CN114058698A

CN114058698A - Application of reagent for detecting exosome miR-6879-5p in preparation of thyroid cancer metastasis detection kit

Info

Publication number: CN114058698A
Application number: CN202010748922.6A
Authority: CN
Inventors: 陈文杰
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2020-07-29
Filing date: 2020-07-29
Publication date: 2022-02-18
Anticipated expiration: 2040-07-29
Also published as: CN114058698B

Abstract

The invention discloses application of a reagent for detecting an exosome miR-6879-5p in preparation of a thyroid cancer lymph node metastasis detection kit, and belongs to the field of cancer diagnosis. In plasma exosomes of patients with thyroid papillary carcinoma lymph node metastasis, the level of miR-6879-5p is significantly higher than in patients with non-lymph node metastasis. According to the discovery, the reagent for detecting the exosome miR-6879-5p is used for preparing the thyroid cancer lymph node metastasis detection kit, whether the thyroid papillary cancer patient has lymph node metastasis can be accurately judged, and the kit has a good application prospect.

Description

Application of reagent for detecting exosome miR-6879-5p in preparation of thyroid cancer metastasis detection kit

Technical Field

The present invention is in the field of cancer diagnosis.

Background

Papillary thyroid carcinoma is the most common pathological subtype of thyroid carcinoma, and the number of patients has increased explosively worldwide in recent years. Most papillary thyroid carcinomas have latent diseases, and have no obvious positive symptom signs and are mostly found during physical examination; and at the time of definitive diagnosis, approximately 30% -80% of patients have incorporated cervical lymph node metastasis. The cervical lymph node metastasis not only increases the operation difficulty and prolongs the incision wound surface, increases the incidence rate of operation complications, but also seriously affects the prognosis of patients.

The existing preoperative diagnosis technology is mainly puncture biopsy under the guidance of high-resolution color ultrasound of the neck, while ultrasonic detection is interfered by various factors, and the accuracy is greatly limited; and the fine needle puncture has bleeding risk and has the risk of tumor implantation metastasis through the puncture needle channel. Therefore, an auxiliary examination means capable of accurately assessing whether metastasis exists in the cervical lymph nodes before an operation is urgently needed, and the auxiliary examination means has important significance for accurate treatment and prognosis evaluation of papillary thyroid cancer.

Exosomes are tiny membrane vesicles secreted by cells and having a diameter of about 40-160 nm, and can be used as communication media between cells to promote recipient cells to generate various physiological or pathological changes by transmitting cell-derived 'goods' (proteins, lipids, nucleic acids and the like). In recent years, the miRNAs in exosomes are the most abundant nucleic acid components, play an important role in tumor metastasis, and can be used as diagnostic markers for early diagnosis of tumors, lymph node metastasis and distant metastasis. For example, the breast cancer cell exosome miR-122 can regulate glucose metabolism of a pre-metastatic part so as to promote tumor metastasis; the cervical cancer cell exosome miR-221-3p can target and silence an oncogene VASH1 so as to promote peritumoral lymphatic vessel hyperplasia and lymph node metastasis; the colon cancer cells can actively transport the cancer-suppressing miR-193a out of the cells through exosomes, and further promote self proliferation and metastasis.

Human miR-6879-5p (MIMAT0027658), a miRNA encoded by human chromosome 11, is currently reported.

Disclosure of Invention

The invention aims to solve the problems that: provides a new application of a reagent for detecting an exosome miR-6879-5p in preparing a thyroid cancer metastasis detection kit.

The technical scheme of the invention is as follows:

application of a reagent for detecting exosome miR-6879-5p in preparation of a thyroid cancer lymph node metastasis detection kit.

The use as described above, said exosomes are exosomes in plasma.

As used herein, the thyroid cancer is papillary thyroid cancer.

The application of the reagent comprises the following steps: gene chip reagent, Northern blot detection reagent, in-situ hybridization reagent or PCR detection reagent.

The application is that the reagent for detecting the exosome miR-6879-5p is a PCR detection reagent and comprises a reverse transcription primer and a quantitative PCR primer;

the sequence of the reverse transcription primer is shown as SEQ ID NO. 1; the sequence of the quantitative PCR primer is shown as SEQ ID NO.2 and SEQ ID NO. 3.

A thyroid cancer lymph node metastasis detection kit comprises a reagent for detecting an exosome miR-6879-5 p.

The kit as described above, wherein the exosomes are exosomes in plasma.

As in the previous kit, the thyroid cancer is papillary thyroid cancer.

The kit comprises the following reagents for detecting the exosome miR-6879-5 p: gene chip reagent, Northern blot detection reagent, in-situ hybridization reagent or PCR detection reagent.

