A piRNA-362733 biomarker for diagnosis or treatment and its application
By screening out piRNA-362733 as a marker and developing its inhibitor, the problems of early diagnosis and treatment of colorectal cancer were solved, effective inhibition of colorectal cancer cell proliferation, invasion and migration, and the treatment effect was improved.
Patent Information
- Application Number
- CN202411370972.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-29
- Publication Date
- 2025-08-19
- Estimated Expiration
- 2044-09-29
AI Technical Summary
The prior art lacks effective early diagnosis and treatment methods to predict and treat invasion and metastasis of colorectal cancer, resulting in the fact that most patients are already in advanced stages at the time of diagnosis, affecting the prognosis.
PiRNA-362733 was screened as a marker and its inhibitor was developed to prepare colorectal cancer therapeutic and diagnostic preparations. Its high expression in colorectal cancer tissues and cells was verified by real-time quantitative PCR. The inhibitors can reduce the proliferation, invasion and migration of colorectal cancer cells.
The piRNA-362733 inhibitor significantly inhibits the proliferation, invasion and migration of colorectal cancer cells, provides a potential tool for early diagnosis and targeted therapy, and improves the efficacy of colorectal cancer treatment.
Smart Images
Figure CN119120701B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology and relates to a new tumor marker piRNA-362733 and its application in predicting, diagnosing and treating colorectal cancer invasion and metastasis. Background Art
[0002] Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related death worldwide. Early-stage CRC lacks specific symptoms and effective screening methods, and most patients are diagnosed in the advanced stage. Distant metastasis severely impacts prognosis. Currently, treatment options other than surgical resection and chemotherapy are ineffective. Therefore, the search for optimized biomarkers and treatments has become a pressing clinical need. Few studies have demonstrated promise in improving early diagnosis, necessitating the identification of novel small molecule targets for early diagnosis and prognosis.
[0003] In recent years, a new class of small, non-coding RNAs, Piwi-interacting RNAs (piRNAs), has been further studied. PiRNAs are typically 24 to 33 nucleotides (nuclotides) in length and specifically bind to Piwi proteins to exert their effects. Because piRNA-encoding genes are found throughout the genome, piRNAs have existed since the early origins of life and have been conserved through natural selection and evolution. Since their discovery, research on piRNAs has advanced rapidly, revealing their crucial roles in germline stem cell differentiation, embryonic development, DNA integrity maintenance, epigenetic regulation, and sex determination. Accumulating evidence indicates that piRNAs are abnormally expressed in various tumors, making them crucial for the early diagnosis and treatment of cancer. Summary of the Invention
[0004] To achieve early diagnosis and targeted treatment of colorectal cancer, the first objective of the present invention is to screen for a piRNA that predicts, diagnoses, and treats colorectal cancer invasion and metastasis. A second objective of the present invention is to provide the use of a marker piRNA inhibitor in the preparation of a colorectal cancer treatment formulation, particularly a formulation that inhibits the invasion and metastasis of human colorectal cancer cells. A third objective of the present invention is to provide a marker piRNA inhibitor as a drug for the treatment of colorectal cancer. A fourth objective of the present invention is to provide the use of a reagent for detecting piRNA-362733 in the preparation of a colorectal cancer diagnostic and / or prognostic formulation.
[0005] To achieve the first objective, the present invention provides a piRNA that is significantly overexpressed in colorectal cancer tissues, wherein the marker is piRNA-362733.
[0006] The second aspect of the present invention is to provide a method for preparing a colorectal cancer treatment agent for use in an agent that inhibits the expression of piRNA-362733, particularly an agent that can reduce the proliferation, invasion, and migration of colorectal cancer cells.
[0007] The third aspect of the present invention aims to provide a preparation for treating colorectal cancer, which is an agent for inhibiting the expression of piRNA-362733. Preferably, the sequence of the piRNA-362733 inhibitor is TTCAACATCAGTCTGATAAGCTACC.
[0008] The fourth aspect of the present invention aims to provide a method for using a reagent for detecting piRNA-362733 in the preparation of a colorectal cancer diagnosis and / or prognosis preparation.
[0009] Furthermore, the colorectal cancer diagnosis and / or prognosis preparation includes a PCR detection reagent, and the primer sequence of the PCR detection reagent is as follows:
[0010] F: 5'-GTAGCTTATCAGACTGATGTTG-3'
[0011] R: 5'-GTCGTATCCAGTGCAGGGTCCGAGGT-3';
[0012] The β-actin internal reference primer sequence is as follows
[0013] F: 5'- GGGAAATCGTGCGTGACATTAAG-3'
[0014] R: 5'-TGGTTGGCGTACAGGTCTTTG-3'.
[0015] This study, using real-time quantitative PCR to expand sample validation, confirmed that piRNA-362733 is highly expressed in colorectal cancer tissues and cells. This piRNA molecule has not been previously discovered in studies of colorectal cancer, other tumor types, or non-tumor diseases, making it highly innovative. Cell-based experiments further validated the mechanism of action of piRNA-362733 in colorectal cancer cell lines, demonstrating that piRNA-362733 inhibitors can inhibit the proliferation, invasion, and migration of colorectal cancer cells.
