A tsrna-sec tca-l35 molecule and application thereof as a diagnostic biomarker and therapeutic target for colorectal cancer

Abstract

This invention discloses a tsRNA-SecTCA-L35 molecule and its application as a diagnostic biomarker and therapeutic target for colorectal cancer, relating to the field of biomedical technology. This invention provides the application of the tsRNA-SecTCA-L35 molecule as a diagnostic biomarker and therapeutic target for colorectal cancer. At the diagnostic level, this molecule, as a novel biomarker, can be sensitively and specifically detected in plasma, tissue, and other samples using techniques such as qRT-PCR, providing a new tool for the early detection and non-invasive diagnosis of colorectal cancer and compensating for the shortcomings of existing biomarkers. At the therapeutic level, by designing analogs related to the function of this molecule and preparing them into drug compositions using liposomes and other carriers, its expression in tumor cells can be effectively upregulated, thereby significantly inhibiting cancer cell proliferation and promoting their death in vitro and in vivo, exhibiting clear anti-tumor activity and possessing significant clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a tsRNA-SecTCA-L35 molecule and its application as a diagnostic biomarker and therapeutic target for colorectal cancer. Background Technology

[0002] Colorectal cancer (CRC) is a common malignant tumor of the digestive system, ranking third in incidence and second in mortality worldwide, posing a serious threat to human health. According to the "Global Cancer Statistics 2020," more than 1.9 million new cases of colorectal cancer are diagnosed globally each year, with more than 900,000 deaths. In my country, with population aging and changes in dietary structure, the incidence of colorectal cancer continues to rise, showing a clear trend towards affecting younger people.

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

[0004] In recent years, the role of non-coding RNA (ncRNA) in tumorigenesis and development has received increasing attention. Among them, short RNA fragments derived from transfer RNA (tRNA) (tsRNAs) are considered another important class of gene expression regulatory molecules after miRNA and lncRNA. Studies have shown that tsRNAs are widely present in various organisms and exhibit high conservation, tissue specificity, and conditional inducibility. They not only participate in basic life processes such as ribosome biosynthesis, cellular stress response, proliferation, and cell death, but are also closely related to various diseases, including malignant tumors.

[0005] Currently, existing research focuses on the roles of miRNAs and lncRNAs in colorectal cancer, but studies on tsRNAs remain relatively limited, especially regarding the functional mechanism of tsRNA-SecTCA-L35 in colorectal cancer and its application as a diagnostic biomarker and therapeutic target, which has not been publicly reported. Therefore, this invention proposes a tsRNA-SecTCA-L35 molecule and its application as a diagnostic biomarker and therapeutic target for colorectal cancer to address the problems existing in the prior art. Summary of the Invention

[0006] To address the aforementioned problems, the present invention aims to propose a tsRNA-SecTCA-L35 molecule and its application as a diagnostic biomarker and therapeutic target for colorectal cancer. By regulating the expression level of tsRNA-SecTCA-L35, the invention aims to solve the problems of limited efficacy, significant side effects, and insufficient targeting of existing colorectal cancer treatments, thereby providing new molecular targets and treatment strategies for the precision diagnosis and treatment of colorectal cancer.

[0007] To achieve the objectives of this invention, the invention is implemented through the following technical solutions: A tsRNA-SecTCA-L35 molecule, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0008] A further improvement is 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 the sample to be tested.

[0009] A further improvement is that the sample to be tested is selected from one of plasma, lymph, puncture fluid, or tissue biopsy specimens.

[0010] A further improvement is that the reagent includes a primer pair for detecting tsRNA-SecTCA-L35 by qRT-PCR, the primer pair containing the sequences shown in SEQ ID NO:2 for reverse transcription and SEQ ID NO:3 for detection.

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

[0012] A further improvement is that the analogue of the tsRNA-SecTCA-L35 molecule is an oligonucleotide molecule whose sequence is designed based on SEQ ID NO.1, and is used to upregulate the expression of tsRNA-SecTCA-L35 in colorectal cancer cells.

[0013] A further improvement is 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 its analogues.

[0014] A further improvement is that the pharmaceutical composition further comprises a pharmaceutically acceptable carrier selected from liposomes, nanoparticles, engineered exosomes, or lentiviral vectors.

[0015] A further improvement is that the drug is configured for use in combination with chemotherapy, targeted therapy, or immunotherapy.

