Application of tRF-Ala-CGC-013 in the preparation of products for predicting lymph node metastasis of esophageal squamous cell carcinoma

Abstract

The present invention discloses the application of tRF‑Ala‑CGC‑013 in the preparation of a product for predicting lymph node metastasis of esophageal squamous cell carcinoma, and the nucleotide sequence of the tRF‑Ala‑CGC‑013 is shown in SEQ ID No.1. The present invention's research shows that tRF‑Ala‑CGC‑013 is highly expressed in esophageal squamous cell carcinoma tissues and plasma samples with lymph node metastasis, and its expression in the plasma of esophageal squamous cell carcinoma patients has certain specificity compared with other cancers with lymph node metastasis. Lymph node metastasis of esophageal squamous cell carcinoma can be relatively accurately predicted by plasma tRF‑Ala‑CGC‑013, with an AUC value of 0.81, which has good specificity and sensitivity. The prediction of the lymph node metastasis status of esophageal squamous cell carcinoma using the biomarkers provided by the present invention can provide clinicians with more detailed disease information so as to formulate more effective personalized prevention and treatment plans.

Description

Technical Field

[0001] The present invention belongs to the technical field of esophageal squamous cell carcinoma diagnosis, and specifically relates to the application of tRF-Ala-CGC-013 in the preparation of a product for predicting lymph node metastasis of esophageal squamous cell carcinoma. Background Art

[0002] As one of the cancers with high morbidity and mortality in my country, esophageal squamous cell carcinoma has brought a heavy burden to the social medical system. Although the application of comprehensive treatment plans such as surgery combined with chemotherapy and immunotherapy in recent years has significantly improved the treatment effect, the overall survival rate of patients still needs to be improved.

[0003] Among them, lymph node metastasis of esophageal squamous cell carcinoma is one of the main reasons for poor prognosis of patients. For patients with advanced esophageal squamous cell carcinoma without distant metastasis but with lymph node metastasis limited to the thoracic cavity, accurate preoperative judgment of whether they have lymph node metastasis can avoid excessive surgical treatment for advanced patients and guide mid-stage patients to receive neoadjuvant therapy, thereby improving patient prognosis. Therefore, accurate preoperative judgment of lymph node metastasis has a great impact on the choice of treatment options for esophageal squamous cell carcinoma.

[0004] However, the current preoperative prediction of esophageal squamous cell carcinoma lymph node metastasis status mainly relies on doctors' experience based on CT imaging. This method is highly subjective, time-consuming and inaccurate. Conventional CT uses the short-axis diameter of lymph nodes ≥1 cm as the basis for metastasis. Existing literature shows that the sensitivity of CT and PET / CT for lymph node metastasis is 50% and 57%, respectively, and the specificity is 83% and 85%, respectively. Foley et al. found that the accuracy, sensitivity and specificity of enhanced CT in detecting esophageal cancer lymph node metastasis were 54.5%, 55.4% and 57.1%, respectively. 18 F-FDG PET / CT is also limited in its role in evaluating the status of mediastinal lymph node metastasis in esophageal squamous cell carcinoma with a diameter of <15 mm. A meta-analysis of 14 retrospective studies showed that in patients with esophageal squamous cell carcinoma who did not receive neoadjuvant therapy before surgery, 18 The sensitivity of F-FDG PET / CT for the assessment of preoperative lymph node metastasis was 57%, with a positive likelihood ratio of 2.5. These data suggest that 18 F-FDG PET / CT is not sensitive and has a low accuracy rate in evaluating esophageal squamous cell carcinoma lymph node metastasis. At the same time, CT and PET / CT also have radiation side effects. Although the commonly used methods such as mediastinoscopy or endobronchial ultrasound lymph node biopsy are reliable, they are invasive operations that can cause a variety of complications and cause great trauma to the body. In addition, these examination methods require that the hospital equipment and doctors reach a high standard, making it difficult to promote homogeneity. Therefore, it is very important to discover and identify biological targets for esophageal squamous cell carcinoma lymph node metastasis.

