Application of miR-106b-5p in screening or preparing pancreatic ductal adenocarcinoma treatment medicine

Abstract

The invention discloses an application of miR-106b-5p in screening or preparing a medicine for treating pancreatic ductal adenocarcinoma. According to the invention, expression of miR-106b-5p is down-regulated by adopting a miRNA inhibitor, miR-NC is taken as a contrast, the miR-106b-5p inhibitor and the contrast miR-NC are respectively transfected with a pancreatic ductal adenocarcinoma PL45 system by applying a Lipofectamine 2000 transfection reagent, then screening is carried out, the PL45 system with stable low expression and normal expression of miR-106b-5p is obtained, in-vitro culture is carried out, and change conditions of proliferation and migration invasion ability of the PL45 system under the two conditions are observed. The result shows that the miR-106b-5p inhibitor can inhibit the proliferation and migration invasion ability of the PL45 system in vitro, and the malignant degree of the pancreatic ductal adenocarcinoma PL45 system can be obviously inhibited by low expression of the miR-106b-5p.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of miR-106b-5p as a biomarker in screening or preparing drugs for 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. The high mortality rate of pancreatic ductal adenocarcinoma 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 miRNA 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] The purpose of this invention is to provide the application of miR-106b-5p in screening or preparing therapeutic drugs for pancreatic ductal adenocarcinoma.

[0005] 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. Figure 1 As shown, this invention utilizes the GEO database with "pancreatic cancer" and "miRNA" as keywords to retrieve the dataset GSE249152 "MiRNA expression profiling in pancreatic cancer cells compared to normal epithelial cells". GEO2R analysis identified abnormally expressed miRNAs in early-stage pancreatic ductal adenocarcinoma tissue, among which miR-106b-5p was one of the significantly highly expressed miRNAs, suggesting that miR-106b-5p may be an oncogene of pancreatic ductal adenocarcinoma. Therefore, substances that promote low expression of miR-106b-5p could serve as therapeutic agents for pancreatic ductal adenocarcinoma.

[0006] The application adopts miRNA inhibitor to down-regulate the expression of miR-106b-5p, takes miR-NC as a control, transfects the pancreatic ductal adenocarcinoma PL45 line with the miR-106b-5p inhibitor and the control miR-NC respectively by using Lipofectamine 2000 transfection reagent, and then carries out screening to obtain the PL45 line with stable low expression and normal expression of miR-106b-5p, in vitro culture, and observation of the change of the proliferation and migration and invasion ability of the PL45 line under the two conditions. The results show that the miR-106b-5p inhibitor can inhibit the proliferation and migration and invasion ability of the PL45 line in vitro P <0.05), and the low expression of miR-106b-5p can obviously inhibit the malignant degree of the pancreatic ductal adenocarcinoma PL45 line P <0.05). BRIEF DESCRIPTION OF DRAWINGS

[0007] Figure 1 It is the data set GSE249152 in the public gene chip database GEO, and the abnormal expression miRNAs in the early cancer tissue of pancreatic ductal adenocarcinoma are obtained by GEO2R analysis, wherein miR-106b-5p is one of the significantly high expression miRNAs.

[0008] Figure 2 It is the OD value statistical graph of MTT detection of cell proliferation ability of the PL45 line after in vitro culture for 3 days (mean ± standard deviation, a vs. Control group, P <0.001; b vs. miR-NC group, P <0.001).

[0009] Figure 3 It is the Hoechst staining nucleus detection result of the migration and invasion cells after Transwell culture for 3 days. Among them: A is the Hoechst staining nucleus of the migration and invasion cells of the three groups, and the scale = 25 microns; B is the statistical graph of the number of migration and invasion cells (mean ± standard deviation, a vs. Control group, P <0.001; b vs. miR-NC group, P <0.001). DETAILED DESCRIPTION

[0010] The preferred embodiments of the application will be described in detail below with reference to the examples. It should be understood that the following examples are given only for the purpose of illustration and are not intended to limit the scope of the application. Those skilled in the art can make various modifications and replacements to the application without departing from the spirit and principles of the application.

