SiRNA for inhibiting p311 gene, composition and use thereof

Abstract

The application relates to the technical field of biological medicine, and discloses siRNA for inhibiting a P311 gene, a composition and application thereof. The siRNA can specifically knock down P311 gene expression in human tumor cells, can be used as an auxiliary component for improving the drug efficacy of gemcitabine, can improve the drug sensitivity of a pancreatic cancer patient to gemcitabine, can reduce the gemcitabine drug resistance of a pancreatic cancer patient in an advanced stage, can improve the prognosis of the pancreatic cancer patient, can prolong the survival period of the pancreatic cancer patient in the advanced stage, and has important significance for the treatment of pancreatic cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a siRNA that inhibits the P311 gene, a composition thereof, and its application. Background Technology

[0002] Pancreatic cancer is one of the most common malignant tumors. In recent years, statistics show that the incidence of pancreatic cancer has shown a significant upward trend. Compared with other types of tumors, pancreatic cancer patients have shorter survival time and higher mortality rate. This is mainly because most pancreatic cancer patients are in the late stage, with a high rate of tumor metastasis. By the time of diagnosis, proximal or distant metastasis has already occurred, and the surgical window period has been missed.

[0003] Among related technologies, palliative chemotherapy is the only treatment option for pancreatic cancer patients, especially those with advanced stages. Gemcitabine, as the only standard first-line treatment for pancreatic cancer, can significantly improve the treatment response rate and overall survival of pancreatic cancer patients. However, drug resistance can develop in patients after only a few weeks of use, which greatly limits the efficacy of gemcitabine chemotherapy.

[0004] Therefore, there is an urgent need to find a composition that can reduce the resistance of pancreatic cancer cells to gemcitabine. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes an siRNA that inhibits the P311 gene, a composition thereof, and its application. This siRNA can effectively reduce the expression of P311 (a neuronal regeneration-related protein), inhibit the epithelial-mesenchymal transition of pancreatic cancer cells, reduce the resistance of pancreatic cancer cells to gemcitabine, and inhibit the growth of subcutaneous tumors of pancreatic cancer, which is of great significance for adjuvant treatment of pancreatic cancer.

[0006] The present invention also proposes a composition for the prevention or treatment of pancreatic cancer, comprising the above-mentioned siRNA.

[0007] This invention also proposes the application of the above-mentioned siRNA in the preparation of drugs for the prevention or treatment of tumors / cancer.

[0008] This invention also proposes the application of the above-mentioned siRNA in the preparation of products that reduce the gemcitabine resistance of pancreatic cancer cells.

[0009] In a first aspect, the present invention provides a siRNA for inhibiting the P311 gene, said siRNA comprising a sense strand and an antisense strand, wherein,

[0010] The nucleotide sequence of the positive strand of the siRNA is: 5'-UGAUAAAUAGCAUGAUUGCAC-3' (SEQ ID NO. 7).

[0011] The nucleotide sequence of the siRNA antisense strand is: 5'-GCAAUCAUGCUAUUUAUCAAA-3' (SEQ ID NO. 8);

[0012] Alternatively, it may be a nucleotide sequence that has more than 80% homology with the sense strand or the antisense strand of the siRNA and has the same function.

[0013] The siRNA according to embodiments of the present invention has at least the following beneficial effects:

[0014] (1) The siRNA of the present invention can knock down the expression of P311, upregulate the expression of E-cadherin protein, downregulate the expression of N-cadherin and Vimentin protein, thereby inhibiting the epithelial-mesenchymal transition of pancreatic cancer cells.

[0015] (2) The siRNA of the present invention can reduce the proliferation ability of pancreatic cancer cells and their resistance to gemcitabine chemotherapy after interfering with the expression of P311, thereby improving the efficacy of gemcitabine.

[0016] (3) The siRNA of the present invention can significantly inhibit the growth of subcutaneous pancreatic cancer tumors mediated by P311 after interfering with P311 expression.

[0017] In some embodiments of the present invention, the P311 gene is selected from the human P311 gene.