[0016] The present invention verifies that piRNA-362733 is upregulated in colorectal cancer tissues and cells, which is statistically significant. The experimental process is rigorous and the results are true and reliable.
[0017] The piRNA-362733 inhibitor provided by the present invention can be used as a targeted therapeutic agent for colorectal cancer, providing a reliable product for improving the therapeutic efficacy of colorectal cancer.
[0018] During the verification process of the present invention, highly expressed piRNA-362733 was first screened out in colorectal cancer tissues. The piRNA-362733 inhibitor can effectively downregulate the expression of piRNA-362733 in colorectal cancer cells. In addition, the piRNA-362733 inhibitor can inhibit the proliferation, invasion and migration of colorectal cancer cells.
[0019] This study, conducted on 60 clinical tissue samples, revealed that piRNA-362733 is highly expressed in colorectal cancer tissue. A piRNA-362733 inhibitor can inhibit the proliferation, invasion, and migration of colorectal cancer cells. This study provides a powerful molecular biology tool for tumor diagnosis and treatment, with profound clinical significance and promising prospects for widespread application.
[0020] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, without limiting the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 A shows the differential piRNA expression analysis between three pairs of colorectal cancer tissues and corresponding normal tissues, and cluster heat map analysis of differentially expressed piRNAs; Figure 1 B is the volcano plot analysis of differentially expressed piRNAs.
[0022] Figure 2 A is the expression analysis of piRNA-362733 in colorectal cancer tissues and normal tissues; Figure 2 B is the expression level of piRNA-362733 in colorectal cancer cells (SW480 and HCT116); Figure 2 C is the expression level of piRNA-362733 after piRNA-NC and piRNA-inhibitor transfection of colorectal cancer cells (SW480 and HCT116). (The two groups were statistically analyzed using the independent sample t-test. represents p<0.01, represents p < 0.001).
[0023] Figure 3 The ROC curve was used to evaluate the sensitivity and specificity of piRNA-362733 for the diagnosis of colorectal cancer. The AUC value was 0.762, the sensitivity was 0.875, and the specificity was 0.649.
[0024] Figure 4 The piRNA-362733 inhibitor inhibits the proliferation, invasion, and migration of colorectal cancer cells HCT116. Figure 4A shows the cell proliferation of HCT116 cell lines after transfection of piRNA-NC and piRNA-inhibitor; Figure 4 B shows the effect of piRNA-362733 inhibitor on the clonogenic ability of HCT116 cells; Figure 4 C shows the effect of piRNA-362733 inhibitor on the invasion and migration ability of HCT116 cells. (The two groups were statistically analyzed using the independent sample t-test. represents p < 0.01). DETAILED DESCRIPTION
[0025] The following examples are intended to further illustrate the present invention, but are not intended to limit the present invention.
[0026] Collection of patient samples
[0027] Sixty normal tissues and 60 cancer tissues were collected from patients undergoing surgery at Guizhou Provincial People's Hospital between 2019 and 2023. All medical records and specimens were collected with informed consent from patients or their families, who signed the relevant informed consent forms and were reviewed by the Ethics Committee of Guizhou Provincial People's Hospital. Tissue RNA was extracted from primary colorectal cancer and normal tissue specimens.
[0028] Example 1: RNA-Seq sequencing to screen differentially expressed piRNAs
[0029] RNA-Seq was performed on colorectal cancer tissues to screen for differentially expressed piRNAs. This portion of the experiment was performed by Shanghai Kangcheng Bioengineering Co., Ltd. The specific steps are as follows: First, total RNA samples were pretreated and cDNA was synthesized. The cDNA template was mixed with Arraystar SYBR® Green qPCR Master Mix, loaded into a microplate, and amplified using an ABI7900 real-time fluorescence quantitative PCR instrument. Significantly overexpressed piRNAs were screened based on a fold change of ≥2 and a p-value <0.05.
[0030] RNA-seq results analysis
[0031] RNA sequencing was performed on total RNA obtained from three pairs of colorectal cancer and normal tissues. Based on the differential expression levels of dysregulated piRNAs (|fold change| ≥ 2 and p value < 0.05), heat maps and volcano plots of piRNA differential expression were plotted ( Figure 1 We selected piRNA-362733, which was the most significantly expressed in colorectal cancer tissues, for qPCR validation.
[0032] Example 2: qPCR detection of piRNA-362733 expression in colorectal cancer tissues and cells
[0033] Extraction and reverse transcription of total RNA from tissues and cell lines
[0034] RNA is extracted from tissues or cells using an RNA extraction kit. After measuring RNA concentration using a NanoDrop, RNA is converted to cDNA using a reverse transcription kit. The synthesized cDNA can be directly used for downstream fluorescence quantitative assays or stored at –20°C.
