CN112646892B

CN112646892B - Group of piRNA biomarkers for early diagnosis of breast cancer and application thereof

Info

Publication number: CN112646892B
Application number: CN202011645563.8A
Authority: CN
Inventors: 洪宏海; 尹萍; 夏勇
Original assignee: Third Affiliated Hospital of Guangzhou Medical University
Current assignee: Third Affiliated Hospital of Guangzhou Medical University
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2022-02-01
Anticipated expiration: 2040-12-30
Also published as: CN112646892A

Abstract

The invention discloses a group of piRNA biomarkers for breast cancer diagnosis and application thereof, belonging to the fields of molecular biology and oncology, wherein the piRNA biomarkers are piR651, piR17458 and piR 20485; wherein the nucleotide sequence of piR651 is shown in SEQ ID NO:1, the nucleotide sequence of piR17458 is shown as SEQ ID NO:2 is shown in the specification; piR20485 has the nucleotide sequence shown in SEQ ID NO:3, respectively. The invention finds that piR651, piR17458 and piR20485 can be used as a breast cancer diagnosis marker in combination, and is applied to breast cancer screening, particularly early breast cancer screening, so as to make up the defects of poor specificity and poor sensitivity of the existing breast cancer diagnosis marker and the early breast cancer diagnosis marker.

Description

Group of piRNA biomarkers for early diagnosis of breast cancer and application thereof

Technical Field

The invention relates to the fields of molecular biology and oncology, in particular to a group of piRNA biomarkers for early diagnosis of breast cancer and application thereof.

Background

Breast cancer is the most common malignancy in women and is one of the most lethal. The cause of breast cancer lethality is not due to the growth of carcinoma in situ itself, but rather distant metastasis. Once breast cancer metastasizes, its five-year survival is only 23%. 70-80% of breast cancer patients found in China are middle and advanced tumor patients. Breast cancer has become a public health problem to be solved and is a hot spot of clinical research. Studies have shown that early diagnosis and surgical resection of breast cancer can effectively improve the quality of life, survival and prognosis of patients. The current diagnosis of breast cancer comprises serum markers, pathological diagnosis, imaging and clinical symptoms, but the indexes are often appeared at a certain stage of disease development, and the diagnosis of the disease is influenced. In addition, the existing breast cancer serum markers also have the defects of poor specificity and insufficient sensitivity. Therefore, the search for serum markers with good specificity and high sensitivity is particularly important for early diagnosis of breast cancer.

piRNA is a small non-coding RNA interacting with Piwi protein, silences gene expression in germ cells through a piRNA-Piwi complex, and researches show that the piRNA is increasingly involved in the occurrence and prognosis of malignant tumors. Many documents report that piRNAs are also present in serum, are closely related to the progression of the disease, and even can serve as diagnostic markers for the disease. However, the expression levels of circulating piRNAs in breast cancer patients and their diagnostic value in breast cancer are not clear.

Disclosure of Invention

The invention aims to provide a group of piRNA biomarkers for breast cancer diagnosis and application thereof, so as to solve the problems in the prior art. The invention finds that piR651, piR17458 and piR20485 in serum can be used as a breast cancer diagnosis marker in combination, and is applied to breast cancer screening, particularly early breast cancer screening, so as to make up the defects of poor specificity and poor sensitivity of the existing breast cancer diagnosis marker and the early breast cancer diagnosis marker.

To achieve the above objects, the present invention provides a set of piRNA biomarkers for breast cancer diagnosis, wherein the piRNA biomarkers are piR651, piR17458 and piR 20485; wherein the nucleotide sequence of piR651 is shown in SEQ ID NO:1, the nucleotide sequence of piR17458 is shown as SEQ ID NO:2 is shown in the specification; piR20485 has the nucleotide sequence shown in SEQ ID NO:3, respectively.

Further, the expression level of the piRNA biomarker is simultaneously down-regulated in the serum of a breast cancer patient.

Further, the breast cancer diagnosis is an early breast cancer diagnosis.

The invention also provides application of the piRNA biomarkers for diagnosing the breast cancer according to any one of the groups in preparation of breast cancer diagnosis products.

Further, the breast cancer diagnosis product detects the level of the piRNA biomarker by using qRT-PCR, blot hybridization, in situ hybridization array hybridization, gene chip, northern hybridization method, ribozyme protection analysis technique, RAKE method, next generation sequencing method, or single molecule sequencing method to diagnose breast cancer.