[0018] In a second aspect, the present invention provides a medicament or composition for the prevention or treatment of pancreatic cancer, the composition comprising: the above-mentioned siRNA; or an expression system capable of expressing the above-mentioned siRNA.

[0019] In some embodiments of the present invention, it further includes: pharmaceutically acceptable carriers and / or excipients; and other active ingredients for the prevention or treatment of tumors.

[0020] In some embodiments of the present invention, pharmaceutically acceptable carriers and / or excipients include, but are not limited to, buffers, emulsifiers, suspending agents, stabilizers, preservatives, saline, excipients, fillers, coagulants and blending agents, surfactants, dispersants, and defoamers.

[0021] In some embodiments of the present invention, the other active ingredients for preventing or treating tumors include: chemotherapeutic agents, radiotherapy agents, or antibody drugs;

[0022] Preferably, the chemotherapeutic agent is gemcitabine.

[0023] In some embodiments of the present invention, the composition is suitable for: direct naked RNA injection, liposome-encapsulated RNA direct injection, protein or polypeptide-encapsulated RNA direct injection, gold-coated RNA gene gun bombardment, bacterial plasmid-carrying RNA expression, or viral RNA expression.

[0024] In some embodiments of the present invention, the drug or composition may be selected from any form of solid, liquid, gel, semi-fluid, or aerosol.

[0025] A third aspect of the present invention provides the use of the above-described siRNA in the preparation of drugs for the prevention or treatment of tumors / cancer.

[0026] According to the application of the embodiments of the present invention, at least the following beneficial effects are achieved: the siRNA of the present invention can knock down the expression of P311, thereby reducing the proliferation ability of tumor cells, and the siRNA of the present invention, when used in combination with gemcitabine, can reduce the resistance of tumor cells to gemcitabine, which helps to provide therapeutic effects.

[0027] In some embodiments of the present invention, the tumor / cancer is pancreatic cancer.

[0028] In a fourth aspect, the present invention provides the use of the above-described siRNA in the preparation of a product that reduces the gemcitabine resistance of pancreatic cancer cells.

[0029] According to the application of the embodiments of the present invention, at least the following beneficial effects are achieved: the combination of the siRNA of the present invention and gemcitabine can reduce the resistance of tumor cells to gemcitabine and help to provide therapeutic effects.

[0030] In some embodiments of the present invention, the cells are pancreatic cancer cells.

[0031] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0032] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0033] Figure 1 This is a comparison of the expression levels of P311 mRNA in pancreatic cancer tissue (T) and paired adjacent normal tissue (N) detected by Real-time PCR in this invention, where the quantification method is log2[T / N].

[0034] Figure 2This image shows the Western blot results of P311 in pancreatic cancer tissue and paired adjacent non-tumor tissue, where C represents pancreatic cancer tissue and N represents adjacent non-tumor tissue.

[0035] Figure 3 The image shows the IHC results of PGAM5 protein expression levels in pancreatic cancer tissue (Tumor) and paired adjacent normal tissue (Non-tumor) in this invention.

[0036] Figure 4 This is a statistical graph showing the protein expression levels of PGAM5 in pancreatic cancer tissue (Tumor) and paired adjacent normal tissue (Non-tumor) in this invention;

[0037] Figure 5 The results of Real-time PCR detection of the mRNA expression levels of P311 in pancreatic cancer cell lines (CAPAN-1 and AsPC-1) derived from metastasis and in situ derived from pancreatic cancer cell lines (BxPC-3 and PANC-1) are presented.

[0038] Figure 6 Western blot analysis was performed on the protein expression level of P311 in the pancreatic cancer cell lines BxPC-3, PANC-1, CAPAN-1, and AsPC-1 of this invention.

[0039] Figure 7 The figure shows the effect of upregulating P311 on the proliferation ability of pancreatic cancer cell lines CAPAN-1 and BXPC-3 detected by CCK8 in this invention.