[0005] Recently, tsRNA (tRNA-derived small RNAs) derived from tRNA have attracted much attention. tsRNA is formed by cleavage of precursor tRNA or mature tRNA, and is divided into two categories according to the cleavage position and length, namely tiRNAs (tRNA halves) and tRFs (tRNA-derived fragments). tRFs are formed by cleavage of specific RNA cleavage enzymes in the cytoplasm, and have the characteristics of high conservation and high stability, and are expected to be used as new tumor markers. In addition, abnormal expression of tRFs is correlated with cancer metastasis, drug resistance, etc.

[0006] The clinical application value of tRFs has been explored in many cancers. Several studies have reported that tRFs can be used for the diagnosis and treatment of tumors, indicating that tRFs may become new diagnostic biomarkers and therapeutic targets for tumors. The latest study confirmed that compared with healthy people, tRF-Gly-CCC-046, tRF-Tyr-GTA-010 and tRF-Pro-TGG-001 were downregulated in the tissues and serum of patients with early breast cancer. These three tRFs can be used as circulating biomarkers for the early diagnosis of breast cancer, with an area under the curve (AUC) of 0.7987. Other studies have found that from tRNA Glu , tRNA Asp , tRNA Gly and tRNA Tyr A new class of tRFs derived from this method disrupts the stability of multiple oncogenic transcripts in breast cancer cells by replacing 3'UTRs on the RNA binding protein YBX1, thereby inhibiting the invasion and metastasis of breast cancer cells, indicating that these tRFs can be used as potential therapeutic targets to inhibit the progression and metastasis of breast cancer. With the continuous advancement of sequencing technology, more and more evidence shows that tRFs can be used as biomarkers for multiple cancers, such as non-small cell lung cancer, colorectal cancer, gastric cancer and prostate cancer, and have broad prospects in the early diagnosis and treatment of cancer.

[0007] At present, tRFs-related research in the field of esophageal squamous cell carcinoma is still in its infancy, and there is no clinically accurate tRF-related research or patent that can be used to predict lymph node metastasis of esophageal squamous cell carcinoma. Therefore, exploring new tumor markers from the perspective of tRFs to predict lymph node metastasis of esophageal squamous cell carcinoma can provide a new means for clinical diagnosis and treatment. Summary of the invention

[0008] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and provide an application of tRF-Ala-CGC-013 in the preparation of a product for predicting lymph node metastasis of esophageal squamous cell carcinoma.

[0009] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0010] The present study found that the expression level of tRF-Ala-CGC-013 in tumor tissue and plasma samples of esophageal squamous cell carcinoma patients with lymph node metastasis (22 cases) was significantly higher than that of esophageal squamous cell carcinoma patients without lymph node metastasis (15 cases), esophageal adenocarcinoma (7 cases), esophageal benign tumors (7 cases) and esophageal adenocarcinoma patients with lymph node metastasis (5 cases). Therefore, tRF-Ala-CGC-013 can be used as a marker to indicate the lymph node metastasis status of esophageal squamous cell carcinoma. In addition, this study found that the expression of tRF-Ala-CGC-013 in tumor tissue and plasma samples of esophageal squamous cell carcinoma (15 cases of lymph node-negative esophageal squamous cell carcinoma and 22 cases of lymph node-positive esophageal squamous cell carcinoma) was positively correlated, and in the plasma samples of esophageal squamous cell carcinoma patients with lymph node metastasis (22 cases) before and after surgery, the level of tRF-Ala-CGC-013 decreased significantly after surgery, further indicating that tRF-Ala-CGC-013 can be used as a marker for lymph node metastasis of esophageal squamous cell carcinoma. Further studies also found that compared with the plasma of gastric cancer (13 cases) and colon cancer (18 cases) patients with lymph node metastasis, the expression of tRF-Ala-CGC-013 in the plasma of esophageal squamous cell carcinoma patients was more prominent, indicating that it has a certain degree of specificity in esophageal squamous cell carcinoma patients. Finally, through ROC curve analysis, the AUC value of tRF-Ala-CGC-013 was 0.81. AUC values ​​exceeding 0.7 can be considered to have good specificity and sensitivity, that is, when plasma tRF-Ala-CGC-013 is used to predict lymph node metastasis of esophageal squamous cell carcinoma, it has good specificity and sensitivity. The above results show that the expression level of tRF-Ala-CGC-013 in plasma can more accurately predict the lymph node metastasis of esophageal squamous cell carcinoma, and therefore tRF-Ala-CGC-013 can be used as an effective marker for predicting lymph node metastasis of esophageal squamous cell carcinoma.

