Blood marker for cervical cancer diagnosis and application thereof

Abstract

The invention discloses a blood marker for cervical cancer diagnosis and application thereof, and belongs to the field of biomarker detection. The blood marker is a serum exosome tRF-16, and the nucleotide sequence of the serum exosome tRF-16 is as shown in SEQ ID NO. 1. By separating serum exosome RNA of a cervical cancer patient and a healthy control group and combining data of a published database, high-specificity and high-sensitivity tsRNA related to the cervical cancer is found, and the diagnostic kit for clinical detection of the cervical cancer is developed. And simpler, more convenient and quicker help is provided for clinical early screening diagnosis and progress early warning of cervical cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomarker detection, and particularly to a blood biomarker for cervical cancer diagnosis and its application. Background Art

[0002] Cervical cancer (CC) is one of the common malignant tumors, seriously threatening health. Although with the progress of cervical cancer screening techniques, such as the combined use of Thinprep Cytologic Test (TCT) and Human Papilloma Virus (HPV) detection, colposcopy, and the wide application of cervical LEEP surgery and conization after Cervical intraepithelial neoplasia (CIN), the global incidence and mortality of CC have decreased significantly by 60%-80%, its overall incidence shows an upward trend. Early CC symptoms are often not obvious. When typical symptoms appear, the disease usually has progressed to the middle and late stages, and the tumor may have invaded surrounding tissues, even with lymph node metastasis or distant metastasis. This not only increases the complexity of surgical treatment but also affects the prognosis of patients. Therefore, early screening and diagnosis of CC are crucial for improving the survival rate of patients, and almost all clinical oncologists agree that finding reliable, accessible, and non- / minimally invasive biomarkers is very necessary for early cancer diagnosis and reducing its mortality.

[0003] Currently, the early screening of CC is mainly based on the combination of TCT and HPV, and subsequent tissue biopsy remains the gold standard for CC diagnosis; however, these detection methods are invasive operations associated with the risk of patient injury, and to some extent, there are also difficulties in sample collection and the situation that the target population cannot be fully universal, especially for patients without sexual life and those with vaginal atresia after menopause. Liquid biopsy (LB) is a method for obtaining real-time tumor heterogeneity information, early detecting metastases, and identifying minimal residual disease by detecting the levels of circulating tumor cells, cell-free circulating tumor DNA, coding and non-coding RNA, and extracellular vesicles (EVs) in blood and other biological fluids. Currently, the commonly used biomarkers for CC screening in clinical practice mainly include serum squamous cell carcinoma antigen (SCC) and Carbohydrate Antigen 125 (CA125), etc., but their low specificity and sensitivity often lead to missed diagnosis of patients, misjudgment of progression, and thus delayed treatment. Therefore, there is an urgent need to explore a more accurate, simple, and minimally invasive method for early diagnosis and screening of CC patients and predicting their progression.

[0004] The role of exosomes in the tumor microenvironment and their potential as biomarkers and therapeutic agents have made them a focus of research in the field of cancer. Studies have shown that exosomes are mainly enriched in short fragments of small RNAs, and transfer RNA-derived small RNAs (tsRNAs) are more enriched than other types of RNAs. Compared with microRNAs (miRNAs), they are more stable in body fluids, blood, and cells. With the development of high-throughput sequencing technology, tsRNAs in exosomes have also become one of the research hotspots. TsRNAs are abnormally expressed in different types of tumors and can act as biomarkers for tumor diagnosis and prognosis.

