TsRNA-SecTCA-L35 molecule and application of tsRNA-SecTCA-L35 molecule as colorectal cancer diagnosis biomarker and treatment target

Abstract

The invention discloses a tsRNA-SecTCA-L35 molecule and application of the tsRNA-SecTCA-L35 molecule as a colorectal cancer diagnosis biomarker and a colorectal cancer treatment target, and relates to the technical field of biological medicine. The invention provides application of the tsRNA-SecTCA-L35 molecule as a colorectal cancer diagnosis biomarker and a treatment target, in the diagnosis level, the molecule as a novel biomarker can be sensitively and specifically detected in samples such as plasma and tissue through qRT-PCR and other technologies, a brand new tool is provided for early discovery and non-invasive diagnosis of colorectal cancer, and the application of the tsRNA-SecTCA-L35 molecule as the colorectal cancer diagnosis biomarker and the treatment target is wide. In the aspect of treatment, by designing an analogue related to the molecular function and preparing the analogue into a pharmaceutical composition of a carrier such as lipidosome, the expression of the analogue in tumor cells can be effectively up-regulated, so that the proliferation of cancer cells is remarkably inhibited in vivo and in vitro, the death of the cancer cells is promoted, and the definite anti-tumor activity is shown; the important clinical application value is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a tsRNA-SecTCA-L35 molecule and application thereof as a colorectal cancer diagnosis biomarker and treatment target. BACKGROUND

[0002] Colorectal cancer (CRC) is a common malignant tumor of the digestive system, ranking third in global incidence and second in mortality, posing a serious threat to human health. According to the data of Global Cancer Statistics 2020, there are more than 1.9 million new cases of colorectal cancer worldwide each year, and more than 900,000 deaths. In China, with the aging of the population and changes in dietary structure, the incidence of colorectal cancer continues to rise and shows a clear trend of youth.

[0003] At present, the clinical treatment of colorectal cancer mainly includes surgical resection, chemotherapy, radiotherapy, targeted therapy and immunotherapy, etc. Although these methods have made certain progress in prolonging the survival of patients, there are still many limitations. For example, traditional chemotherapeutic drugs (such as oxaliplatin, fluorouracil, etc.) have limited efficacy in some patients and are often accompanied by significant side effects; targeted drugs (such as bevacizumab, cetuximab, etc.) can improve the prognosis of some patients, but their efficacy depends on the screening of specific molecular markers and are prone to drug resistance. Therefore, it is urgent to explore new molecular targets and treatment strategies to improve the treatment effect of colorectal cancer and the quality of life of patients.

[0004] In recent years, the role of non-coding RNA (ncRNA) in the occurrence and development of tumors has been increasingly concerned. Among them, short RNA fragments derived from transfer RNA (tRNA) (transfer RNA-derived small RNAs, tsRNAs) are considered to be another important gene expression regulatory molecule after miRNA and lncRNA. Studies have shown that tsRNAs exist widely in various organisms, with high conservation, tissue specificity and conditional inducibility. They not only participate in ribosome biosynthesis, cell stress response, proliferation and death, etc. basic life processes, but also are closely related to a variety of diseases including malignant tumors.

[0005] At present, more studies have focused on the role of miRNA and lncRNA in colorectal cancer, but the study of tsRNA is still relatively limited, especially the functional mechanism of tsRNA-SecTCA-L35 in colorectal cancer and its application as a diagnostic marker and therapeutic target, which has not been reported. Therefore, the present application proposes a tsRNA-SecTCA-L35 molecule and its application as a colorectal cancer diagnostic biomarker and therapeutic target to solve the problems in the prior art. SUMMARY

[0006] In view of the above problems, the purpose of the present application is to propose a tsRNA-SecTCA-L35 molecule and its application as a colorectal cancer diagnostic biomarker and therapeutic target, by regulating the expression level of tsRNA-SecTCA-L35, aiming to solve the problems of limited efficacy, large side effects and insufficient targeting of existing colorectal cancer treatment methods, thereby providing new molecular targets and treatment strategies for precise diagnosis and treatment of colorectal cancer.

[0007] In order to achieve the purpose of the present application, the present application realizes the following technical solutions: A tsRNA-SecTCA-L35 molecule, the nucleotide sequence of the tsRNA-SecTCA-L35 molecule is shown as SEQ ID NO: 1.

[0008] Further improvement lies in that the tsRNA-SecTCA-L35 molecule is used for preparing a kit for diagnosing colorectal cancer, and the kit comprises reagents for detecting the expression level of tsRNA-SecTCA-L35 in a to-be-tested sample.