[0011] Tumor-derived tRFs in plasma are a new type of biomarker that is stable, minimally invasive, and can be monitored in real time, which can greatly improve the sensitivity and specificity of disease diagnosis. In this regard, the successful development of the plasma biomarker tRF-Ala-CGC-013 will play an important role in predicting lymph node metastasis of esophageal squamous cell carcinoma. This technical solution helps clinicians quickly and accurately understand the patient's condition and take more personalized prevention and treatment plans in a timely manner.

[0012] Therefore, the present invention provides the use of tRF-Ala-CGC-013 in preparing a product for predicting lymph node metastasis of esophageal squamous cell carcinoma, and the nucleotide sequence of the tRF-Ala-CGC-013 is shown in SEQ ID No. 1: ATCCCCGGCATCTCCACCA.

[0013] The present invention also provides the use of the preparation for detecting the expression level of the above-mentioned tRF-Ala-CGC-013 in the preparation of a product for predicting lymph node metastasis of esophageal squamous cell carcinoma. The detection of the expression level of tRF-Ala-CGC-013 can be achieved by conventional technical means in the art, including but not limited to high-throughput sequencing technology, real-time quantitative PCR technology or microfluidic biochip technology, etc. Among them, real-time quantitative PCR technology has the advantages of being simple and easy to repeat.

[0014] Specifically, the high-throughput sequencing method is as follows: total plasma RNA is extracted, and the purified RNA is sequenced using rtStar TM tRF&tiRNA pretreatment kit was used to remove genetic modifications. tRF-seq library was constructed by RNA ligation, cDNA synthesis, PCR amplification and other methods to obtain DNA fragments of corresponding length. DNA fragments were subjected to 50 cycles on the NextSeq system and base analysis was performed using Solexa pipeline. Sequencing quality was checked and trimmed reads were aligned with reference sequence bases.

[0015] The real-time quantitative PCR method was as follows: total plasma RNA was extracted and the All-in-One TM miRNA detection kit, using poly (A) tailing method for real-time quantitative PCR detection of tRF. Amplification was performed on the Roche Light Cycler 480 system, and U6 RNA was selected as the internal control of tissue samples, while the plasma expression level was homogenized to miR-16. -ΔΔCt The expression of relevant tRFs was detected by the method, and each sample was tested three times.

[0016] Microfluidic biochip detection: 1) Preparation of microfluidic biochip: The microfluidic biochip consists of a PDMS layer and a functional substrate. The functional substrate is constructed by GO and PLL locally assembled on a clean glass substrate. GO is used to fix the DNA probe labeled with FAM. Once the target RNA hybridizes with the probe, the DNA-RNA duplex will be released, and then the fluorescently labeled DNA-RNA duplex will be collected using PLL. The PDMS layer consists of a control layer and a flow layer, and a control valve is used to guide the directional flow of the liquid in the microchannel. The flow layer includes microchannels and microcavities. The microchannels are aligned with the GO side of the functional substrate to facilitate the fixation of DNA probes and capture of RNA, while the microcavities are aligned with the PLL side for fluorescent signal detection of DNA-RNA duplexes. 2) Quantification of tRF concentration in samples by biochip: In order to verify the detection process of the microfluidic bioassay chip, a DNA probe with FAM fluorescence (10 -4 M, 2 μL) was adsorbed on the GO surface, and then the target RNA (10 -9M, 2 μL) was loaded into the microchannel and incubated for 25 minutes, and then the DNA probe that combined with the target RNA to form a duplex was suspended on the GO surface, and the DNA-RNA duplex was washed to the PLL end of the channel with PBS buffer so that the duplex was adsorbed by PLL. Finally, the substrate was scanned for FAM fluorescence with a laser scanner with a wavelength of 532 nm, and a linear standard curve of fluorescence intensity-different concentrations was generated for the synthetic sequence of the target RNA, and the fluorescence intensity was analyzed using GenePix Pro 7.0 software.