[0005] Different types of tsRNAs have multiple biological functions and are involved in various physiological and pathological processes through multiple mechanisms. The dysregulation of tsRNA expression is closely related to the occurrence and development of tumors. According to their specific functions, they can act as oncogenes or tumor suppressors, affecting key cellular processes such as cell proliferation, apoptosis, metastasis, chemoresistance, and immune regulation. They can also regulate the malignant progression of tumors through multiple mechanisms, including gene expression regulation, epigenetic modification regulation, protein translation regulation, alternative splicing regulation, etc. A number of studies have shown that tsRNAs play important roles in the occurrence and development of malignant tumors such as breast cancer, gastric cancer, gallbladder cancer, and non-small cell lung cancer. Given that there is no sufficient experimental research on tsRNAs in the field of cervical cancer screening and diagnosis, it is undoubtedly of great potential scientific research value to deeply explore the differential expression and mechanism of action of tsRNAs in the peripheral blood exosomes of cervical cancer patients. However, there is no existing technology on the research of tRF-16 in cervical cancer. Summary of the Invention

[0006] The object of the present invention is to provide a blood biomarker for cervical cancer diagnosis and its application to solve the problems existing in the above-mentioned prior art. By isolating serum exosome RNAs from cervical cancer patients and healthy control groups and combining the data of publicly published databases, the present invention discovers a highly specific and sensitive tsRNA related to cervical cancer and develops a diagnostic kit for cervical cancer that can be used for clinical detection, providing a more convenient and rapid help for the early screening diagnosis and progress warning of cervical cancer in clinical practice.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a blood biomarker for cervical cancer diagnosis, and the blood biomarker is serum exosome tRF-16, and the nucleotide sequence of the serum exosome tRF-16 is shown in SEQ ID NO.1.

[0009] The present invention also provides the use of a reagent for detecting the blood biomarker in the preparation of a kit for early screening and progression warning of cervical cancer.

[0010] Optionally, the reagent includes a primer pair for amplifying serum exosomal tRF-16, and the primer pair consists of a forward primer shown in SEQ ID NO.2 and a reverse primer shown in SEQ ID NO.3.

[0011] The primer pair is used for real-time quantitative fluorescence PCR detection of the serum exosomal tRF-16.

[0012] Optionally, the reagent further includes a reverse transcription stem-loop primer for the serum exosomal tRF-16, and the nucleotide sequence of the reverse transcription stem-loop primer is as shown in SEQ ID NO.4.

[0013] The present invention also provides a kit for early screening and progression warning of cervical cancer, including a reagent for detecting the blood biomarker.

[0014] Optionally, the reagent includes a primer pair for amplifying serum exosomal tRF-16, and the primer pair consists of a forward primer shown in SEQ ID NO.2 and a reverse primer shown in SEQ ID NO.3.

[0015] Optionally, the reagent further includes a reverse transcription stem-loop primer for the serum exosomal tRF-16, and the nucleotide sequence of the reverse transcription stem-loop primer is as shown in SEQ ID NO.4.

[0016] Optionally, it further includes a primer pair for amplifying an internal reference gene.

[0017] Optionally, the internal reference gene is the U6 gene.

[0018] Optionally, the primer pair for amplifying the internal reference gene consists of a forward primer shown in SEQ ID NO.5 and a reverse primer shown in SEQ ID NO.6.

[0019] The present invention discloses the following technical effects:

[0020] By separating serum exosomal RNA from cervical cancer patients and healthy control groups and combining the data in the publicly published database, the present invention discovers a tsRNA with high specificity and sensitivity related to cervical cancer and develops a diagnostic kit for cervical cancer that can be used for clinical detection, providing a more convenient and rapid assistance for the early screening diagnosis and progression warning of cervical cancer in clinical practice. Brief Description of the Drawings

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0022] Figure 1 Volcano plot (a) and heat map (b) of differentially expressed tsRNAs in serum exosomes of the cervical cancer group and the healthy control group;

[0023] Figure 2 Box plot of the true expression levels of significantly dysregulated tsRNAs in serum exosomes of the cervical cancer group and the healthy control group;

[0024] Figure 3 Volcano plot (a) and heat map (b) of differentially expressed tsRNAs in cervical cancer tissues and control tissues in the TCGA database;