[0009] Further improvement lies in that the to-be-tested sample is selected from one of plasma, lymph, puncture fluid or tissue biopsy specimen.

[0010] Further improvement lies in that the reagent comprises a primer pair for detecting tsRNA-SecTCA-L35 by qRT-PCR, and the primer pair comprises sequences shown in a reverse transcription stem loop primer SEQ ID NO: 2 and a detection primer SEQ ID NO: 3.

[0011] A tsRNA-SecTCA-L35 molecule as a colorectal cancer diagnostic biomarker and therapeutic target.

[0012] Further improvement lies in that the analog of the tsRNA-SecTCA-L35 molecule is an oligonucleotide molecule, the sequence of which is designed based on SEQ ID NO. 1, and is used for up-regulating the expression of tsRNA-SecTCA-L35 in colorectal cancer cells.

[0013] Further improvement lies in that the tsRNA-SecTCA-L35 molecule is applied to preparation of a therapeutic target drug as a colorectal cancer diagnostic biomarker, and the drug is a pharmaceutical composition containing the tsRNA-SecTCA-L35 or an analog thereof.

[0014] Further improvement lies in that the pharmaceutical composition further comprises a pharmaceutically acceptable carrier selected from one of a liposome, a nanoparticle, an engineered exosome or a lentivirus vector.

[0015] Further improvement lies in that the drug is configured to be used in combination with chemotherapy, targeted therapy or immunotherapy.

[0016] The application has the following beneficial effects: the application provides application of the tsRNA-SecTCA-L35 molecule as a colorectal cancer diagnostic biomarker and therapeutic target, in the diagnosis aspect, the molecule is a novel biomarker, can be sensitively and specifically detected in plasma, tissue and other samples through qRT-PCR and other technologies, provides a new tool for early detection and non-invasive diagnosis of colorectal cancer, and makes up for the deficiency of existing markers, in the treatment aspect, an analog (such as an oligonucleotide molecule) related to the function of the molecule is designed and prepared into a drug composition of a liposome carrier, which can effectively up-regulate the expression of the molecule in tumor cells, thereby significantly inhibiting the proliferation of cancer cells and promoting the death of the cancer cells in vivo and in vitro, and exhibits clear anti-tumor activity, realizes the dual application of the same tsRNA molecule in colorectal cancer diagnosis and treatment, forms a closed-loop strategy from diagnosis discovery to targeted therapy, provides a new molecular target and comprehensive solution for precision medicine of colorectal cancer, and has important clinical application value and market prospect. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description.

[0018] Figure 1 is a volcano plot of differential tsRNAs of 12 cancer and paracancer tissue samples in the embodiment of the application; Figure 2 is a hierarchical clustering plot of differential tsRNAs of colorectal cancer and paracancer tissue samples in the embodiment of the application; Figure 3 is a tsRNA-SecTCA-L35 cleavage difference plot of cancer and paracancer in a colorectal cancer sample in the embodiment of the application; Figure 4 is a CCK8 growth curve schematic diagram of a colorectal cancer cell line after overexpression of the tsRNA-SecTCA-L35 in the embodiment of the application; Figure 5is a schematic diagram of the colony formation experiment of the colorectal cancer cell line after overexpression of tsRNA-SecTCA-L35 in the embodiment of the present application. Figure 6 is a schematic diagram of the death detection of the colorectal cancer cell line after overexpression of tsRNA-SecTCA-L35 in the embodiment of the present application. Figure 7 is a schematic diagram of the influence of tsRNA-SecTCA-L35 analogues on tumor size in the animal model in the embodiment of the present application. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0020] The Scutellaria baicalensis Georgi extract in the present embodiment is obtained by commercial purchase from Shaanxi Xintianyu Biotechnology Co., Ltd.

[0021] Embodiment 1 In the present embodiment, sequence identification and expression analysis of tsRNA-SecTCA-L35 are performed by clinical samples and high-throughput sequencing technology, and the specific steps are as follows: 1. Clinical sample collection In cooperation with the oncology department of the hospital, 6 patients with colorectal cancer diagnosed by pathology were selected in strict accordance with the ethical norms, and cancer tissues and paired normal tissues (more than 5 cm away from the cancer tissues) were collected simultaneously during the operation, a total of 12 samples. All patients did not receive any anti-tumor treatment before operation, and signed the informed consent form. The basic clinical information of the patients is shown in Table 1.

[0022] Table 1. List of patient clinical data

[0023] 2. RNA extraction and pretreatment Total RNA was extracted from the tissues using miRNeasy Mini Kit. In order to accurately detect tsRNA, the total RNA was pretreated using rtStar™ tRF and tsRNA pretreatment kit, including removing 3'-cP, modifying 5'-OH terminal phosphorylation, removing internal modification (m1A, m1G, m3C, etc.), so that the RNA has 3'-OH and 5'-P terminal, thereby ensuring the accuracy of subsequent primer ligation and reverse transcription.

