CN111778335A - tsRNA marker and probe for detecting cancer tissue and paracarcinoma tissue of pancreatic cancer and application of tsRNA marker and probe - Google Patents
tsRNA marker and probe for detecting cancer tissue and paracarcinoma tissue of pancreatic cancer and application of tsRNA marker and probe Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and discloses a tsRNA marker for detecting cancer tissues and paracarcinoma tissues of pancreatic cancer, and application thereof in pancreatic cancer prognosis and survival judgment. The tsRNA marker is tRF-Pro-AGG-004 and/or tRF-Leu-CAG-002. The marker can be used for preparing a kit for pancreatic cancer monitoring and prognosis evaluation.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a tsRNA marker and a probe for detecting cancer tissues and paracarcinoma tissues of pancreatic cancer and application thereof.
Background
Pancreatic cancer is one of the most fatal types of malignancy, with a 5-year survival rate of only around 5% and median survival times of less than half a year. Therefore, it is important for pancreatic cancer patients to be able to quickly and quickly determine the prognosis effect after a certain treatment, so as to screen the optimal treatment scheme. At present, one of the most common pathological indexes in tumor treatment is the stage of tumor, but a large amount of clinical data show that tumor patients in the same stage have significant treatment effects on the same treatment mode. Therefore, screening sensitive and effective biomarkers, and carrying out treatment prognosis evaluation on pancreatic cancer patients at an early stage is an important way for improving the treatment effect of pancreatic cancer patients and prolonging the survival period of patients.
Non-coding small RNA is a hotspot in the field of tumor molecular biology research in recent years, and the development of RNA high-throughput sequencing technology helps people to discover and identify various novel non-coding small RNAs, wherein one type of non-coding small RNAs is named as tRNA (tRNA-derived small RNAs, tsRNAs). tsRNA is about 18-40nt long and is derived from tRNA maturates or precursors. Recent studies have found that tsRNA is involved in the regulation of a variety of physiological and pathological functions, including stress, translational regulation, neurological diseases, viral infections, epigenetics, and the like. The tsRNA is closely related to the occurrence and development of tumors, and the tsRNA is reported to be abnormally expressed in lung cancer, chronic lymphocytic leukemia, breast cancer and acute myelocytic leukemia and is closely related to the occurrence and development of the tumors.
The molecular phenotype and characteristics of tumor tissue can provide more information about tumor treatment, prognosis evaluation, and survival judgment. If specific tsRNAs in pancreatic cancer tissues can be screened from the pancreatic cancer tumor tissues excised by the operation to be used as biomarkers, the treatment and survival prognosis of patients after the operation excision can be evaluated according to the expression of the tsRNAs, and the treatment effect of pancreatic cancer patients can be obviously improved.
Disclosure of Invention
In order to solve the technical problems, the invention mainly aims to provide a tsRNA marker and a probe for detecting cancer tissues and paracarcinoma tissues of pancreatic cancer and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a tsRNA marker for detecting cancer tissues and paracarcinoma tissues of pancreatic cancer, wherein the tsRNA marker is any one or combination of the following sequences:
tRF-Pro-AGG-004：SEQ ID No.1；
tRF-Leu-CAG-002：SEQID No.2。
the invention provides an in situ hybridization probe, which detects the tsRNA markers in the cancer tissues and the paracarcinoma tissues for detecting pancreatic cancer; sequences SEQ ID No.3 and/or SEQ ID No. 4. The in situ hybridization probes are 3 'and 5' labeled Locked Nucleic Acid (LNA) oligonucleotide probes from Exiqon.
The invention provides application of the tsRNA marker and the in situ hybridization probe in preparation of a pancreatic cancer diagnosis kit.
The invention provides a kit for pancreatic cancer diagnosis and prognosis judgment, which is used for detecting the expression quantity of one or more of tRF-Pro-AGG-004 and/or tRF-Leu-CAG-002 in cancer tissues and paracarcinoma tissues of pancreatic cancer.
In a further technical scheme, the kit contains the in situ hybridization probe.
In a further technical scheme, the kit also comprises reagents and enzymes commonly used in the original-impurity reaction. Comprises stationary liquid, confining liquid, hybridization liquid, eluent, color development liquid and the like.
The invention has the beneficial effects that:
the tsRNAs marker provided by the invention has the advantages as a marker for pancreatic cancer diagnosis:
(1) different from the traditional protein biomarkers, the tissue tsRNAs are novel biomarkers, are stable in expression, easy to detect and accurate in quantification, can greatly improve the sensitivity and specificity of disease diagnosis, are beneficial to prognosis diagnosis of pancreatic cancer, and provide reference for development of other disease biomarkers.
(2) The tissue tsRNAs kit is a comprehensive and systematic prognosis judgment and monitoring kit, can be used for auxiliary diagnosis of pancreatic cancer patients, is helpful for reflecting the disease state of pancreatic cancer patients, and provides better support for clinical treatment.