[0025] Figure 4 Venn diagram (a) of differentially expressed tsRNAs in the TCGA database and differentially expressed tsRNAs in 6 serum exosome samples, and position and sequence of tRF-16 (b);

[0026] Figure 5 Relative expression levels of tRF-16 in serum exosomes of cervical cancer patients at different stages (a), and its relationships with the pathological differentiation degree grading (b) and lymph node metastasis status of cervical cancer patients (c);

[0027] Figure 6 Receiver operating characteristic curve of the diagnostic effect of tRF-16 for cervical cancer (a) and receiver operating characteristic curve of the diagnostic effect of tRF-16 combined with CA-125 and SCC for cervical cancer (b). Detailed implementation manners

[0028] Now, the various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0031] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0032] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0033] The present invention analyzes the copy differences and expression differences of tsRNAs between cervical cancer patients and healthy controls through high-throughput sequencing, screens and verifies the serum exosomal tsRNAs with significant differences in 35 serum samples of cervical cancer patients and 12 healthy control samples by RT-qPCR technology, and combines the differential tsRNA expression profiles obtained from the TCGA database to finally screen the serum exosomal diagnostic marker tRF-16 related to cervical cancer. On this basis, a cervical cancer diagnostic kit is developed, and the kit includes stem-loop primers for tsRNA (used in the reverse transcription process), upstream and downstream primers (used in qRT-PCR), II 1st Strand cDNA Synthesis Kit reverse transcription kit (Vazyme, China); PCR reaction reagents: AceQ Universal SYBR qPCR Master Mix (Vazyme, China), etc.

[0034] In the present invention, GraphPad Prism 8 software was used to perform statistical analysis on the experimental data. For RT-qPCR data, it was presented in the form of mean ± standard deviation (SD) to intuitively reflect the central tendency and dispersion degree of the experimental results. In terms of statistical tests, non-parametric Mann-Whitney test was used for RT-qPCR data to avoid affecting the accuracy of the results due to the data not conforming to the normal distribution. For other experimental data, t-test was used for comparison. DESeq2 package in R software (version 4.3.2) was used for differential expression analysis, and pROC package was used for receiver operating characteristic curve analysis. In all statistical analyses, P<0.05 was used as the criterion for judging that the difference was statistically significant.

[0035] The relevant sequences of tRF-16-RPM830D (tRF-16) used in the examples of the present invention are shown in Table 1. In Table 1, F represents the upstream primer; RT represents the reverse transcription stem-loop primer; R represents the downstream primer.

[0036] Table 1 Relevant sequences of tRF-16-RPM830D (tRF-16)

[0037]

[0038] Example 1 Screening and verification of tsRNA

[0039] 1. Collection of clinical samples

[0040] The clinical samples were from the patients diagnosed and treated in the Affiliated Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital) from 2022 to 2023, a total of 55 cases, including 39 cervical cancer samples and 16 healthy control samples. All patients understood and signed the informed consent form.

[0041] Inclusion criteria: (1) Aged between 50-65 years old; (2) Clear pathological diagnosis; (3) Without any other history of malignant tumors; (4) The patients did not receive any anti-tumor treatment before surgery.

[0042] Exclusion criteria: (1) Severe cardiovascular and cerebrovascular diseases or lung diseases; (2) Recurrent cases; (3) Those who had received anti-tumor treatments such as chemotherapy, immunotherapy or radiotherapy before surgery; (4) Combined with other malignant tumors. All healthy controls were strictly screened to ensure that their blood biochemical indexes and tumor marker test results were within the normal range, and other related diseases had been excluded.

[0043] 2. Pretreatment of clinical serum specimens

[0044] For the above-mentioned clinical samples, 10 mL of fresh peripheral venous blood was collected using a vacuum blood collection device as the clinical blood sample, and placed in an anticoagulant tube containing Ethylene Diamine Tetraacetic Acid (EDTA). To avoid hemolysis, it was left standing at room temperature for 30 min. Subsequently, it was centrifuged for 10 min (3500 g, 4 °C) to remove blood cells and debris. The upper light yellow transparent serum was immediately aliquoted into sterile EP tubes and stored at -80 °C for a long time (avoid repeated freezing and thawing).