[0024] 3. High-throughput sequencing and bioinformatics analysis After pretreatment, the RNA of each sample was ligated to 3' and 5' small RNA adapters and transcribed into cDNA using Superscript II reverse transcriptase (Invitrogen, USA). Subsequently, the cDNA was synthesized and amplified using Illumina proprietary RT primers and amplification primers. The sequencing library (about 135-160 bp) was selected using an automatic gel cutter and identified using an Agilent Bioanalyzer 2100. The PCR products were sequenced on an Illumina NextSeq instrument. The raw data was subjected to adapter sequence removal using Cutadapt and filtering of fragments with a length < 15 nt; subsequently, the sequences were aligned to mature tRNA and precursor tRNA sequences in the GtRNAdb database using NovoAlign; unaligned sequences were further aligned to a transcriptome database (including miRNA, mRNA, rRNA, snRNA, snoRNA, and piRNA), and one mismatch was allowed for tRNA alignment. Sequences with a count less than 2 were removed. The expression level of tsRNA was represented as transcripts per million (TPM). Sequences with a TPM lower than 1 (below the 50th percentile) were filtered. Differential expression analysis was performed using a paired sample t-test, and P values were corrected using the FDR method. The screening criteria were: fold change ≥ 2, and FDR < 0.1. According to the source and length, tsRNA was divided into six categories: 5'tiRNA, 3'tiRNA, tRF-5, tRF-3, tRF-1, and i-tRF.

[0025] As shown in Figure 1 , there were a large number of significantly different tsRNAs in colorectal cancer and paracancerous tissues, and Figure 2 The results showed that the differential tsRNAs could well distinguish colorectal cancer from paracancerous tissues, suggesting that tsRNA had the potential for colorectal cancer diagnosis. Among them, Figure 3 The results showed that the cleavage mode of tsRNA-SecTCA-L35 in colorectal cancer tissue was significantly different from that in paracancerous tissue. This result clearly showed that the expression level of tsRNA-SecTCA-L35 was closely related to the occurrence of colorectal cancer, and could be used as an effective diagnostic biomarker and could be used for the development of a diagnostic kit.

[0026] Example 2 This example constructs a diagnostic kit for detecting tsRNA-SecTCA-L35, and the specific steps are as follows: 1. Primer design According to the sequence of tsRNA-SecTCA-L35 (SEQ ID NO: 1), a specific qRT-PCR primer pair was designed and synthesized, which contains the sequence shown in SEQ ID NO: 2 and SEQ ID NO: 3. The primer pair of the control gene U6 is shown in SEQ ID NO: 4 and SEQ ID NO: 5. The sequence information is shown in Table 2 below.

[0027] Table 2 Sequence information

[0028] 2. qRT-PCR detection: (1) Extract total RNA from the tissue or plasma sample to be tested and reverse transcribe it into cDNA.

[0029] (2) Configure the qRT-PCR reaction system, as shown in Table 3 below.

[0030] Table 3 qRT-PCR reaction system

[0031] (3) Run the program on the real-time fluorescence quantitative PCR instrument, and the reaction conditions are shown in Table 4 below.

[0032] Table 4 qRT-PCR reaction conditions

[0033] (4) Result analysis: Calculate the expression amount of tsRNA-SecTCA-L35 in the sample to be tested relative to the control or normal control by comparing the Ct value (2^–ΔΔCt method). Significant down-regulation of the expression amount indicates a risk of colorectal cancer.

[0034] This example demonstrates that the kit containing the specific primer pair can be used to detect the biomarker level in various samples, thereby achieving the auxiliary diagnosis of colorectal cancer.

[0035] Example 3 This example verifies the therapeutic function (cell level) of the tsRNA-SecTCA-L35 analog, and the specific steps are as follows: 1. Cell culture and transfection Human colorectal cancer cell line SW1116 was cultured in RPMI-1640 medium containing 10% fetal bovine serum, and the oligonucleotide molecule (analog) designed based on SEQ ID NO: 1, which can up-regulate the expression of endogenous tsRNA-SecTCA-L35, was transfected into SW1116 cells using Lipofectamine RNAiMax transfection reagent. The culture and cell treatment of NCM460 cells were consistent with SW1116 cells.

[0036] 2. Functional experiment Cell proliferation experiment (CCK-8 method): detection at 0, 24, 48, and 72 hours after transfection, as shown in Figure 4 Compared with the negative control group, the proliferation ability of the cells in the experimental group transfected with the analog was significantly inhibited (P<0.05).