(3) The invention adopts a strict design and evaluation system, and adopts an in-situ hybridization technology to detect the tissue tsRNAs of 60 pancreatic cancer patients at the initial stage, and carries out statistical analysis; ensures the application of the tissue tsRNAs biomarkers and the diagnostic kit, and provides a method and a strategic reference for the development of other disease biomarkers.
(4) The system of the invention researches the role of the tissue tsRNAs in the pancreatic cancer prognosis survival evaluation, and reveals the clinical value of the tissue tsRNAs in the pancreatic cancer diagnosis and prognosis. Therefore, the invention obtains the tsRNAs markers of pancreatic cancer tissues; through the development and application of the tissue tsRNAs biomarkers and the diagnosis kit, the prognosis evaluation of pancreatic cancer can be more convenient and feasible, and a foundation is provided for clinical treatment.
Drawings
FIG. 1 is a graph showing in situ hybridization results for expression levels of tRF-Pro-AGG-004 and tRF-Leu-CAG-002 in pancreatic cancer patient cancers and paracarcinoma tissues.
FIG. 2 shows the expression levels of 2 tsRNAs in 60 patients with pancreatic cancer and in tissues adjacent to the cancer.
FIG. 3 shows the expression of 2 tsRNAs in tissues of 30 patients with pancreatic cancer with longer survival time and 30 patients with pancreatic cancer with shorter survival time.
FIG. 4 is a survival curve analyzing the correlation of 2 tsRNAs with the survival of pancreatic cancer patients.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
Examples
A tsRNA marker for detecting cancer tissues and paracarcinoma tissues of pancreatic cancer, wherein the tsRNA marker is any one or combination of the following sequences:
tRF-Pro-AGG-004：SEQ ID No.1；
tRF-Leu-CAG-002：SEQ ID No.2。
the invention provides an in situ hybridization probe, which detects the tsRNA markers in the cancer tissues and the paracarcinoma tissues for detecting pancreatic cancer; sequences SEQ ID No.3 and/or SEQ ID No. 4. The in situ hybridization probes are 3 'and 5' labeled Locked Nucleic Acid (LNA) oligonucleotide probes from Exiqon.
The invention provides application of the tsRNA marker and the in situ hybridization probe in preparation of a pancreatic cancer diagnosis kit.
The invention provides a kit for pancreatic cancer diagnosis and prognosis judgment, which is used for detecting the expression quantity of one or more of tRF-Pro-AGG-004 and/or tRF-Leu-CAG-002 in cancer tissues and paracarcinoma tissues of pancreatic cancer.
The kit contains the in situ hybridization probe. The kit also comprises reagents and enzymes commonly used in the original-impurity reaction. Comprises stationary liquid, confining liquid, hybridization liquid, eluent, color development liquid and the like.
The invention firstly collects cancer and tissue samples beside the cancer of 60 pancreatic cancer patients, respectively detects the samples through In Situ Hybridization (ISH) experiments, discovers a group of tsRNA tissue markers highly related to the pancreatic cancer, develops a kit applicable to clinical diagnosis of the pancreatic cancer based on the tsRNA tissue markers, and provides a basis for screening and early diagnosis of the pancreatic cancer.
Specifically, the technical solution to the problem of the present invention includes:
(1) collecting cancer and paracarcinoma tissue samples of pancreatic cancer patients;
(2) and respectively carrying out tissue section and detecting the expression of tRF-Pro-AGG-004 and tRF-Leu-CAG-002 by a hybridization method.
(3) Based on the above method, a kit was developed.
The present inventors collected standard-meeting pancreatic cancer tissue samples according to SOP, collected complete pathology data (including age, sex, type of pathology, WHO grading, TMN staging, survival time, etc.), and then tested using in situ hybridization methods.
Specifically, the method comprises the following parts:
(1) collecting a patient sample: cases of pancreatic cancer that were diagnosed imagewise, confirmed pathologically; in the study, 60 standard pancreatic cancer patient tissue samples are adopted for study.
(2) Pancreatic cancer and tissues adjacent to the cancer were sectioned using cryosectioning.
(3) In Situ Hybridization (ISH) process
Firstly, slicing and fixing tissues;
② digesting the sample by protease;
thirdly, performing hybridization reaction and washing by using an LNA fluorescent probe of the company Exiqon, and performing immunodetection and color reaction;
and fourthly, detecting and comparing the expression change of the tsRNA in the cancer of the pancreatic cancer patient and the tissue sample beside the cancer.
(4) Preparation of pancreatic cancer tsRNA diagnostic kit
Firstly, detecting tRF-Pro-AGG-004 and tRF-Leu-CAG-002 in cancer and tissues beside the cancer of a pancreatic cancer patient by an ISH technology, and taking the detected result as an index for assisting pancreatic cancer prognosis judgment. On the basis, a pancreatic cancer diagnosis kit is developed, and the kit comprises two tsRNA (ribonucleic acid) original-hybrid probes, and reagents such as a fixing solution, a sealing solution, a hybridization solution, an eluent, a developing solution and the like.