[0045] 3. Extraction of serum exosomes:

[0046] ① Take 2 mL of the serum sample thawed at room temperature, add 2 mL of Phosphate Buffered Saline (PBS) solution, centrifuge at 2000 g for 10 min to remove cells, dead cells, etc.; ② Transfer the supernatant to a new centrifuge tube and centrifuge for 30 min (16500 g, 4 °C) to remove cell debris; ③ Transfer the supernatant to an ultracentrifuge tube and centrifuge for 120 min (100000 g, 4 °C), reserve 2 mL of the supernatant in the tube, and remove the remaining liquid with a pipette; ④ Add 4 mL of PBS solution to the supernatant, gently pipette to mix well to wash the exosomes, centrifuge for 70 min (100000 g, 4 °C), and remove the supernatant; ⑤ Repeat step 4 at least 4 times to thoroughly wash the exosomes; ⑥ Resuspend the exosomes with 100 μL of PBS solution and store at -80 °C for later use.

[0047] 4. Screening of differentially expressed tsRNAs by high-throughput sequencing of small RNAs

[0048] (1) Sample preparation and RNA extraction

[0049] Randomly select serum exosomes from 4 cervical cancer patients and 4 healthy controls, extract total RNA of serum exosomes using the Trizol method, and recover small RNA fragments by PAGE electrophoresis.

[0050] (2) Library construction and prediction sequencing

[0051] Adapter ligation: Use specific primers to ligate 3′ and 5′ end adapters to small RNA molecules.

[0052] Library preparation: Based on the Agilent 2100 Bioanalyzer, quality detection and construction of the tsRNA library were carried out.

[0053] High-throughput sequencing: 50 bp single-end sequencing (SE50) was performed on the Illumina NextSeq platform. All sequencing and library construction work were completed by Aksomics (Shanghai, China).

[0054] (3) Data analysis

[0055] The raw sequencing data was subjected to quality control, alignment, and normalization. Significantly differentially expressed tsRNAs between the cervical cancer group and the healthy control group were screened through differential expression analysis, as Figure 1 shown.

[0056] Figure 1 showed that through small RNA high-throughput sequencing analysis of the serum exosomes of 4 cervical cancer patients and 4 healthy controls, a total of 70 tsRNAs differentially expressed in the serum exosomes of cervical cancer patients were found, including 36 down-regulated tsRNAs and 34 up-regulated tsRNAs.

[0057] 5. Verification of the levels of differentially expressed tsRNAs in serum exosome samples by RT-qPCR technology

[0058] (1) Total RNA of serum exosomes from all clinical samples was extracted, and reverse transcription of tsRNAs was performed using the II 1st Strand cDNA Synthesis Kit reverse transcription kit (Vazyme, China) to synthesize cDNA (stem-loop primers were used in this process), obtaining cDNA samples;

[0059] (2) RT-qPCR detection was performed using the AceQ Universal SYBR qPCR Master Mix kit (Vazyme, China) (upstream and downstream primers were used in this process), and U6 snRNA was used as an internal reference to correct the expression levels of tsRNAs, and 18 tsRNAs with the most significant differential expression in the high-throughput sequencing results were detected.

[0060] Through RT-qPCR verification, a total of 6 tsRNAs (tRF-15-V29K9U, tRF-16-RPM830D, tRF-18-HRERXFD2, tRF-22-WE8SPOX52, tRF-24-R29P4P9LH9, and tRF-30-87R8WP9N1EWJ) were screened, and their expression levels were consistent with the sequencing results and showed statistically significant differences, indicating that these tsRNAs have potential application value in the screening and diagnosis of CC( Figure 2 ).

