A micromolecule RNA-19b-3p and its application in the treatment of pancreatic ductal adenocarcinoma.

Abstract

This invention relates to the field of biomedical technology, specifically to a micromolecule RNA-19b-3p and its application in the treatment of pancreatic ductal adenocarcinoma. The micromolecule RNA-19b-3p has the nucleotide sequence shown in SEQ ID NO:1. This invention discloses its use in the preparation of a medicament for treating pancreatic ductal adenocarcinoma, achieved by inhibiting the expression and / or activity of the micromolecule RNA-19b-3p. The medicament comprises a substance capable of reducing the expression or activity of the micromolecule RNA-19b-3p. This substance is its inhibitor, suppressor, antagonist, or antisense oligonucleotide. The medicament is used to inhibit the proliferation, migration, and / or invasion of pancreatic ductal adenocarcinoma cells. This invention provides a novel biomarker and therapeutic intervention target for pancreatic ductal adenocarcinoma, clarifies the feasibility of targeted therapy, and possesses clear translational potential.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a micromolecule RNA-19b-3p and its application in the treatment of pancreatic ductal adenocarcinoma. Background Technology

[0002] The global incidence and mortality rates of pancreatic ductal adenocarcinoma have been rising steadily in recent years. In 2020, there were approximately 495,773 new cases and 466,003 deaths worldwide. It is projected that by 2030, pancreatic ductal adenocarcinoma will become the second leading cause of cancer-related deaths globally. Its high mortality rate is primarily due to late-stage diagnosis and the lack of effective treatments. Despite breakthroughs in early diagnostic technologies (such as imaging biomarkers and ctDNA testing), survival rates have not significantly improved; therefore, exploring effective treatment methods is of paramount importance.

[0003] Dysregulation of microRNA-19b-3p (miR-19b-3p) expression plays a central role in the occurrence, progression, and metastasis of pancreatic ductal adenocarcinoma. Therefore, identifying key miRNAs that influence the malignancy of pancreatic ductal adenocarcinoma cells could become novel therapeutic targets, potentially leading to breakthroughs in the treatment of this cancer. Summary of the Invention

[0004] To address the current lack of highly effective diagnostic biomarkers and novel therapeutic targets for pancreatic ductal adenocarcinoma (PDAC), this invention provides a small molecule RNA-19b-3p and its application in the treatment of pancreatic ductal adenocarcinoma.

[0005] The specific technical solution is as follows: First, the present invention relates to the use of a micromolecule RNA-19b-3p in the preparation of a medicament for treating pancreatic ductal adenocarcinoma, wherein the micromolecule RNA-19b-3p has a nucleotide sequence as shown in SEQ ID NO: 1.

[0006] SEQ ID NO. 1: UGUGCAAAUCCAUGCAAAACUGA.

[0007] Bioinformatics analysis based on the public gene expression database (GEO, dataset GSE249152) revealed that miR-19b-3p was significantly highly expressed in pancreatic ductal adenocarcinoma tissues (see Experiment 1 in the detailed implementation plan). Figure 1 The presence of this gene suggests it may be an oncogene and could serve as a potential intervention target. Its differential expression also suggests its potential as a diagnostic biomarker.

[0008] Furthermore, the intended use is achieved by inhibiting the expression and / or activity of the micromolecule RNA-19b-3p. In vitro functional experiments confirmed that downregulating its expression with miR-19b-3p inhibitors significantly reduced the proliferation, migration, and invasion abilities of pancreatic ductal adenocarcinoma PL45 cells (P<0.05) (see Experiment 1 in the detailed embodiments). Figure 2 , Figure 3 This result directly proves that "inhibiting miR-19b-3p" is a specific and effective technical means to achieve therapeutic effects.

[0009] Secondly, the present invention also provides a medicament for treating pancreatic ductal adenocarcinoma, the medicament comprising a substance capable of reducing the expression or activity of the micromolecular RNA-19b-3p. The "miR-19b-3p inhibitor" used in the experiment is a specific form of this substance, which successfully reduced the expression of the target miRNA and produced a therapeutic effect, demonstrating the feasibility of pharmaceutical compositions containing such substances.