(5) Data analysis
All statistical tests were performed using GraphPad Prism software 7 (san diego, CA). Data are presented as mean ± SEMs. P <0.05 is statistically significant for the differences. The normality and the equal variance of samples between groups were evaluated using the Shapiro-Wilk test and the Brown-Forsythe test, respectively. When the normality and variance were equal between the groups, the analysis was performed using the t-test.
The following is a further description of the invention:
the invention respectively detects 60 cases of pancreatic cancer patient cancer tissues and matched paracancerous tissues by using an in-situ hybridization method, as shown in figure 1, tRF-Pro-AGG-004 and tRF-Leu-CAG-002 are found to be obviously increased in pancreatic cancer tissue samples, the expression levels of 2 tsRNAs in 60 cases of pancreatic cancer patient cancers and paracancerous tissues are analyzed, and the statistical result is shown in figure 2.
Then, 60 patients were divided into two groups according to the pathological information data of the patients: the short-survival group and the long-survival group (30 cases each) were analyzed for differences in expression of tRF-Pro-AGG-004 and tRF-Leu-CAG-002 between the two groups. The results showed that the expression levels of tRF-Pro-AGG-004 and tRF-Leu-CAG-002 were relatively high in the tissues of patients with shorter survival, while the expression levels of tRF-Pro-AGG-004 and tRF-Leu-CAG-002 were lower in the tissues of patients with longer survival, and the results are shown in FIG. 3.
Further analyzing the effect of the above two tsRNAs on prognosis, the survival curve analysis result shows that the expression of tRF-Pro-AGG-004 and tRF-Leu-CAG-002 in pancreatic cancer tissues is significantly related to the survival time of patients, and the result is shown in FIG. 4.
Based on the above results, a kit useful for diagnosis of pancreatic cancer was prepared, which contained a probe for detecting tRF-Pro-AGG-004 and tRF-Leu-CAG-002, which stably existed and could be detected in the tissues of patients, and other detection reagents.
The related diagnostic kit consisting of the tsRNAs markers, the probes, the reagents and the like is favorable for prognosis evaluation of pancreatic cancer, provides support for clinically detecting the disease state of a patient, and is favorable for early treatment of the patient and improvement of the survival rate of the patient with pancreatic cancer.
SEQUENCE LISTING
<110> Wang Yanbo
<120> tsRNA markers and probes for detecting cancer tissues and tissues beside cancer of pancreatic cancer and application thereof
<130>/
<160>4
<170>PatentIn version 3.5
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5'-DIG-gagaatcata cccctagacc aacgagcc- DIG-3' 28
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5'-DIG-ggtgtcagga gtgggat- DIG-3' 17
Claims (8)
1. The tsRNA marker for detecting the cancer tissues and the paracarcinoma tissues of the pancreatic cancer is any one or combination of the following sequences:
tRF-Pro-AGG-004：SEQ ID No.1；
tRF-Leu-CAG-002：SEQ ID No.2。
2. an in situ hybridization probe for detecting tsRNA markers in cancerous tissue and paracancerous tissue of pancreatic cancer as claimed in claim 1; sequences SEQ ID No.3 and/or SEQ ID No. 4.
3. Use of the tsRNA marker of claim 1 in the preparation of a pancreatic cancer diagnostic kit.
4. Use of the in situ hybridization probe of claim 2 in the preparation of a pancreatic cancer diagnostic kit.
5. A kit for diagnosis and prognosis of pancreatic cancer, wherein the kit is used to detect the expression level of one or more of the tsRNA markers of claim 1 in cancerous tissues and paracancerous tissues of pancreatic cancer.
6. The kit of claim 5, wherein the kit comprises the in situ hybridization probe of claim 2.
7. The diagnostic kit of claim 6, wherein the kit further comprises reagents and enzymes commonly used in promiscuous reactions.
8. The diagnostic kit according to claim 7, wherein the kit comprises a fixative, a blocking solution, a hybridization solution, an elution solution, and a color-developing solution.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112522262A (en) * | 2020-11-12 | 2021-03-19 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Pancreatic cancer-associated tRF and application thereof |
| CN113308530A (en) * | 2021-05-24 | 2021-08-27 | 嘉兴市第一医院 | Blood tsRNA marker for herpes zoster, preparation and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108048460A (en) * | 2018-02-01 | 2018-05-18 | 浙江大学 | A kind of New molecular marker and its application in preparing for the kit of head and neck cancer diagnosis and prognosis |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108048460A (en) * | 2018-02-01 | 2018-05-18 | 浙江大学 | A kind of New molecular marker and its application in preparing for the kit of head and neck cancer diagnosis and prognosis |
Non-Patent Citations (1)
| Title |
|---|
| JUNPENG WANG等: "Expression Profiling of Exosomal miRNAs Derived from the Peripheral Blood of Kidney Recipients with DGF Using High-Throughput Sequencing", 《BIOMED RESEARCH INTERNATIONAL》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112522262A (en) * | 2020-11-12 | 2021-03-19 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Pancreatic cancer-associated tRF and application thereof |
| CN113308530A (en) * | 2021-05-24 | 2021-08-27 | 嘉兴市第一医院 | Blood tsRNA marker for herpes zoster, preparation and application |
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