[0061] 6. Screening of differential genes in the TCGA database

[0062] The sequencing data of tsRNAs from 307 tumor tissues and 3 control tissues were obtained using the TCGA database, and differential analysis was performed on them. By analyzing the sequencing data of cervical cancer in the TCGA database, a total of 88 differentially expressed tsRNAs were identified, among which 62 were up-regulated and 26 were down-regulated( Figure 3 ).

[0063] Intersection analysis was performed on the tsRNAs with significant differential expression between the two groups identified in the TCGA database, the sequencing data of tsRNAs in the serum exosomes of the present invention, and the RT-qPCR validation set, and the final target serum tsRNA marker was screened out as serum exosome tRF-16( Figure 4 ).

[0064] 7. Expand clinical sample verification

[0065] The expression level of the screened serum exosome marker (tRF-16) and its relationship with the FIGO stage and tissue differentiation degree of cervical cancer patients were verified in the serum samples of the remaining 35 cervical cancer patients and 12 healthy control samples using RT-qPCR technology, and the results are as Figure 3 shown.

[0066] As Figure 5 known, tRF-16 was significantly increased in cervical cancer patients, and its expression level was closely related to the FIGO stage and tissue differentiation degree of the patients.

[0067] 8. Analyze the clinical value of differential tsRNAs in the diagnosis of cervical cancer

[0068] Receiver operating characteristic curve analysis found that the AUC of tRF-16 for the diagnosis of cervical cancer was 0.7369( Figure 6 a)), and the AUC of the combined tumor markers CA125 and SCC for the diagnosis of cervical cancer was 0.9595( Figure 6 b)).

[0069] Example 2 Construction of a diagnostic kit for cervical cancer serum exosome tsRNA

[0070] The kit includes a stem-loop primer for tRF-16 (SEQ ID NO.4, used in the reverse transcription process), upstream and downstream primers (SEQ ID NO.2-3, used in qRT-PCR), II 1st Strand cDNA Synthesis Kit reverse transcription kit (Vazyme, China); PCR reaction reagents: AceQ Universal SYBR qPCR Master Mix (Vazyme, China), etc.

[0071] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the spirit of the design of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A blood biomarker for cervical cancer diagnosis, characterized in that, The blood biomarker is serum exosomal tRF-16, and the nucleotide sequence of the serum exosomal tRF-16 is as shown in SEQ ID NO.

1.

2. Use of a reagent for detecting the blood biomarker according to claim 1 in the preparation of a kit for early screening and progression warning of cervical cancer.

3. The application according to claim 2, wherein The reagent comprises a primer pair for amplifying the serum exosomal tRF-16, and the primer pair consists of a forward primer shown in SEQ ID NO.2 and a reverse primer shown in SEQ ID NO.

3.

4. The application according to claim 3, characterized in that, The reagent further comprises a reverse transcription stem-loop primer for the serum exosomal tRF-16, and the nucleotide sequence of the reverse transcription stem-loop primer is as shown in SEQ ID NO.

4.

5. A kit for early screening and progression warning of cervical cancer, characterized in that, Comprising a reagent for detecting the blood biomarker according to claim 1.

6. The kit according to claim 5, characterized in that, The reagent comprises a primer pair for amplifying the serum exosomal tRF-16, and the primer pair consists of a forward primer shown in SEQ ID NO.2 and a reverse primer shown in SEQ ID NO.

3.

7. The kit according to claim 6, wherein The reagent further comprises a reverse transcription stem-loop primer for the serum exosomal tRF-16, and the nucleotide sequence of the reverse transcription stem-loop primer is as shown in SEQ ID NO.

4.

8. The kit according to claim 5, characterized in that, Further comprising a primer pair for amplifying an internal reference gene.

9. The kit according to claim 8, characterized in that, The internal reference gene is the U6 gene.

10. The kit according to claim 9, wherein The primer pair for amplifying the internal reference gene consists of a forward primer shown in SEQ IDNO.5 and a reverse primer shown in SEQ ID NO.6.

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