CN114058696A

CN114058696A - Application of miR-519e-5p as target point for detecting or treating thyroid papillary carcinoma metastasis at remote position

Info

Publication number: CN114058696A
Application number: CN202010747826.XA
Authority: CN
Inventors: 雷建勇; 李根棚; 陈文杰; 李志辉; 朱精强; 魏涛
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: CN114058696B

Abstract

The invention discloses application of miR-519e-5p as a target for detecting or treating distal metastasis of papillary thyroid cancer, and belongs to the field of cancer detection and treatment. The technical scheme mainly comprises the application of a reagent for detecting the exosome miR-519e-5p in preparing a papillary thyroid carcinoma distant metastasis detection kit and the application of an artificial mimic of miR-519e-5p or miR-519e-5 in preparing a drug for inhibiting papillary thyroid carcinoma metastasis. The kit can simplify the detection process and relieve the pain of patients; the medicine can obviously inhibit the mobility, the invasiveness and the dryness of thyroid cancer cells, and has great application potential.

Description

Application of miR-519e-5p as target point for detecting or treating thyroid papillary carcinoma metastasis at remote position

Technical Field

The present invention is in the field of cancer detection and treatment.

Background

In recent years, global data indicate that the incidence of thyroid cancer presents a rapid increase, mainly due to an explosive increase in the pathological type of Papillary Thyroid Carcinoma (PTC). Although the prognosis of PTC is good, the 10-year survival rate can reach 90%, once distant organ metastasis of lung, bone, brain, liver and the like occurs (the distant metastasis rate is 6-20%), the survival rate of patients is remarkably reduced, and the 10-year survival rate is only 51.2% at most. How to effectively control and treat the distant PTC metastasis is an urgent problem to be solved in thyroid surgery.

Exosomes (Exosomes) are vesicle-like bodies of uniform size, about 40-160nm in diameter secreted extracellularly by cells, which can specifically carry a variety of bioactive molecules, including lipids, proteins, RNAs and DNA. Researches in recent years show that exosome miRNAs play an important role in distant organ metastasis, for example, exosome miR-19a can target PTEN to promote tumor brain metastasis, liver cancer exosome miR-1247-3p can be taken by tumor-related fibroblasts to promote tumor distant metastasis, bladder cancer exosome-23 b promotes primary tumor angiogenesis and lung metastasis, and colon cancer exosome-23 b or miR-193a promotes tumor liver metastasis.

miR-519e-5p (MIMAT0002828) is transcribed from human chromosome 19, is found to be related to acute myocardial infarction and some adverse events (such as preeclampsia, premature rupture of fetal membranes, spontaneous preterm birth and the like) during pregnancy, and has no report that miR-519e-5p is related to thyroid cancer.

Disclosure of Invention

The invention aims to solve the problems that: provides the application of miR-519e-5p as a target point for detecting or treating the distant metastasis of papillary thyroid cancer.

The technical scheme of the invention is as follows:

a kit for detecting papillary thyroid carcinoma distant metastasis, which comprises a reagent for detecting exosome miR-519e-5 p.

According to the kit, the reagent for detecting the exosome miR-519e-5p is as follows: gene chip reagent, Northern blot reagent or quantitative PCR reagent.

Application of a reagent for detecting exosome miR-519e-5p in preparation of a kit for detecting thyroid papillary carcinoma distant metastasis.

As for the use described previously, the distant metastasis is: the reagent for detecting the exosome miR-519e-5p is as follows: gene chip reagent, Northern blot reagent or quantitative PCR reagent.

The application of the artificial mimics of miR-519e-5p or miR-519e-5 in preparing medicines for inhibiting thyroid papillary carcinoma metastasis.

The artificial mimic of miR-519e-5p is miRNA mimics for the aforementioned application.

The artificial mimic of miR-519e-5p is miRNA agomir for the aforementioned application.

A drug for inhibiting papillary thyroid carcinoma metastasis takes an artificial mimic of miR-519e-5p or miR-519e-5 as an active ingredient.

As the medicine, the artificial mimic of miR-519e-5p is miRNA mimics.

The artificial mimic of miR-519e-5p is miRNA agomir as the medicine.

The term "artificial mimic of miR-519e-5 p" refers to: the sequence comprises a nucleic acid with the length within 40nt (or 40bp) of the sequence of miR-519e-5p or the DNA form (U base is changed into T base) of the sequence.