[0037] Cloning experiment: after transfection, cells were inoculated at low density and cultured for 1-2 weeks, as shown in Figure 5 The number of cell clones in the analog transfection group was significantly less than that in the control group (P<0.05).

[0038] Cell death detection (Annexin V-FITC / PI double staining method): flow cytometry detection showed that, as shown in Figure 6 The cell death rate (22.4%) in the analog transfection group was significantly higher than that in the control group (15.4%) (P<0.01).

[0039] This example demonstrates that the oligonucleotide molecule (analog) designed based on SEQ ID NO: 1 can up-regulate the function of tsRNA-SecTCA-L35, and then prepare a pharmaceutical composition containing the analog, which can inhibit proliferation and promote death to play a role in treating colorectal cancer.

[0040] Example 4 This example verifies the therapeutic function of the tsRNA-SecTCA-L35 analog (at the animal level), and the specific steps are as follows: 1. Establishment of animal model HCT116 colorectal cancer cells were subcutaneously inoculated into the axillary of BALB / c nude mice to establish a tumor xenograft model.

[0041] 2. Drug treatment When the tumor volume was about 100 mm 3 , the mice were randomly divided into groups (n=7), the experimental group was injected with a mixture of tsRNA-SecTCA-L35 analog (i.e. a pharmaceutical composition) wrapped by liposomes, and the control group was injected with an equal amount of blank preparation, once every three days, for a total of 6 times.

[0042] The results are shown in Figure 7As shown, compared with the control group, the tumor growth of the tsRNA-SecTCA-L35 analogue treatment group was significantly inhibited, and no significant decrease in mouse body weight was observed during the experiment, indicating that it has effective tumor inhibition effect and good safety, and this animal experiment model itself can also be used to evaluate the combined use effect of the drug with chemotherapy (such as oxaliplatin), targeted therapy or immunotherapy drugs.

[0043] This example demonstrates the application of tsRNA-SecTCA-L35 analogue in the preparation of drugs for treating colorectal cancer at the whole animal level, and shows its specific drug preparation form and potential combined application scenarios.

[0044] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A tsRNA-SecTCA-L35 molecule, characterized in that: The nucleotide sequence of the tsRNA-SecTCA-L35 molecule is shown in SEQ ID NO:

1.

2. The tsRNA-SecTCA-L35 molecule according to claim 1, characterized in that: The tsRNA-SecTCA-L35 molecule is used to prepare a kit for diagnosing colorectal cancer, the kit containing reagents for detecting the expression level of tsRNA-SecTCA-L35 in a sample to be tested.

3. The reagent kit for diagnosing colorectal cancer according to claim 2, characterized in that: The sample to be tested is selected from one of the following: plasma, lymph, puncture fluid, or tissue biopsy specimen.

4. The reagent kit for diagnosing colorectal cancer according to claim 2, characterized in that: The reagent includes a primer pair for detecting tsRNA-SecTCA-L35 by qRT-PCR, the primer pair comprising the sequences shown in SEQ ID NO:2 for reverse transcription and SEQ ID NO:3 for detection.

5. Application of a tsRNA-SecTCA-L35 molecule as a diagnostic biomarker and therapeutic target for colorectal cancer.

6. The application of the tsRNA-SecTCA-L35 molecule as a diagnostic biomarker and therapeutic target for colorectal cancer according to claim 5, characterized in that: The analogues of the tsRNA-SecTCA-L35 molecule are oligonucleotide molecules whose sequences are designed based on SEQ ID NO.1 and are used to upregulate the expression of tsRNA-SecTCA-L35 in colorectal cancer cells.

7. The application of the tsRNA-SecTCA-L35 molecule as a diagnostic biomarker and therapeutic target for colorectal cancer according to claim 5, characterized in that: The tsRNA-SecTCA-L35 molecule is used as a diagnostic biomarker for colorectal cancer in the preparation of therapeutic target drugs, wherein the drug is a pharmaceutical composition containing tsRNA-SecTCA-L35 or an analogue thereof.

8. The application of the tsRNA-SecTCA-L35 molecule as a diagnostic biomarker and therapeutic target for colorectal cancer according to claim 7, characterized in that: The pharmaceutical composition further comprises a pharmaceutically acceptable carrier selected from liposomes, nanoparticles, engineered exosomes, or lentiviral vectors.

9. The application of the tsRNA-SecTCA-L35 molecule as a diagnostic biomarker and therapeutic target for colorectal cancer according to claim 5, characterized in that: The drug is formulated for use in combination with chemotherapy, targeted therapy, or immunotherapy.

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