[0040] Figure 8 The graph shows the changes in gemcitabine resistance in cells after treatment with gemcitabine (20 nM) using crystal violet staining. Con represents the control group, and P311 represents the group with upregulated P311.

[0041] Figure 9 The graph shows the apoptosis and necrosis of cells after treatment with gemcitabine (20 nM) by Annexin V / PI flow cytometry. Con represents the control group, and P311 represents the group with upregulated P311.

[0042] Figure 10 This is a statistical chart showing the expression levels of P311 mRNA in cancer cells after treatment with different siRNAs according to the present invention.

[0043] Figure 11 Western blot analysis of the expression levels of EMT markers E-cadherin, N-cadherin and Vimentin proteins after P311 overexpression;

[0044] Figure 12 The results show the expression levels of EMT marker molecules E-cadherin, N-cadherin, and Vimentin mRNA after P311 overexpression in this invention.

[0045] Figure 13 The expression levels of EMT markers E-cadherin, N-cadherin, and Vimentin proteins in the stable pancreatic cancer cell lines CAPAN-1 and BXPC-3, which overexpress P311 in this invention, were detected by Western Blot after siRNA interference with P311 expression.

[0046] Figure 14 The figure shows the effect of siRNA treatment on the proliferation of pancreatic cancer cell lines CAPAN-1 and BXPC-3. Con represents the control group, P311 represents the high expression P311 group, and P311+siRNA represents the high expression P311 plus siRNA treatment group.

[0047] Figure 15 The crystal violet staining results show the changes in gemcitabine resistance in cells after siRNA interference with P311 expression.

[0048] Figure 16 Flow cytometry was used to detect apoptosis and necrosis of cells after siRNA interference with P311 expression using Annexin V / 7-AAD.

[0049] Figure 17 The number and distribution of bioluminescent cells in nude mice after subcutaneous injection of LUC-stable transfected control (Con), P311 high expression group (P311), and P311 overexpression plus siRNA interference group (P311+siRNA) cells.

[0050] Figure 18 Comparison of subcutaneous tumor size in nude mice from the control group, the P311 high expression group, and the P311 overexpression plus siRNA interference group;

[0051] Figure 19 The figure shows the statistical distribution of subcutaneous tumor size in nude mice in the control group, the P311 high expression group, and the P311 overexpression plus siRNA interference group. Detailed Implementation

[0052] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0053] In the description of this invention, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0054] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0055] Example 1: Study on the expression of P311 in pancreatic cancer and its functional mechanism affecting invasion and metastasis

[0056] (I) Upregulation of P311 expression in pancreatic cancer tissues

[0057] To investigate the expression of P311 in pancreatic cancer tissues, this invention analyzed the TCGA-PAAD dataset and found that the mRNA level of P311 protein was upregulated in pancreatic cancer tissues compared to adjacent normal tissues. Simultaneously, real-time PCR results from 15 pairs of fresh pancreatic cancer tissues (cancer and adjacent normal tissues) surgically resected at Sun Yat-sen University Cancer Center also showed that the mRNA level of P311 was upregulated in pancreatic cancer, specifically as follows... Figure 1 As shown.

[0058] Further Western blot analysis was performed on the P311 protein levels in these 15 pairs of freshly frozen pancreatic cancer tissues and adjacent normal tissues. The Western blot results are as follows: Figure 2 As shown, the results indicate that the expression level of P311 protein is increased in pancreatic cancer tissue compared to adjacent normal tissue. Next, immunohistochemical staining was performed on tissue microarrays containing 80 pancreatic cancer (cancer and adjacent) samples, and the results are as follows... Figure 3 and Figure 4 As shown, the protein expression level of P311 is higher in most pancreatic cancer tissues compared with normal adjacent tissues.

[0059] The above results indicate that P311 expression is upregulated in pancreatic cancer, including at both the mRNA and protein levels, suggesting that transcriptional activation of P311 expression occurs in pancreatic cancer tissues.

[0060] (II) P311 is highly expressed in metastatic pancreatic cancer cell lines.