The term "miRNA mimics" refers to: the function of the small-segment double-stranded miRNA designed and synthesized aiming at the mature body of the miRNA is the same as that of the mature body of the miRNA, and the small-segment double-stranded miRNA can up-regulate the content of the corresponding miRNA in cells, and is also called as an miRNA mimic; the miRNA agomir is a product which is added with chemical modification on the basis of miRNA mimics to enhance stability.

The term "distant metastasis" refers to: the cancer cells are carried to other parts of the whole body through the blood system, and the same tumor phenomenon occurs.

The invention provides that the miR-519e-5p can be used for diagnosing and treating thyroid cancer distant metastasis for the first time. The kit can simplify the diagnosis process of the thyroid cancer distant metastasis, improve the detection speed and relieve the pain of patients; the medicine of the invention can obviously inhibit the migration, the invasiveness and the dryness of thyroid cancer cells, and is a medicine with great potential.

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: expression levels of miRNAs in metastatic and non-metastatic groups.

FIG. 2: ROC curve.

FIG. 3: miR-519e-5p expression quantity in different cells and after miR-mimics transfection; nthv-ori 3-1 refers to a thyroid-normal cell line; miR-MC refers to miRNA mimics control; miR-IC refers to miRNA inhibitor control.

FIG. 4: the result of the scratch test; the mapping distance (% of 0H) is calculated as: (0 h-48 h cell spacing)/0 h cell spacing.

FIG. 5: transwell experimental results.

FIG. 6: and (5) three-dimensional balling detection experimental results.

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 (short for 'Stem loop primer')

The sequence (SEQ ID NO.1) is:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGAAAGT。

(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'-GCGCTTCTCCAAAAGGGAGC-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 from a container with a volume of not less than 10ml to EDTA anticoagulation tube, centrifuging at 1800g for 10min at 4 deg.C, transferring the supernatant to another EDTA tube, freezing at-80 deg.C in a freezing tube at 3000g for 15min at 4 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 known not to have distant metastasis, the level of exosome miR-519e-5p in the detected object is significantly higher, which indicates that the detected object has high risk of having the distant 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-519e-5p of the papillary thyroid cancer patient known not to have distant metastasis is more than 3.5, the patient is indicated to have the possibility of having the distant metastasis of papillary thyroid cancer; in contrast, the patient did not develop distant metastasis of papillary thyroid carcinoma.

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

The advantageous effects of the present invention are further illustrated in the form of experimental examples.

Experimental example 1 correlation of exosome miR-519e-5p with distant metastasis of papillary thyroid carcinoma

PTC plasma exosomes were extracted from 27 distant metastases (group M1) and 81 non-distant metastases (group M0) patient plasma samples using exosome isolation kit ExoQuick-TCTM. The ExoReasysSerum/Plasma Maxi Kit extracts exosome miRNAs. Real-time fluorescent quantitative PCR detects the expression of some miRNAs, and cel-39-3p is used as an external reference.

The information of the detected exosome miRNAs is shown in table 3.

TABLE 3 miRNA information

Primers for detecting miR-519e-5p are shown in example 1, and primers for detecting other miRNAs in Table 3 are shown in Table 4.

TABLE 4 primers of interest

Relative quantification results show that miR-519e-5p has a very significant difference between the M0 and M1 groups, with higher levels in the M1 group (fig. 1); the ROC curve results showed that the area under the miR-519e-5p curve was maximal (AUC ═ 0.854) (fig. 2), and the sensitivity was 74.1% and the specificity was 96.3% when the relative copy number (calculated as in example 1) was 3.5.

And (4) conclusion: the far metastasis of papillary thyroid carcinoma is identified with the best accuracy by miR-519e-5 p.

Experimental example 2 migration experiment of miR-519e-5p inhibition PTC cell line

In the experimental example, miR-519e-5p mimics or miR-519e-5p inhibitor are transfected by two papillary thyroid cancer cell lines, namely TPC-1(BRAF wild type) and K1(BRAF mutant type), and then the influence of the papillary thyroid cancer cell lines on PTC migration, invasion and cell dryness is detected through expression quantity detection, scratch experiment, transwell experiment and three-dimensional balling.

miRNA mimics are artificial mimics of mirnas that can increase miRNA levels in vivo or in cells. In the embodiment, the sequence of miR-519e-5p mimics is as follows: 5'-UUCUCCAAAAGGGAGCACUUUC-3' (SEQ ID NO.25)

The miRNA inhibitor is an inhibitor of miRNA, and can reduce the level of miRNA in vivo or in cells. In this example, the sequence of miR-519e-5p inhibitor is: 5'-GAAAGUGCUCCCUUUUGGAGAA-3' (SEQ ID NO. 26).