[0017] Furthermore, the preparation is a high-throughput sequencing preparation, a real-time quantitative PCR detection preparation or a microfluidic biochip detection preparation.

[0018] Furthermore, the real-time quantitative PCR detection preparation contains amplification primers and internal reference primers for detecting tRF-Ala-CGC-013.

[0019] Furthermore, the amplification primers include an upstream primer and a downstream primer, and the nucleotide sequences thereof are shown in SEQ ID No. 2: 5'-GATCCCCGGCATCTCCACCA-3' and SEQ ID No. 3: 5'-GTGCAGGGTCCGAGGT-3', respectively.

[0020] The present invention also provides a detection kit for predicting lymph node metastasis of esophageal squamous cell carcinoma, wherein the detection kit contains a preparation for detecting the expression of tRF-Ala-CGC-013.

[0021] Furthermore, the detection kit also contains a microfluidic biochip.

[0022] Furthermore, the preparation includes real-time quantitative PCR amplification primers and internal reference primers for detecting tRF-Ala-CGC-013; the amplification primers include upstream primers and downstream primers, and their nucleotide sequences are shown in SEQ ID No.2 and SEQ ID No.3, respectively.

[0023] Furthermore, the detection sample of the detection kit is an esophageal squamous cell carcinoma tumor tissue sample or a plasma sample.

[0024] Furthermore, the detection sample of the detection kit is a plasma sample.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention provides the use of tRF-Ala-CGC-013 as a marker for predicting lymph node metastasis of esophageal squamous cell carcinoma in the preparation of a product for predicting lymph node metastasis of esophageal squamous cell carcinoma. The present invention shows that tRF-Ala-CGC-013 is highly expressed in esophageal squamous cell carcinoma tissue and plasma samples with lymph node metastasis. Compared with gastric cancer and colon cancer patients with lymph node metastasis, tRF-Ala-CGC-013 is expressed in the plasma of esophageal squamous cell carcinoma patients with certain specificity. Therefore, the tRF-Ala-CGC-013 can be used as a marker for predicting whether lymph nodes in esophageal squamous cell carcinoma are metastatic. The lymph node metastasis of esophageal squamous cell carcinoma can be relatively accurately predicted by plasma tRF-Ala-CGC-013, and after ROC curve analysis, the AUC value of tRF-Ala-CGC-013 is 0.81, which has good specificity and sensitivity. The use of the biomarkers provided by the present invention to predict the status of lymph node metastasis in esophageal squamous cell carcinoma can provide clinicians with more detailed disease information so as to formulate more effective personalized prevention and treatment plans. In addition, the technical solution also provides new ideas for the prevention and treatment of other similar diseases, and has broad application prospects and market value. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Figure 2 is the expression of tRF-Ala-CGC-013 in tumor tissues and plasma of patients with esophageal cancer; A is the expression correlation of tRF-Ala-CGC-013 in plasma of patients with esophageal squamous cell carcinoma without lymph node metastasis (LN-negative ESCC), patients with esophageal squamous cell carcinoma with lymph node metastasis (LN-positive ESCC), patients with esophageal adenocarcinoma without lymph node metastasis (LN-negative EAD), patients with benign esophageal tumor (benign tumor) and patients with esophageal adenocarcinoma with lymph node metastasis (LN-positive EAD); B is the expression correlation of tRF-Ala-CGC-013 in plasma of patients with esophageal squamous cell carcinoma without lymph node metastasis (LN-negative ESCC), patients with esophageal squamous cell carcinoma with lymph node metastasis (LN-positive ESCC), patients with esophageal adenocarcinoma without lymph node metastasis (LN-negative The expression correlation of esophageal adenocarcinoma with lymph node metastasis (LN-positive EAD) and esophageal benign tumor (benign tumor) was detected in the tumor tissues of patients with esophageal adenocarcinoma and esophageal adenocarcinoma with lymph node metastasis (LN-positive EAD).