[0061] This section examined the expression of P311 in different pancreatic cancer cell lines. Among them, CAPAN-1 is a pancreatic cancer cell line derived from liver metastasis, AsPC-1 is a pancreatic cancer cell line derived from ascites metastasis, while BxPC-3 and PANC-1 are pancreatic cancer cell lines derived from in situ pancreatic cancer.

[0062] Real-time PCR results confirmed that the mRNA expression level of p311 in metastatic pancreatic cancer cell lines (CAPAN-1 and AsPC-1) was higher than that in orthotopic pancreatic cancer cell lines (BxPC-3 and PANC-1), specifically as follows: Figure 5 As shown; meanwhile, the Western blot results show (specifically as follows) Figure 6 As shown in the figure, the protein expression level of P311 in metastatic pancreatic cancer cell lines (CAPAN-1 and AsPC-1) is higher than that in orthotopic pancreatic cancer cell lines (BxPC-3 and PANC-1).

[0063] The above results indicate that P311 is highly expressed in metastatic pancreatic cancer cell lines compared to in situ pancreatic cancer cell lines, further suggesting that P311 is associated with the metastatic potential of pancreatic cancer.

[0064] (III) High expression of P311 promotes the proliferation of pancreatic cancer cells and resistance to gemcitabine chemotherapy.

[0065] To further investigate the effect of P311 upregulation on tumor progression in pancreatic cancer, P311 was overexpressed in pancreatic cancer cell lines CAPAN-1 and BXPC-3. Then, CCK8 cell proliferation assay, crystal violet staining assay, and Annexin V / PI flow cytometry assay were performed to detect apoptosis.

[0066] The CCK8 cell proliferation results are as follows: Figure 7 As shown, the results indicated that P311 overexpression significantly enhanced the proliferation of pancreatic cancer cells. After treatment with the pancreatic cancer chemotherapy drug gemcitabine (20 nM) for 48 h, cells were collected, and crystal violet staining was used to detect changes in gemcitabine resistance. The results are shown below. Figure 8 As shown, the results indicate that P311 overexpression significantly enhances the resistance of pancreatic cancer cells to gemcitabine. Annexin V / PI flow cytometry results of apoptosis assays are shown below. Figure 9 As shown, P311 overexpression can significantly reduce gemcitabine-induced apoptosis and necrosis.

[0067] Example 2: siRNA Design and Screening

[0068] (I) siRNA Design

[0069] Based on the above research, this embodiment designs and synthesizes four siRNA sequences that can specifically knock down the expression of the P311 gene in human tumor cells, according to the basic principles of siRNA target sequences. These are P311-siRNA#1, P311-siRNA#2, P311-siRNA#3, and P311-siRNA#4 sequences. The sense and antisense strand base sequences of P311-siRNA#1 are as follows:

[0070] P311-siRNA#1 positive strand: 5'-UUUCUUCUUAUACUUGAUGAU-3' (SEQ ID NO.1);

[0071] P311-siRNA#1 antisense strand: 5'-CAUCAAGUAUAAGAAGAAACU-3' (SEQ ID NO.2).

[0072] The base sequences of the sense and antisense strands of P311-siRNA#2 are as follows:

[0073] P311-siRNA#2 positive strand: 5'-AUGAGUUUCAAACUACUUCUA-3' (SEQ ID NO.3);

[0074] P311-siRNA#2 antisense strand: 5'-GAAGUAGUUUGAAACUCAUUG-3' (SEQ ID NO.4).

[0075] The sense and antisense strand base sequences of P311-siRNA#3 are as follows:

[0076] P311-siRNA#3 positive strand: 5'-AACAACAUUCCAUGAGUAGAU-3' (SEQ ID NO.5);

[0077] P311-siRNA#3 antisense strand: 5'-CUACUCAUGGAAUGUUGUUGU-3' (SEQ ID NO.6).