[0016] The beneficial effects of this invention are as follows: This invention provides the application of tsRNA-SecTCA-L35 molecule as a diagnostic biomarker and therapeutic target for colorectal cancer. At the diagnostic level, this molecule, as a novel biomarker, can be sensitively and specifically detected in plasma, tissue, and other samples using techniques such as qRT-PCR, providing a new tool for the early detection and non-invasive diagnosis of colorectal cancer and making up for the shortcomings of existing biomarkers. At the therapeutic level, by designing analogs (such as oligonucleotide molecules) with functions related to this molecule and preparing them into drug compositions using liposomes and other carriers, its expression in tumor cells can be effectively upregulated, thereby significantly inhibiting cancer cell proliferation and promoting their death in vitro and in vivo, exhibiting clear anti-tumor activity. This realizes the dual application of the same tsRNA molecule in the diagnosis and treatment of colorectal cancer, forming a closed-loop strategy from diagnostic discovery to targeted therapy, providing a new molecular target and comprehensive solution for precision medicine of colorectal cancer, and has significant clinical application value and market prospects. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0018] Figure 1 These are differential tsRNA volcano plots of 12 cancer and adjacent normal tissue specimens in this embodiment of the invention; Figure 2 This is a hierarchical clustering diagram of differentially expressed tsRNAs between colorectal cancer and adjacent tissue specimens in this embodiment of the invention; Figure 3 This is a diagram showing the difference in tsRNA-SecTCA-L35 cleavage between cancerous and adjacent normal tissue samples in an embodiment of the present invention. Figure 4 This is a schematic diagram of the growth curve of the colorectal cancer cell line CCK8 after tsRNA-SecTCA-L35 overexpression in an embodiment of the present invention; Figure 5This is a schematic diagram of the colony formation experiment of colorectal cancer cell lines after tsRNA-SecTCA-L35 overexpression in an embodiment of the present invention; Figure 6 This is a schematic diagram illustrating the detection of colorectal cancer cell line death after tsRNA-SecTCA-L35 overexpression in an embodiment of the present invention. Figure 7 This is a schematic diagram illustrating the effect of tsRNA-SecTCA-L35 analogs on tumor size in an animal model in an embodiment of the present invention. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1 This embodiment uses clinical samples and high-throughput sequencing technology to identify the sequence and analyze the expression of tsRNA-SecTCA-L35. The specific steps are as follows: 1. Clinical Sample Collection In collaboration with the hospital's oncology department and strictly adhering to ethical guidelines, we selected 6 patients with pathologically confirmed colorectal cancer. During the surgery, we simultaneously collected cancerous tissue and paired adjacent normal tissue (>5cm from the cancerous tissue), totaling 12 samples. All patients had not received any anti-tumor treatment before the operation and had signed informed consent forms. The basic clinical information of the patients is shown in Table 1.

[0021] Table 1 Summary of Patient Clinical Data

[0022] 2. RNA extraction and pretreatment Total RNA was extracted from tissues using the miRNeasy Mini Kit. To accurately detect tsRNA, the total RNA was pretreated using the rtStar™ tRF and tsRNA pretreatment kit, including removal of 3'-cP, modification of 5'-OH end phosphorylation, and removal of internal modifications (m1A, m1G, m3C, etc.) to ensure that the RNA has 3'-OH and 5'-P ends, thereby ensuring the accuracy of subsequent primer ligation and reverse transcription.

[0023] 3. High-throughput sequencing and bioinformatics analysis After pretreatment, RNA from each sample was ligated to 3' and 5' small RNA adapters and transcribed into cDNA using Superscript II reverse transcriptase (Invitrogen, USA). The cDNA was then synthesized and amplified using Illumina's proprietary RT primers and amplification primers. Sequencing libraries (approximately 135–160 bp) were selected using an automated gel cutter and identified using an Agilent Bioanalyzer 2100. PCR products were sequenced on an Illumina NextSeq instrument. Raw data were processed using Cutadapt to remove adapter sequences and filter fragments <15 nt in length; then, NovoAlign was used to align to mature tRNA and precursor tRNA sequences in the GtRNAdb database; unaligned sequences were then aligned to transcriptome databases (including miRNA, mRNA, rRNA, snRNA, snoRNA, and piRNA). One mismatch was allowed in tRNA alignments. Sequences with a count less than 2 were discarded. tsRNA expression levels are expressed as total markers per million (TPM). Sequences with a TPM below 1 (below the 50th quantile) were filtered. Differential expression analysis employed paired-samples t-tests, with p-values ​​corrected using the FDR method. The screening criteria were: fold change ≥ 2 and FDR < 0.1. Based on origin and length, tsRNAs were classified into six categories: 5'tiRNA, 3'tiRNA, tRF-5, tRF-3, tRF-1, and i-tRF.

[0024] like Figure 1 As shown, a large number of significantly different tsRNAs exist between colorectal cancer and adjacent tissues, and Figure 2 The results showed that differentially expressed tsRNAs could effectively distinguish colorectal cancer from adjacent tissues, suggesting that tsRNAs have the potential for colorectal cancer diagnosis. Among them, Figure 3 The results showed that the cleavage pattern of tsRNA-SecTCA-L35 in colorectal cancer tissues differed significantly from that in adjacent normal tissues. This result clearly indicates that the expression level of tsRNA-SecTCA-L35 is closely related to the occurrence of colorectal cancer and can serve as an effective diagnostic biomarker, which can be used to develop diagnostic kits.

