patients by detecting the expression level of TESC in the tumor tissues of patients with papillary thyroid microcarcinoma to be tested, and can be used for clinical auxiliary diagnosis of papillary thyroid microcarcinoma, and can take relevant measures for patients. It provides an effective basis for specific treatment measures or decision-making, and has a good prospect for clinical application.

sequence listing

<110> West China Hospital of Sichuan University

<120> A screening kit for metastatic screening of papillary thyroid microcarcinoma

<130> GY026-18P1353

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212>DNA

<213> TESC-F（Artificial Sequence）

<400> 1

tcaaccccat ccgatccaaa 20

<210> 2

<211> 20

<212>DNA

<213> TESC-R（Artificial Sequence）

<400> 2

atctcagctt ctccttccgg 20

Claims (8)

1. a kind of metastatic kit for screening of the small papillary carcinoma of thyroid gland, it is characterised in that: it includes optional for examining Survey the reagent of the expression of TESC in the small papillary carcinoma tissue of thyroid gland.

2. kit for screening according to claim 1, it is characterised in that: the small papillary carcinoma tissue of detection thyroid gland The reagent of middle TESC expression is PCR detection reagent.

3. kit for screening according to claim 2, it is characterised in that: PCR detection reagent includes SEQ ID Primer pair shown in NO.1~2.

4. kit for screening according to claim 1, it is characterised in that: the small papillary carcinoma tissue of detection thyroid gland The reagent of middle TESC expression is Western-blot detection method reagent.

5. the reagent of the expression of TESC is preparing the small mamillary of thyroid gland in the detection small papillary carcinoma tissue of thyroid gland Purposes in the metastatic screening agent of cancer.

6. purposes according to claim 5, it is characterised in that: TESC in the small papillary carcinoma tissue of detection thyroid gland The reagent of expression is PCR detection reagent.

7. purposes according to claim 6, it is characterised in that: PCR detection reagent includes NO.1~2 SEQ ID Shown primer pair.

8. purposes according to claim 5, it is characterised in that: TESC in the small papillary carcinoma tissue of detection thyroid gland The reagent of expression is Western-blot detection method reagent.