[0078] The base sequences of the sense and antisense strands of P311-siRNA#4 are as follows:

[0079] P311-siRNA#4 positive strand: 5'-UGAUAAAUAGCAUGAUUGCAC-3' (SEQ ID NO.7);

[0080] P311-siRNA#4 antisense strand: 5'-GCAAUCAUGCUAUUUAUCAAA-3' (SEQ ID NO.8).

[0081] (II) Detection of siRNA inhibition of P311 expression

[0082] 1. siRNA transfection in cells

[0083] The siRNAs of this invention are all synthesized by Sangon Biotech and act directly on the mRNA of the target gene P311. The conversion relationship of the 21bp long siRNA is: 1 OD duplex = 3.0 nmols = 40 μg; 1 OD of siRNA is resuspended in 150 μL LEPCH2O and dissolved to obtain a 20 μM stock solution.

[0084] siRNA storage: Store the dry siRNA powder at -80℃. Before use, prepare a 20μM stock solution using the provided RNase-free H2O or sterile ddH2O, and aliquot and store. All pipette tips that come into contact with the siRNA should be imported RNase-free tips. During the experiment, the siRNA should be kept on ice. After use, store at -20℃. Before transfection, place the siRNA and Lipofectamin 2000 transfection reagent on ice and bring them into the cell culture room for transfection. The specific transfection steps are as follows.

[0085] (1) Cell Culture Preparation

[0086] Cell culture was performed using DMEM medium supplemented with 10% serum. Cells were cultured in a 5% CO2 incubator at 37°C. Cells were passaged when the cell density reached 80-90%.

[0087] (2) siRNA transfection

[0088] ① The day before transfection, inoculate 8 × 10⁸ cells per well of a 12-well plate. 4 Add 1 mL of DMEM medium containing FBS (Fetal Bovine Serum) but without antibiotics to the cells;

[0089] ② After culturing for 24 hours, wait until the cell density reaches 50%–60% before use;

[0090] ③ Half an hour before transfection, replace the culture medium of the cells in the 12-well plate with serum-free culture medium and place it in a CO2 incubator;

[0091] ④ Dilute 1 μL of 20 μM siRNA stock solution (i.e., 25 pmol of siRNA) with 50 μL of Opti-MEM medium, and add it to the cells to a final concentration of 50 nM. Mix gently.

[0092] ⑤ Mix lipofectamin 2000 reagent, dilute 1 μL of lipofectamin 2000 reagent with 50 μL of Opti-MEM medium, mix gently, and let stand at room temperature (25±2℃) for 5 min;

[0093] ⑥ Mix the diluted siRNA and diluted lipofectamin 2000 reagent, mix gently, and incubate at room temperature for 20 minutes to form the siRNA / lipofectamin 2000 complex; add 100 μL of the siRNA / lipofectamin 2000 complex to the pre-transfected starved cells, and gently shake the culture plate back and forth and side to side.

[0094] ⑦ Six hours after transfection, remove the culture supernatant from the well, add 1 ml of DMEM medium without antibiotics containing 10% FBS, and put it back into the CO2 incubator to continue culturing. Depending on the cell status, decide whether to change the medium before testing the transfection effect.

[0095] ⑧ The siRNA silencing effect can be detected 48h to 72h after cell transfection.

[0096] 2. Quantitative Real-Time PCR Detection

[0097] Cells were collected after transfection, RNA was extracted, reverse transcribed, and real-time quantitative PCR was performed to detect the expression level of P311 mRNA in cancer cells after treatment with different siRNAs.

[0098] Test results as follows Figure 11 As shown in the results, P311-siRNA#4 has the most significant effect in inhibiting the expression of P311 mRNA. Therefore, in this invention, P311-siRNA#4 was selected as the silencing siRNA for P311 and subsequent experiments were conducted.

