CN113943800A - Papillary thyroid carcinoma iodine resistance screening kit - Google Patents
Papillary thyroid carcinoma iodine resistance screening kit Download PDFInfo
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- CN113943800A CN113943800A CN202010689343.9A CN202010689343A CN113943800A CN 113943800 A CN113943800 A CN 113943800A CN 202010689343 A CN202010689343 A CN 202010689343A CN 113943800 A CN113943800 A CN 113943800A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The invention discloses application of a reagent for detecting exosome miR-15a-5p in preparation of a papillary thyroid carcinoma iodine resistance screening kit, and belongs to the field of cancer diagnosis. In plasma exosomes of patients with iodine-resistant papillary thyroid carcinoma, miR-15a-5p levels are significantly lower than in patients with non-iodine-resistant papillary thyroid carcinoma. According to the discovery, the reagent for detecting the exosome miR-15a-5p is used for preparing the papillary thyroid carcinoma iodine resistance screening kit, and has good application prospect.
Description
Technical Field
The present invention is in the field of cancer diagnosis.
Background
Papillary Thyroid Carcinoma (PTC) is the most common pathological subtype of thyroid cancer, and traditional treatment (surgery, TSH inhibition and radioiodine 131) has resulted in a 10-year survival rate of PTC of up to 90%. However, as the disease progresses, 6-20% of PTCs gradually develop tissue organ metastasis and subsequent papillary thyroid cancer iodine resistance (abbreviated as "PTC iodine resistance"), i.e., ineffective radioiodine 131 treatment. PTC iodine resistance seriously affects patient quality of life and survival rate. Studies have shown that PTC transferred patients who take radioiodine have a 10-year survival rate of about 56% while radioiodine resistant patients have a 10-year survival rate of only 10%. The PTC iodine resistant patient is identified in advance, so that personalized treatment can be performed in time, and the survival rate of the patient is improved.
PTC iodine resistance is in much the same way as tumor resistance, and is essentially "drug resistant" to radioactive iodine 131. In recent years, the role of exosomes in drug resistance of malignant tumors is gradually revealed, for example, kidney cancer exosomes can promote the generation of sorafenib drug resistance by transducing lncARSR, breast cancer exosomes can regulate the activity of drug resistance-related pathways, and exosomes miR-1290 and miR-375 can be used as predictive markers of castration-resistant prostate cancer.
miR-15a-5p (MIMAT0000068) is miRNA expressed by chromosome 13, and research shows that: miR-15a-5p can be related to the development of Non-Small Cell Lung cancer and can be used as a diagnostic marker of the Non-Small Cell Lung cancer (Significance of miR-15a-5p and CNKSR3 as Novel diagnostic Biomarkers in Non-Small Cell Lung cancer. anticancer Agents Med chem.2018; 18(12): 1695-1701.); the miR-15a-5p can also be used as a diagnostic marker of Colorectal adenocarcinoma, and the expression level of the miR-15a-5p in Colorectal adenocarcinoma tissues is obviously improved (miR-15a-5p, A Novel diagnostic Biomarker Biomarker, and diagnosis Current Colorological Adenococcus. mol Diagn. 2017; 21(4): 453-. However, no report about the correlation between miR-15a-5p and PTC iodine resistance exists at present.
Disclosure of Invention
The invention aims to solve the problems that: provides application of a reagent for detecting exosome miR-15a-5p in preparation of a papillary thyroid carcinoma iodine resistance screening kit and provides the papillary thyroid carcinoma iodine resistance screening kit.
The technical scheme of the invention is as follows:
application of a reagent for detecting exosome miR-15a-5p in preparation of a thyroid cancer iodine resistance screening kit.
The use as described above, said exosomes are exosomes in plasma.
As used herein, the thyroid cancer is papillary thyroid cancer.
As the application, the reagent for detecting the exosome miR-15a-5p is as follows: gene chip reagent, Northern blot detection reagent, in-situ hybridization reagent or PCR detection reagent.
The application of the method is that the reagent for detecting the exosome miR-15a-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 iodine resistance screening kit comprises a reagent for detecting an exosome miR-15a-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-15a-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-15a-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.