[0010] Furthermore, the substance is an inhibitor, suppressor, antagonist, or antisense oligonucleotide of the micromolecule RNA-19b-3p. In Experiment 1, a commercially available miRNA inhibitor (a chemically modified antisense oligonucleotide) was used as a representative substance, achieving significant technical effects (inhibition of proliferation, migration, and invasion), verifying the effectiveness of the technical approach, and demonstrating that such substances are effective components for preparing therapeutic drugs.

[0011] Furthermore, the drug is used to inhibit the proliferation, migration, and / or invasion of pancreatic ductal adenocarcinoma cells. This invention, using the miR-19b-3p inhibitor, clearly demonstrated a beneficial effect of "inhibiting the proliferation, migration, and invasion of the PL45 lineage" at the cellular level (P<0.001), providing direct experimental evidence for this use.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Discovery of novel diagnostic and therapeutic targets: For the first time, miR-19b-3p was found to be significantly highly expressed in pancreatic ductal adenocarcinoma, and it was verified that it is a key factor driving the malignant behavior of cancer cells, providing new biomarkers and therapeutic intervention targets for this disease.

[0013] 2. The feasibility of targeted therapy has been clarified: In vitro experiments have confirmed that inhibiting the expression of miR-19b-3p can significantly reduce the proliferation, migration and invasion of cancer cells, which provides direct experimental evidence for the development of therapeutic drugs (such as inhibitors, antisense oligonucleotides, etc.) targeting it.

[0014] 3. Possesses clear translational potential: This invention also lays the molecular foundation for the development of targeted therapies (such as targeted drugs) for pancreatic ductal adenocarcinoma, and is of great value in addressing the pain points of difficult clinical diagnosis and limited treatment options for this disease. Attached Figure Description

[0015] Figure 1 A schematic diagram of the GEO2R analysis results of dataset GSE249152 in the public gene chip database GEO; Figure 2 A statistical graph showing the OD values ​​of PL45 cells after 3 days of in vitro culture, as determined by MTT assay to assess cell proliferation. Figure 3 This is a schematic diagram showing the Hoechst nuclear staining results of migrating and invading cells after 3 days of Transwell culture. Where: A shows the Hoechst-stained nuclei of three groups of migrating and invasive cells, scale bar = 25 micrometers; B is a statistical graph of the number of migrating and invasive cells. Detailed Implementation

[0016] The technical solution of the present invention will be described in detail below through experiments, but the scope of protection of the present invention is not limited thereto.

[0017] Experiment 1: Discovery of miR-19b-3p and its functional validation in pancreatic ductal adenocarcinoma This experiment aims to demonstrate that miR-19b-3p is a potential oncogene for pancreatic ductal adenocarcinoma and its rationale as a therapeutic target.

[0018] 1. Bioinformatics screening identifies key miRNAs The Gene Expression Omnibus (GEO) database is a gene expression database created and maintained by the National Center for Biotechnology Information (NCBI) in the United States. The GEO database contains high-throughput gene expression data submitted by research institutions worldwide. Using the GEO database with the keywords "pancreatic cancer" and "miRNA," the dataset GSE249152, "MiRNA expression profiling in pancreatic cancer cells compared to normal epithelial cells," was retrieved. GEO2R analysis identified abnormally expressed miRNAs in early-stage pancreatic ductal adenocarcinoma tissue, among which miR-19b-3p was one of the significantly highly expressed miRNAs (see [link to relevant documentation]). Figure 1The above analysis suggests that miR-19b-3p may act as an oncogene involved in the development and progression of pancreatic ductal adenocarcinoma. Therefore, substances that can reduce (inhibit) its expression could serve as potential therapeutic agents for pancreatic ductal adenocarcinoma.

[0019] 2. In vitro functional experiments to verify target function 2.1 Cell Culture and Transfection The human pancreatic ductal adenocarcinoma PL45 cell line used in this experiment was purchased from Shanghai Jinyuan Biotechnology Co., Ltd. Cells were cultured in a 37°C incubator with 5% CO2 in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin.