[0028] Figure 2Figure 2 is the correlation expression pattern of tRF-Ala-CGC-013 in esophageal squamous cell carcinoma tissue and plasma samples; A is the expression correlation analysis result of tRF-Ala-CGC-013 in tumor tissue and plasma samples in esophageal squamous cell carcinoma population; B is the change of tRF-Ala-CGC-013 expression level in plasma samples of esophageal squamous cell carcinoma patients with positive lymph node metastasis before and after surgery.

[0029] Figure 3 The expression of tRF-Ala-CGC-013 in the plasma of patients with esophageal squamous cell carcinoma (ESCC), gastric cancer, and colon cancer with lymph node metastasis; A is the result of real-time quantitative PCR analysis, and B is the result of ROC curve analysis. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0031] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0032] Example 1 Expression of tRF-Ala-CGC-013 in tumor tissues and plasma of patients with esophageal squamous cell carcinoma, esophageal adenocarcinoma, and esophageal benign tumors

[0033] The present invention first studies the expression of tRF-Ala-CGC-013 (sequence as shown in SEQ ID No. 1) in tumor tissue and plasma of patients with esophageal squamous cell carcinoma, esophageal adenocarcinoma, and esophageal benign tumors. The specific research samples are tumor tissue and plasma samples of patients with esophageal squamous cell carcinoma without lymph node metastasis (15 cases), patients with esophageal squamous cell carcinoma with lymph node metastasis (22 cases), patients with esophageal adenocarcinoma (7 cases), patients with esophageal benign tumors (7 cases), and patients with esophageal adenocarcinoma with lymph node metastasis (5 cases).

[0034] The specific experimental steps are as follows:

[0035] RNA extraction: Total plasma RNA (including small RNA) was extracted using the miRNeasy plasma kit, and RNA was extracted from tumor tissue using TRIzol reagent. The extracted RNA was eluted with RNase-free water, and the purity and concentration were tested using a Nanodrop 2000 spectrophotometer. The optical density ratio of all RNA samples was between 1.8 and 2.0.

[0036] Real-time quantitative PCR: Using All-in-One from Genecopeia TMmiRNA detection kit, real-time quantitative PCR detection of tRF by poly(A) tailing method. Amplification was performed on the Roche Light Cycler 480 system, U6 RNA was selected as the internal control of tissue samples, and plasma expression levels were homogenized to miR-16.

[0037] Adoption 2 -ΔΔCt The expression of relevant tRF was detected by the method, and each sample was tested three times. The sequence information of all primers is shown in Table 1 below:

[0038] Table 1

[0039]

[0040]

[0041] Through the above experimental steps, the samples were subjected to real-time quantitative PCR analysis of tRF-Ala-CGC-013. Figure 1 A and Figure 1 B shows that tRF-Ala-CGC-013 is significantly highly expressed in tumor tissues and plasma of esophageal squamous cell carcinoma with lymph node metastasis, and can be used as a marker for lymph node metastasis in esophageal squamous cell carcinoma.

[0042] Example 2 Correlation of tRF-Ala-CGC-013 expression in esophageal squamous cell carcinoma tissue and plasma samples

[0043] Referring to the experimental steps described in Example 1, real-time quantitative PCR analysis of tRF-Ala-CGC-013 was performed on esophageal squamous cell carcinoma tissue and plasma samples, and the esophageal squamous cell carcinoma tissue and plasma samples were specifically tumor tissue and plasma samples of patients with lymph node-negative esophageal squamous cell carcinoma (15 cases) and lymph node-positive esophageal squamous cell carcinoma (22 cases). According to the analysis results, Figure 2 A shows that the expression of tRF-Ala-CGC-013 in esophageal squamous cell carcinoma tumor tissue and plasma is positively correlated, indicating that there is a certain correlation between the expression of tRF-Ala-CGC-013 in plasma and the expression in esophageal squamous cell carcinoma tissue.