The inventor finds through research that the level of miR-15a-5p in plasma exosomes of patients with thyroid papillary cancer iodine resistance is very significantly lower than that of patients without iodine resistance. By detecting the level of plasma exosome miR-15a-5p, patients with iodine-resistant and non-iodine-resistant papillary thyroid carcinoma can be effectively distinguished.
According to the discovery, the reagent for detecting the exosome miR-15a-5p is used for preparing the papillary thyroid carcinoma iodine resistance screening 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: comparing the relative expression amount of miRNA; Non-RAIR, Non-iodine resistant; RAIR, iodine resistance; ns, no significant difference; very significant differences (p < 0.001).
Detailed Description
Example 1: kit of the invention
This example is described with reference to a PCR screening 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’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCACAAA-3’(SEQ ID NO.1)。
(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'-GGGTAGCAGCACATAATGG-3' (SEQ ID NO. 2);
reverse primer (SEQ ID NO. 3): 5'-CTCAACTGGTGTCGTGGA-3' (SEQ ID NO. 3).
(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 patients with confirmed thyroid papillary carcinoma, collecting blood from EDTA anticoagulation tube with volume not less than 10ml, centrifuging at 1800g and 10min at 4 deg.C, transferring supernatant into another EDTA tube, collecting supernatant at 3000g and 15min at 4 deg.C, and storing in a freezing tube at-80 deg.C for refrigeration. 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.
Compared with the average level of exosome miR-15a-5p of a patient suffering from non-iodine-resistant papillary thyroid cancer, if the level of exosome miR-15a-5p of the patient to be detected is higher, the possibility of iodine resistance is high.
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 validation
In the experimental example, the level of the exosome miR-15a-5p in a clinical plasma sample is detected, and the levels of miR-500a-5p, miR-1296-5p and miR-3120-5p are used as controls. Wherein miR-500a-5p, miR-1296-5p and miR-3120-5p are miRNAs which have been reported to be related to both sodium iodine transporter (SLC5A5, NIS) and tumor.
1. Method of producing a composite material
Plasma samples of 21 iodine-tolerant PTC patients and plasma samples of 42 non-iodine-tolerant PTC patients were randomly selected, exosome RNA was extracted by using an exosome separation kit ExoQuick-TCTM, plasma exosome miR-15a-5p levels were detected by using the kit and method of example 1, the primers in example 1 were replaced with primers for miR-500a-5p, miR-1296-5p and miR-3120-5p, and the plasma exosome miR-500a-5p, miR-1296-5p and miR-3120-5p levels were detected. And then adopting an external reference Cel-miR-39 to quantify the miRNAs.
The primer sequences of the miR-500a-5p, miR-1296-5p and miR-3120-5p are shown in Table 3.
TABLE 3 primer sequences for miR-4707-3p, miR-4749-5p and miR-8058
2. Results
As shown in FIG. 1, there was a very significant difference in the relative expression of miR-15a-5p in the iodine-resistant and non-iodine-resistant groups (p <0.001), while miR-500a-5p, miR-1296-5p and miR-3120-5p did not have a significant difference in the two groups.
In conclusion, exosome miR-15a-5p is highly related to papillary thyroid cancer iodine resistance, and a reagent for detecting exosome miR-15a-5p level in plasma can be used for preparing a kit for detecting papillary thyroid cancer iodine resistance.
SEQUENCE LISTING
<110> Sichuan university Hospital in western China
<120> thyroid papillary carcinoma iodine resistance screening kit
<130> GYKH1673-2020P0110389CC
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<170> PatentIn version 3.5
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Claims (10)
1. Application of a reagent for detecting exosome miR-15a-5p in preparation of a thyroid cancer iodine resistance screening 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-15a-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-15a-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.
6. A thyroid cancer iodine resistance screening kit is characterized in that: the kit comprises a reagent for detecting the exosome miR-15a-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-15a-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-15a-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.
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| CN202010689343.9A CN113943800A (en) | 2020-07-16 | 2020-07-16 | Papillary thyroid carcinoma iodine resistance screening kit |
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| Application Number | Priority Date | Filing Date | Title |
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