[0020] The expression of miR-19b-3p was downregulated using a miR-19b-3p inhibitor (an antisense oligonucleotide, anti-miR-19b-3p), with a negative control sequence (miR-NC) used as a transfection control. Both the miR-19b-3p inhibitor (anti-miR-19b-3p) and the negative control (miR-NC) were purchased from Thermo Fisher Scientific. Cell transfection was performed using Invitrogen's Lipofectamine 2000 transfection reagent, following the manufacturer's instructions. The transfection concentration of both the miRNA inhibitor and the control was 5 nanomoles / liter.

[0021] The groups were set as follows: blank control group (PL45 cells without any treatment), miR-NC negative control group, and miR-19b-3p inhibitor transfection group.

[0022] 2.2 MTT assay for cell proliferation PL45 cells and transfected cells were loaded at a rate of 1x10 5 Seeds were planted at a density of 1000 cells / mL in 24-well plates, and 2 mL of DMEM medium containing 10% FBS was added to each well. After incubation at 37°C and 5% CO2 for 72 hours, the MTT assay kit (Beyotime) was followed according to the instructions. Finally, the optical density (OD) of each well was measured at 570 nm using a microplate reader.

[0023] 2.3 Transwell assay to detect cell migration and invasion capabilities Cells were spaced at 1x10 5Cells were seeded at a density of 10 cells / mL in the upper chamber of a Transwell chamber containing serum-free DMEM medium, while the lower chamber contained DMEM medium with 20% FBS as a chemotactic agent. After culturing at 37°C in a 5% CO2 incubator for 72 hours, cells that had migrated to the lower chamber membrane were fixed with 4% paraformaldehyde for 15 minutes and stained with Hoechst (1:3000 dilution) in the dark for 30 minutes. The number of migrated cells was then observed and counted under a fluorescence microscope.

[0024] 2.4 Statistical Analysis SPSS 21.0 statistical software was used to analyze the data. The obtained measurement data conformed to a normal distribution after Shapiro-Wilk test and were expressed as mean ± standard deviation. One-way ANOVA was used for pairwise comparisons among the three groups. P A value <0.05 was considered statistically significant.

[0025] 2.5 Experimental Results The results are as follows Figure 2 , Figure 3 As shown. Compared with the blank control group and the miR-NC group, by Figure 2 It can be seen that the experimental group transfected with miR-19b-3p inhibitor showed a significant decrease in the proliferation capacity (OD value) of PL45 cells. Figure 3 middle Figure 3 (A) and Figure 3 (B) indicates that the number of migrating and invading cells was significantly reduced. One-way ANOVA showed that the differences between groups were statistically significant (P<0.05). Specifically, a vs. Control group, P<0.001; b vs. miR-NC group, P<0.001.

[0026] 3. Experimental Conclusions This experiment demonstrates that inhibiting miR-19b-3p expression can significantly reduce the proliferation, migration, and invasion of pancreatic ductal adenocarcinoma PL45 cells, thus validating its effectiveness as a therapeutic target at the functional level.

[0027] Unless otherwise specified, all technologies mentioned above refer to existing technologies.

[0028] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. The use of a small molecule RNA-19b-3p in the preparation of a medicament for treating pancreatic ductal adenocarcinoma, characterized in that, The micromolecule RNA-19b-3p has a nucleotide sequence as shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that, The intended use is achieved by inhibiting the expression and / or activity of the micromolecule RNA-19b-3p.

3. A drug for treating pancreatic ductal adenocarcinoma, characterized in that, The drug contains a substance that can reduce the expression or activity of the micromolecule RNA-19b-3p.

4. The drug according to claim 3, characterized in that, The substance is an inhibitor, suppressor, antagonist, or antisense oligonucleotide of the micromolecule RNA-19b-3p.

5. The drug according to any one of claims 3 or 4, characterized in that, The drug is used to inhibit the proliferation, migration, and / or invasion of pancreatic ductal adenocarcinoma cells.

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