The kit is characterized in that the reagent for detecting the exosome miR-6879-5p is a PCR detection reagent and comprises a reverse transcription primer and a quantitative PCR primer;

The inventor finds that the level of miR-6879-5p in plasma exosomes of patients with thyroid papillary carcinoma lymph node metastasis is remarkably higher than that of patients without lymph node metastasis. By detecting the level of plasma exosome miR-6879-5p, the papillary thyroid carcinoma patients with lymph node metastasis and lung metastasis can be effectively distinguished, the sum of sensitivity and specificity can reach more than 170%, and the detection level is very high in the practice of using a single marker for disease diagnosis.

According to the discovery, the reagent for detecting the exosome miR-6879-5p is used for preparing the thyroid cancer lymph node metastasis detection kit, and has good application prospect.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1: ROC curve of relation of miR-6879-5p and papillary thyroid carcinoma.

Detailed Description

Example 1 kit of the invention

This example is described with reference to a PCR assay kit.

1. Composition of the kit

1.1 reverse transcription reagents

(1) MicroRNA Stem-loop reverse transcription primer

The sequence (SEQ ID NO.1) is:

5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTCTCC-3’。

(2) other conventional reagents required for reverse transcription

Comprises dNTP, 5 x buffer and M-MLV reverse transcriptase; may be a reverse transcription reagent which is conventional in the art.

1.2 real-time fluorescent quantitative PCR reagent

(1) Quantitative PCR primer

The sequences are respectively as follows:

forward primer (SEQ ID NO. 2): 5'-CAGGGCAGGGAAGGTG-3', respectively;

reverse primer (SEQ ID NO. 3): 5'-GTGCAGGGTCCGAGGT-3' are provided.

(2) Real-time fluorescent quantitative PCR (polymerase chain reaction) premix solution

TB Green Premix Ex Taq II (Tli RNaseH Plus) from TaKaRa.

2. Method of use of the kit of the invention

2.1 plasma exosome RNA extraction

Collecting blood with blood amount not less than 10ml for papillary thyroid carcinoma patient, placing in EDTA anticoagulation tube, centrifuging at 1800g, 10min and 4 deg.C, transferring the supernatant into another EDTA tube, performing centrifugation at 3000g, 15min and 4 deg.C, collecting supernatant, storing in freezing tube, and refrigerating at-80 deg.C. When detection is required, plasma exosomes are extracted using ultracentrifugation methods conventional in the art and resuspended in 200ul PBS. And extracting exosome RNA.

2.2 reverse transcription

Reverse transcription of mirnas is performed using a reverse transcription reagent. The reverse transcription reaction system is shown in Table 1.

TABLE 1 reverse transcription reaction System

Reaction procedure: 16 ℃ for 10 minutes, 42 ℃ for 55 minutes, 85 ℃ for 5 minutes, and 4 ℃.

2.3 real-time fluorescent quantitative PCR

And detecting the reverse transcription DNA template by using a real-time fluorescent quantitative PCR reagent. The PCR reaction system is shown in Table 2.

TABLE 2 PCR reaction System

Reaction procedure: 95 ℃ for 2 min, (95 ℃ for 15 sec, 72 ℃ for 30 sec) 5 cycles, (95 ℃ for 15 sec, 60 ℃ for 1 min) 40 cycles to collect fluorescence, 10 ℃ for 10 min.

2.4 preliminary judgment

When compared with papillary thyroid cancer patients without known lymph node metastasis, the level of exosome miR-6879-5p in the tested object is remarkably higher, which indicates that the tested object has high risk of lymph node metastasis of papillary thyroid cancer.

2.5 further judgment

(1) Relative copy number calculation

After the real-time fluorescence quantification is finished, the target (including an experimental group and a control group) miRNAs are quantified by adopting the external reference Cel-miR-39, and a quantitative formula 2 is used according to the Ct value of the detection^-ΔΔCtRelative expression levels (relative copy number) were calculated.

The general sequence of the exoginseng Cel-miR-39 is as follows: 5'-UCACCGGGUGUAAAUCAGCUUG-3' (SEQ ID NO. 4); primers were synthesized by QIAGEN (MIRCURY LNA miRNA PCR Starter Kit, cat # 339320) with the following sequences:

FP(SEQ ID NO.5)：5’-GCGCTCACCGGGTGTAAA-3’；

RP(SEQ ID NO.6)：5’-GTGCAGGGTCCGAGGT-3’。

calculating the formula:

2^-ΔΔCt＝2^{- (Delta Ct test group-Delta Ct control group)}＝2^{- [ (Experimental group Gene Ct-Experimental group reference Gene Ct) - (control group Gene Ct-control group reference Gene Ct)]}。

(2) Interpretation

When the relative copy number of the detected object relative to the exosome miR-6879-5p of the papillary thyroid cancer patient without the known lymph node metastasis is more than 2.74, the patient is indicated to have the lymph node metastasis of papillary thyroid cancer; in contrast, the patient did not develop lymph node metastasis from papillary thyroid carcinoma.