The results are as follows:

FIG. 3 shows the expression quantity of miR-519e-5p in different cells and the expression quantity of miR-519e-5p after miR-519e-5p mimic or inhibitor transfection. The expression quantity of miR-519e-5p in different cells is different, the miR-519e-5p mimics can improve the miR-519e-5p level, and the miR-519e-5p inhibitor can reduce the miR-519e-5p level.

Fig. 4 shows the results of the scratch test. Under the influence of miR-519e-5p mimics, the level of miR-519e-5p is improved, and the migration distance of thyroid papillary carcinoma cells K1 is reduced; under the influence of miR-519e-5p inhibitor, the level of miR-519e-5p is reduced, and the migration distance of the TPC-1 of papillary thyroid carcinoma cells is increased.

FIG. 5 shows the results of a transwell experiment. Under the influence of miR-519e-5p mimics, the miR-519e-5p level is improved, and the invasiveness of thyroid papillary carcinoma cells K1 is reduced; under the influence of miR-519e-5p inhibitor, the level of miR-519e-5p is reduced, and the invasiveness of thyroid papillary carcinoma cells TPC-1 is improved.

Fig. 6 shows the results of the three-dimensional balling test. Under the influence of miR-519e-5p mimics, the miR-519e-5p level is improved, and the cell dryness of thyroid papillary carcinoma cells K1 is reduced; under the influence of miR-519e-5p inhibitor, the miR-519e-5p level is reduced, and the cell dryness of thyroid papillary carcinoma cells TPC-1 is improved.

The results of this experimental example show that: by increasing the level of miR-519e-5p, the migration capability, invasiveness and dryness of papillary thyroid carcinoma cells can be inhibited, and the metastasis of papillary thyroid carcinoma cells can be inhibited.

In conclusion, the plasma exosome miR-519e-5p level of the papillary thyroid carcinoma distant metastasis patient is higher than that of a non-metastasis patient, and a reagent for detecting the plasma exosome miR-519e-5p can be used for preparing a papillary thyroid carcinoma distant metastasis detection kit; the miR-519e-5p can inhibit the migration ability, invasiveness and dryness of papillary thyroid carcinoma, and the artificial mimic of miR-519e-5p can be used for preparing medicines and inhibiting the metastasis of papillary thyroid carcinoma.

SEQUENCE LISTING

<110> Sichuan university Hospital in western China

Application of miR-519e-5p as target for detecting or treating distant metastasis of papillary thyroid carcinoma

<130> GYKH1673-2020P0110392CC

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Claims

1. A kit for detecting the distant metastasis of papillary thyroid carcinoma is characterized in that: the kit comprises a reagent for detecting the exosome miR-519e-5 p.

2. The kit of claim 1, wherein: the reagent for detecting the exosome miR-519e-5p is as follows: gene chip reagent, Northern blot reagent or quantitative PCR reagent.

3. Application of a reagent for detecting exosome miR-519e-5p in preparation of a kit for detecting thyroid papillary carcinoma distant metastasis.

4. Use according to claim 3, characterized in that: the distant metastasis is: the reagent for detecting the exosome miR-519e-5p is as follows: gene chip reagent, Northern blot reagent or quantitative PCR reagent.

Use of an artificial mimic of miR-519e-5p or miR-519e-5 in the preparation of a medicament for inhibiting papillary thyroid carcinoma metastasis.

6. Use according to claim 5, characterized in that: the artificial mimic of the miR-519e-5p is miRNA mimics.

7. Use according to claim 5, characterized in that: the artificial mimic of miR-519e-5p is miRNA agomir.

8. A drug for inhibiting metastasis of papillary thyroid carcinoma cells, which comprises: the medicine takes an artificial mimic of miR-519e-5p or miR-519e-5 as an active ingredient.

9. The medicament of claim 8, wherein: the artificial mimic of the miR-519e-5p is miRNA mimics.

10. The medicament of claim 8, wherein: the artificial mimic of miR-519e-5p is miRNA agomir.