Furthermore, the breast cancer diagnosis product is a kit, a PCR detection reagent or a chip.

The invention discloses the following technical effects: the invention firstly discovers a new application of the combination of the serum piR651, the serum piR17458 and the serum piR20485 as a breast cancer diagnostic marker and an early-stage breast cancer diagnostic marker. Specifically, we identified for the first time that piR651, piR17458 and piR20485 were present in the serum of breast cancer patients, at levels significantly lower than those of normal healthy people. In addition, ROC curve analysis results show that piR651, piR17458 and piR20485 in serum can be used as breast cancer diagnosis markers in combination and applied to breast cancer screening, particularly early breast cancer screening, so as to make up the defects of poor specificity and insufficient sensitivity of the existing breast cancer diagnosis markers and early breast cancer diagnosis markers.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.

FIG. 1 is a graph of the levels of piR651, piR17458 and piR20485 in sera from breast cancer patients;

FIG. 2 shows the results of ROC curve analysis of piR651, piR17458 and piR20485 in serum.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in further detail below.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available and the experimental methods used are conventional in the art.

Example 1 qRT-PCR detection of piR651, piR17458 and piR20485 levels in serum of breast cancer patients

Human serum from breast cancer patients and healthy persons was obtained from the third hospital affiliated to Guangzhou medical university. Serum was centrifuged at 10000rpm for 10min at high speed to remove cell debris. RNA was extracted using the QIAGEN MIRNeasy Serum/Plasma Kit, reverse transcription was performed using the MiScript II RT Kit, and real-time fluorescent PCR was performed using the MiScript SYBR Green PCR Kit.

And (3) PCR reaction system: 10. mu.l of 2X SYBR Green PCR mixture, 2. mu.l of universal primers, 4. mu.l of RNase free water, 2. mu.l of cDNA template and 2. mu.l of corresponding specific piRNA primers. Specific primer sequences are shown in Table 1.

PCR reaction procedure: 15min at 95 ℃; at 94 ℃ for 15s, at 55 ℃ for 30s, and at 70 ℃ for 30s, for a total of 40 amplification cycles.

TABLE 1 qRT-PCR primers

The results of piRNA expression analysis using small RNA U6 as a reference are shown in FIG. 1, and piR651, piR17458 and piR20485 levels in the serum of breast cancer patients are significantly lower than those of healthy people, compared with those of healthy people.

Example 2ROC curve analysis of serum piR651, piR17458 and piR20485 in combination for application value in breast cancer diagnosis

The SPSS17.0 software is adopted to analyze the application value of piR651, piR17458 and piR20485 in the breast cancer diagnosis. The results are shown in fig. 2, and the ROC curve analysis result shows that the AUC area of the combination of piR651, piR17458 and piR20485 in breast cancer diagnosis is 0.8113, which indicates that the combination of serum piR651, piR17458 and piR20485 has better clinical value in breast cancer diagnosis; meanwhile, ROC curve analysis shows that the sensitivity and the specificity of the combination of piR651, piR17458 and piR20485 to breast cancer diagnosis are 67.7% and 84.8% respectively; the 95% confidence interval is 0.3-0.7, and the method has the characteristics of good specificity and high sensitivity.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Sequence listing

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Claims

1. The application of a group of piRNA biomarkers for breast cancer diagnosis in the preparation of breast cancer diagnosis products is characterized in that: the piRNA biomarkers are piR651, piR17458, and piR 20485;

wherein, the nucleotide sequence of piR651 is shown as SEQ ID NO. 1, and the nucleotide sequence of piR17458 is shown as SEQ ID NO. 2; piR20485 has the nucleotide sequence shown in SEQ ID NO. 3;

(ii) the expression level of the piRNA biomarker is simultaneously down-regulated in the serum of a breast cancer patient; the breast cancer diagnosis is an early breast cancer diagnosis.

2. Use according to claim 1, characterized in that: the breast cancer diagnosis product detects the level of the piRNA biomarker by using qRT-PCR, imprinting hybridization, in situ hybridization array hybridization, gene chip, northern hybridization method, ribozyme protection analysis technique, RAKE method, secondary sequencing method, or single molecule sequencing method to diagnose breast cancer.

3. Use according to claim 1 or 2, characterized in that: the breast cancer diagnosis product is a kit, a PCR detection reagent or a chip.