[0035] qRT-PCR experimental steps and reaction procedures
[0036] (1) The reaction system is as follows:
[0037]
[0038] (2) The reaction procedure is as follows:
[0039]
[0040] (3) Export the reaction results and calculate the Ct value, ΔCt value and 2 -ΔΔCt The specific calculation method is as follows:
[0041] The Ct value was calculated by ABI 7500 Manager software; ΔCt1 = Ct of the gene to be tested in the experimental group - Ct of the reference gene in the experimental group; ΔCt2 = Ct of the gene to be tested in the control group - Ct of the reference gene in the control group; ΔΔCt = ΔCt1 - ΔCt2; 2 -ΔΔCt Calculated by formula.
[0042] piRNA-362733 is highly expressed in colorectal cancer tissues. In addition, compared with normal intestinal epithelial cells NCM460, piRNA-362733 is significantly more highly expressed in colorectal cells (SW460 and HCT116). The use of piRNA-362733 inhibitors can reduce the expression of colorectal cells SW460 and HCT116. Figure 2 AC).
[0043] Example 3: Inhibitory effect of piRNA-362733 inhibitor on colorectal cancer cells
[0044] 1. Selection of piRNA-362733 Inhibitors
[0045] Based on the sequence of piRNA-362733, a specific piRNA-362733 inhibitor (Shanghai Sangon Biotechnology Co., Ltd.) was designed. Its sequence is: TTCAACATCAGTCTGATAAGCTACC.
[0046] 2. piRNA-362733 Inhibitor Transfection
[0047] Colorectal cancer cell lines SW480 and HCT116 cells were placed in 24-well plates and transfected with Lipo3000 transfection reagent (purchased from Thermo Fisher). 25 pmol of piRNA-362733 inhibitor was added to each well. Cellular RNA was extracted 72 hours later, and the expression of piRNA-362733 was detected by real-time fluorescence quantitative PCR.
[0048] 3. CCK-8 assay for cell proliferation
[0049] HCT116 colorectal cancer cells were suspended in the wells of a 96-well plate, with 100 μL added to each well in triplicate. The plate was incubated in a 5% CO2, 37°C incubator until the cells completely covered the bottom of the well. 10 μL of CCK-8 solution was added to each well, and the plate was gently tapped to mix the cells and the solution. The cells were incubated in the cell culture incubator for 4 hours. The absorbance of each cell group was calculated using a microplate reader at 450 nm (note that the absorbance of wells containing culture medium and CCK8 solution but no cells should be subtracted from the results).
[0050] 4. Plate colony formation ability test
[0051] Well-growing HCT116 colorectal cancer cells from each group were trypsinized and collected. The cell pellet was resuspended in complete culture medium to form a single-cell suspension. The cell suspension was serially diluted and seeded into six-well plates according to the optimal number of cells found in pre-experimental experiments. The plated six-well plates were then incubated at 37°C, 5% CO2, and 95% humidity for two weeks. Cell culture was terminated when visible cell colonies were observed. After removing the six-well plates from the incubator, the cells were gently washed twice with PBS, fixed with methanol for 15 minutes, air-dried at room temperature, and stained with Giemsa stain for 30 minutes, followed by air-drying at room temperature. The colony formation rate was calculated as follows: number of colonies / number of cells seeded × 100%.
[0052] 5. Transwell assay
[0053] piRNA-NC or piRNA-inhibitor were transfected into colorectal cancer cells HCT116 for 48 h, and 250 μL of 2×10 5 Cell resuspension (in FBS-free medium) was added to the upper chamber of a Transwell insert, and medium containing 10% FBS was added to the lower chamber. Incubate at 37°C, 5% CO₂ for 24 hours. Cells in the upper chamber were scraped off and the cells were rinsed twice with PBS. Fix the cells with 4% paraformaldehyde for 10 minutes, discard the formaldehyde, and rinse twice with PBS. Observe and count cells under a microscope for statistical analysis.
[0054] The experimental results showed that piRNA-362733 inhibitors can significantly inhibit the proliferation, invasion and migration of colorectal cancer cells HCT116 ( Figure 4 The results suggest that piRNA-362733 inhibitors have an inhibitory effect on colorectal cancer cells.
Claims
1. Use of a preparation for inhibiting the expression level of piRNA-362733 in the preparation of a drug for treating colorectal cancer; the preparation for inhibiting the expression level of piRNA-362733 comprises: piRNA-362733 inhibitor; the piRNA-362733 inhibitor sequence is TTCAACATCAGTCTGATAAGCTACC.
2. Use of a reagent for detecting piRNA-362733 in the preparation of a diagnostic and / or prognostic preparation for colorectal cancer; the reagent for detecting piRNA-362733 includes a reagent for detecting piRNA-362733 expression; the reagent for detecting piRNA-362733 expression includes a PCR reagent, and the PCR primer sequence is: F: 5'-GTAGCTTATCAGACTGATGTTG-3' R: 5'-GTCGTATCCAGTGCAGGGTCCGAGGT-3'.