There are also many reagents for detecting mirnas, such as: gene chip reagents, Northern blot detection reagents, in situ hybridization reagents and the like are conventional alternative forms of the kit.

In order to further illustrate the beneficial effects of the present invention, the present invention also provides the following experimental examples.

Experimental example 1 clinical verification

1. Method of producing a composite material

120 plasma samples of papillary thyroid carcinoma were randomly selected, 60 samples of which had lymph node metastasis (metastatic group) and 60 samples of which had not had lymph node metastasis (control group), exosome RNA was extracted, plasma exosome miR-6879-5p levels were detected by the kit and method of example 1, and miR-4707-3p, miR-4749-5p and miR-8058 primers were replaced with the primers of miR-4707-3p, miR-4749-5p and miR-8058 in example 1, and the plasma exosome miR-4707-3p, miR-4749-5p and miR-8058 levels were detected. And then adopting an external reference Cel-miR-39 to quantify the miRNAs.

The primer sequences of the miR-4707-3p, miR-4749-5p and miR-8058 are shown in Table 3.

TABLE 3 primer sequences for miR-4707-3p, miR-4749-5p and miR-8058

And calculating the sensitivity and specificity of the exosome miR-6879-5p to the prediction of the lymph node metastasis of papillary thyroid carcinoma by adopting an ROC curve and AUC, and calculating an optimal threshold value.

2. Results

As shown in Table 4, there was a very significant difference in the relative copy number of miR-6879-5p in the metastatic group and the control group (p <0.001), while miR-4707-3p, miR-4749-5p and miR-8058 did not have a significant difference in the two groups.

Table 4 relative copy number of exosome miRNAs as shown by real-time fluorescent quantitative PCR

The ROC curve shows that the AUC value is up to 0.941; when the relative copy number threshold was chosen to be 2.74, the sensitivity of predicting lymph node metastasis was 93.3% and the specificity was 83.3% (FIG. 1).

In conclusion, the exosome miR-6879-5p is highly related to thyroid papillary carcinoma lymph node metastasis, and a reagent for detecting the level of exosome miR-6879-5p in plasma can be used for preparing a kit for detecting thyroid papillary carcinoma lymph node metastasis.

SEQUENCE LISTING

<110> Sichuan university Hospital in western China

<130> GYKH1673-2020P0110390CC

<160> 15

<170> PatentIn version 3.5

<210> 1

<211> 50

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacctctcc 50

<210> 2

<211> 16

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

cagggcaggg aaggtg 16

<210> 3

<211> 16

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

gtgcagggtc cgaggt 16

<210> 4

<211> 22

<212> RNA

<213> human (Homo sapiens)

<400> 4

ucaccgggug uaaaucagcu ug 22

<210> 5

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

gcgctcaccg ggtgtaaa 18

<210> 6

<211> 16

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

gtgcagggtc cgaggt 16

<210> 7

<211> 50

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacagaacc 50

<210> 8

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

ataccgagcc cgccccag 18

<210> 9

<211> 16

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

gtgcagggtc cgaggt 16

<210> 10

<211> 50

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacgatgcc 50

<210> 11

<211> 16

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

tgcggggaca ggccag 16

<210> 12

<211> 16

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 12

gtgcagggtc cgaggt 16

<210> 13

<211> 50

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 13

gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacttatgg 50

<210> 14

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 14

gcgcgcctgg actttgatc 19

<210> 15

<211> 16

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 15

gtgcagggtc cgaggt 16

Claims

1. Application of a reagent for detecting exosome miR-6879-5p in preparation of a thyroid cancer lymph node metastasis detection kit.

2. Use according to claim 1, characterized in that: the exosomes are exosomes in plasma.

3. Use according to claim 1, characterized in that: the thyroid cancer is papillary thyroid cancer.

4. Use according to claim 1, characterized in that: the reagent for detecting the exosome miR-6879-5p is as follows: gene chip reagent, Northern blot detection reagent, in-situ hybridization reagent or PCR detection reagent.

5. Use according to claim 1, characterized in that: the reagent for detecting the exosome miR-6879-5p is a PCR detection reagent and comprises a reverse transcription primer and a quantitative PCR primer;

6. A thyroid cancer lymph node metastasis detection kit is characterized in that: the kit comprises a reagent for detecting the exosome miR-6879-5 p.

7. The kit of claim 6, wherein: the exosomes are exosomes in plasma.

8. The kit of claim 6, wherein: the thyroid cancer is papillary thyroid cancer.

9. The kit of claim 6, wherein: the reagent for detecting the exosome miR-6879-5p is as follows: gene chip reagent, Northern blot detection reagent, in-situ hybridization reagent or PCR detection reagent.

10. The kit of claim 6, wherein: the reagent for detecting the exosome miR-6879-5p is a PCR detection reagent and comprises a reverse transcription primer and a quantitative PCR primer;