[0044] Example 3 Correlation of tRF-Ala-CGC-013 expression in plasma samples before and after esophageal squamous cell carcinoma surgery

[0045] Referring to the experimental steps described in Example 1, in the population of esophageal squamous cell carcinoma with lymph node metastasis (22 cases), the plasma samples of the same patients with esophageal squamous cell carcinoma before and after surgery were analyzed by real-time quantitative PCR for tRF-Ala-CGC-013. Figure 2B shows that the expression of tRF-Ala-CGC-013 in the plasma after surgery of esophageal squamous cell carcinoma with lymph node metastasis is significantly lower than that in the plasma before surgery, indicating that the expression change of tRF-Ala-CGC-013 in plasma is partly derived from the release of tumor.

[0046] Example 4 Expression of tRF-Ala-CGC-013 in the plasma of patients with esophageal squamous cell carcinoma, gastric cancer, and colon cancer with lymph node metastasis

[0047] Referring to the experimental steps described in Example 1, plasma samples of patients with esophageal squamous cell carcinoma with lymph node metastasis (22 cases) and patients with gastric cancer (13 cases) and colon cancer (18 cases) with lymph node metastasis were subjected to real-time quantitative PCR analysis of tRF-Ala-CGC-013. Figure 3 As shown in A, tRF-Ala-CGC-013 is significantly highly expressed in the plasma of patients with esophageal squamous cell carcinoma with lymph node metastasis, and there is no significant difference in expression in other cancers with lymph node metastasis, indicating that tRF-Ala-CGC-013 has a certain specificity in patients with esophageal squamous cell carcinoma with lymph node metastasis. Figure 3 As can be seen from B, the AUC value of tRF-Ala-CGC-013 is 0.81, indicating that plasma tRF-Ala-CGC-013 has good specificity and sensitivity when predicting esophageal squamous cell carcinoma lymph node metastasis.

[0048] The above technical solution is only one embodiment of the present invention. Based on the understanding of the application method and principle of the present invention, those skilled in the art can make various types of deformation and improvement, and are not limited to the method described in the above specific embodiment of the present invention. Therefore, the above method is only preferred and not restrictive. As long as the expression of tRF-Ala-CGC-013 is detected to predict the status of lymph node metastasis of esophageal squamous cell carcinoma, it is within the scope of protection of this patent.

Claims

1. Use of a preparation for detecting the expression level of tRF-Ala-CGC-013 in the preparation of a product for predicting lymph node metastasis of esophageal squamous cell carcinoma, characterized in that: The nucleotide sequence of the tRF-Ala-CGC-013 is shown in SEQ ID No. 1, and the preparation contains amplification primers and internal reference primers for detecting tRF-Ala-CGC-013.

2. The use according to claim 1, characterized in that: The amplification primers include an upstream primer and a downstream primer, and the nucleotide sequences thereof are shown as SEQ ID No.2 and SEQ ID No.3 respectively.

3. A detection kit for predicting lymph node metastasis of esophageal squamous cell carcinoma, characterized in that: The invention relates to a preparation for detecting the expression level of tRF-Ala-CGC-013, wherein the preparation comprises real-time quantitative PCR amplification primers and internal reference primers for detecting tRF-Ala-CGC-013; the real-time quantitative PCR amplification primers comprise upstream primers and downstream primers, and the nucleotide sequences thereof are shown in SEQ ID No.2 and SEQ ID No.3, respectively.

4. The detection kit according to claim 3, characterized in that It also contains a microfluidic biochip.

5. The detection kit according to claim 3, characterized in that The detection sample of the detection kit is an esophageal squamous cell carcinoma tumor tissue sample or a plasma sample.

6. The detection kit according to claim 5, characterized in that The detection sample of the detection kit is a plasma sample.

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