[0011] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0012] The materials, reagents, etc. used in the following examples can be obtained commercially unless otherwise specified. Example 1

[0013] The in vitro observation of the malignant degree of pancreatic ductal adenocarcinoma in this embodiment uses human PL45 cell lines.

[0014] In this embodiment, the expression of miR-106b-5p is down-regulated using miRNA inhibitors. The PL45 line without any treatment is used as a control. The miR-106b-5p inhibitor and the control miR-NC are applied to the pancreatic ductal adenocarcinoma PL45 line using the Lipofectamine 2000 transfection reagent, respectively, and then screened. The PL45 line with stable low expression and normal expression of miR-106b-5p is obtained, cultured in vitro, and the changes in the proliferation and migration and invasion abilities of the PL45 line under the two conditions are observed.

[0015] The specific process is as follows: 1. PL45 line culture and miRNA inhibitor transfection 1) The PL45 line is from Shanghai Jin Yuan Biotechnology Co., Ltd. and is cultured in a 37°C incubator with 5% CO2. The culture medium is DMEM (Gibco Company) with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin.

[0016] 2) The miR-106b-5p inhibitor and miR-NC are from Thermo Fisher Scientific. The cell transfection uses the Lipofectamine 2000 transfection reagent from Invitrogen. The transfection steps are performed according to the instructions. The concentration of the miRNA inhibitor is 5 nanomoles per liter.

[0017] 2. In vitro culture and MTT detection The PL45 line and the transfected PL45 line are inoculated in each well of a 24-well culture plate at a density of 1×10 6 The 24-well culture plate is placed in a 37°C, 5% CO2 incubator with saturated humidity for culture. After 72 hours, the proliferation ability of the cells is detected according to the instructions of the MTT kit (Bi Yun Tian). Finally, the OD value of each well is measured at 570 nm using an enzyme marker.

[0018] 3. In vitro Transwell culture and Hoechst nuclear staining detection The PL45 line and the transfected PL45 line are inoculated in each well of a 24-well culture plate at a density of 1×10 6Cells were seeded at a density of [number] cells / mL in the upper chamber of a Transwell incubator and cultured in serum-free DMEM medium. Then, DMEM medium containing 20% ​​FBS was added to the lower chamber. The cells were incubated in a humidified incubator at 37 °C and 5% CO2. After 72 hours of culture, cells that had migrated to the lower surface of the upper chamber were fixed with 4% paraformaldehyde for 15 minutes, stained with Hoechst (1:3000) in the dark for 30 minutes, and the number of migrating and invading cells was observed and counted under a fluorescence microscope.

[0019] 4. Statistical processing 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 is considered statistically significant.

[0020] The results are as follows Figure 2 , 3 As shown, compared with the blank control group and the miR-NC group, the cell proliferation capacity of PL45 cells transfected with miR-106b-5p inhibitor was significantly reduced, and the number of migrating and invading cells was significantly decreased. One-way ANOVA results showed that the differences between groups were statistically significant. P <0.05). a vs. Control group, P <0.001; b vs. miR-NC group, P <0.001.

Claims

1. Use of a substance for detecting miR-106b-5p in the preparation of a diagnostic reagent for pancreatic ductal adenocarcinoma.

2. A diagnostic agent for diagnosing or aiding in the diagnosis of pancreatic ductal adenocarcinoma, characterized by: The substance for detecting miR-106b-5p.

3. Use of miR-106b-5p as a marker in the development of a diagnostic reagent for pancreatic ductal adenocarcinoma.

4. Use of miR-106b-5p as a marker in the development of a therapeutic drug for pancreatic ductal adenocarcinoma.

5. Use of a substance for reducing the expression of miR-106b-5p in the preparation of a therapeutic drug for pancreatic ductal adenocarcinoma.

6. Use of a substance for reducing the expression of miR-106b-5p in the preparation of a drug for inhibiting the proliferation and / or migration of pancreatic ductal adenocarcinoma cells.

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