[0025] Example 2 This embodiment describes the construction of a diagnostic kit for detecting tsRNA-SecTCA-L35. The specific steps are as follows: 1. Primer design Based on the sequence of tsRNA-SecTCA-L35 (SEQ ID NO:1), a specific qRT-PCR primer pair was designed and synthesized. This primer pair contains the sequences shown in SEQ ID NO:2 for reverse transcription and SEQ ID NO:3 for detection. The internal reference gene U6 was used as a control, with primer pairs shown in SEQ ID NO:4 and SEQ ID NO:5. Sequence information is shown in Table 2 below.

[0026] Table 2 Sequence Information

[0027] 2. qRT-PCR detection: (1) Extract total RNA from the tissue or plasma sample to be tested and reverse transcribe it into cDNA.

[0028] (2) Configure the qRT-PCR reaction system as shown in Table 3 below.

[0029] Table 3 qRT-PCR reaction system

[0030] (3) Run the program on the real-time fluorescence quantitative PCR instrument. The reaction conditions are shown in Table 4 below.

[0031] Table 4 qRT-PCR reaction conditions

[0032] (4) Results analysis: The expression level of tsRNA-SecTCA-L35 in the test samples was calculated relative to the internal control or normal control by comparing Ct values ​​(2^–ΔΔCt method). Significant downregulation of expression level suggests the risk of colorectal cancer.

[0033] This embodiment demonstrates that a kit containing specific primer pairs can be used to detect biomarker levels in a variety of samples, thereby enabling the auxiliary diagnosis of colorectal cancer.

[0034] Example 3 This embodiment verifies the therapeutic function (cellular level) of tsRNA-SecTCA-L35 analogues, 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. Using Lipofectamine RNAiMax transfection reagent, oligonucleotide molecules (analytes) designed based on SEQ ID NO:1 that upregulate endogenous tsRNA-SecTCA-L35 expression were transfected into SW1116 cells. The culture and treatment of NCM460 cells were the same as for SW1116 cells.

[0035] 2. Functional Experiment Cell proliferation assay (CCK-8 assay): Detection was performed at 0, 24, 48, and 72 hours post-transfection. Figure 4 As shown, compared with the negative control group, the cell proliferation capacity of the experimental group transfected with the analog was significantly inhibited (P<0.05).

[0036] Colony formation assay: After transfection, cells are seeded at low density and cultured for 1–2 weeks, such as… Figure 5 As shown, the number of cell clones formed in the analog transfection group was significantly less than that in the control group (P<0.05).

[0037] Cell death detection (Annexin V-FITC / PI double staining): Flow cytometry analysis showed, for example... Figure 6 As shown, the cell death rate in the analogue transfection group (22.4%) was significantly higher than that in the control group (15.4%) (P<0.01).

[0038] This embodiment demonstrates that oligonucleotide molecules (analogs) designed based on SEQ ID NO:1 can upregulate the function of tsRNA-SecTCA-L35, thereby preparing a pharmaceutical composition containing the analog to treat colorectal cancer by inhibiting proliferation and promoting cell death.

[0039] Example 4 This embodiment verifies the therapeutic function of tsRNA-SecTCA-L35 analogues (at the animal level), and the specific steps are as follows: 1. Establishment of animal models HCT116 colorectal cancer cells were subcutaneously inoculated into the axilla of BALB / c nude mice to establish a xenograft model.

[0040] 2. Drug therapy When the tumor volume reached approximately 100 mm³, the mice were randomly divided into groups (n=7). The experimental group was injected with a mixture of tsRNA-SecTCA-L35 analogues (i.e., a drug composition) encapsulated in liposomes, while the control group was injected with an equal amount of blank preparation. The drugs were administered once every three days for a total of 6 times.

[0041] The results are as follows Figure 7As shown, compared with the control group, tumor growth in the tsRNA-SecTCA-L35 analog treatment group was significantly inhibited, and no significant decrease in mouse weight was observed during the experiment, indicating that it has effective tumor-suppressing effects and good safety. This animal experimental model itself can also be used to evaluate the combined use of this drug with chemotherapy (such as oxaliplatin), targeted therapy or immunotherapy drugs.

[0042] This embodiment demonstrates the application of tsRNA-SecTCA-L35 analogs in the preparation of drugs for treating colorectal cancer at the whole animal level, and shows its specific pharmaceutical formulation and potential combination application scenarios.

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of a reagent for detecting the expression level of tsRNA-SecTCA-L35 in a test sample in the preparation of a kit for diagnosing colorectal cancer, characterized in that: The nucleotide sequence of the tsRNA-SecTCA-L35 molecule is shown in SEQ ID NO:

1. The reagent includes a primer pair for detecting tsRNA-SecTCA-L35 by qRT-PCR, the primer pair containing the sequences shown in the stem-loop primer SEQ ID NO:2 for reverse transcription and the detection primer SEQ ID NO:3.

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