[0099] Example 3: Application of siRNA in inhibiting epithelial-mesenchymal transition in pancreatic cancer cells

[0100] Epithelial-mesenchymal transition (EMT) is the process by which tumor cells transform from epithelial-like to mesenchymal-like cells, acquiring the ability to invade and migrate. It plays a crucial role in tumor cell chemoresistance. In this study, Western blotting and RT-PCR were used to detect changes in EMT-related molecular markers (E-cadherin, N-cadherin, and Vimentin proteins) after siRNA interference with P311 in pancreatic cancer cell lines CAPAN-1 and BXPC-3. The Western blotting method is as follows:

[0101] ① Electrophoresis: Add the sample to the gel wells. Start with a voltage of 60V. When the stacking gel appears as a straight line, increase the voltage to 120V until the bromophenol blue appears.

[0102] ② Transfer: Soak the PVDF membrane in methanol, then equilibrate it with transfer buffer. Arrange the membrane in a "sandwich" configuration: filter paper-membrane-gel-filter paper. Pay attention to the positive and negative electrodes.

[0103] ③ Sealing: Place the transferred membrane into milk diluted with 5% TBST and shake on a shaker for 2 hours.

[0104] ④ Primary antibodies (E-cadherin, N-cadherin, and Vimentin antibodies): Dilute the primary antibodies 1:1000 with milk diluted with 5% TBST. Incubate at room temperature for 2 hours or overnight at 4°C.

[0105] ⑤ Secondary antibody (HRP-labeled rabbit or mouse antibody): Dilute the secondary antibody 1:2000 with milk diluted with 5% TBST. Incubate at room temperature for 1 hour.

[0106] ⑥ Illumination: Use luminescent liquid (liquid A + liquid B), first with super exposure, then with standard exposure. Take a picture and save the image for later use.

[0107] The amplification method for real-time PCR is as follows:

[0108] (1) RNA extraction

[0109] ① Prepare large, medium and small imported sterilizing pipette tips and 1.5ml imported EP tubes, and wipe the table clean with alcohol swabs; add 1ml of Trizol to each well of the cell plate, let it stand at room temperature for 3 minutes, then transfer it to a 1.5ml imported EP tube and let it stand at room temperature for 3 minutes.

[0110] ② Add 200 μL of chloroform to each EP tube, shake vigorously for 15 seconds, let stand at room temperature for 3 minutes, and centrifuge at 4℃ and 12000g for 15 minutes.

[0111] ③ Transfer the upper aqueous phase (400 μL) to a new 1.5 ml imported EP tube, add an equal volume (400 μL) of isopropanol, mix well, let stand at room temperature for 10 min, centrifuge at 4℃ for 12000 g × 10 min, and remove the supernatant.

[0112] ④ Add 1 mL of pre-cooled 75% ethanol to resuspend the precipitate, then centrifuge at 4°C for 7500 g for 5 min and discard the supernatant.

[0113] ⑤ Dry in a 37℃ oven, add 25μL of DEPC water or RNase-free water, place in a 50-60℃ water bath, dissolve for 15 minutes, and centrifuge for later use.

[0114] (2) Reverse transcription

[0115] The reverse transcription system (10 μL) in this embodiment is shown in Table 1, and the reverse transcription program is shown in Table 2.

[0116] Table 1: Reverse Transcription System

[0117] reagents content MgCl 2μL 10x RT buffer 1μL RNase-free water 3.75μL dNTP mixture 1μL RNase inhibitor 0.25μL AMV 0.5μL Oligo-dT 0.5μL RNA 1μg

[0118] Table 2: Reverse transcription program (1 cycle)

[0119] temperature time 30℃ 10min 42℃ 30min 99℃ 5min 5℃ 5min

[0120] (3) Real-time PCR reaction

[0121] First, dilute the primers as follows: 10 μL primer (forward) + 10 μL primer (reverse) + 80 μL ddH2O = 100 μL; then dilute the reverse transcription product template 8 times with ddH2O for later use. The real-time PCR reaction system is shown in Table 3, with 3-4 replicates per group.

[0122] Table 3: Real-time PCR reaction system (20 μL system)

[0123] reagents content SYBR 10μL Primer (forward + reverse) 0.8μL Rox 0.4μL cDNA 2μL <![CDATA[ddH2O]]> 6.8μL

[0124] Test results as follows Figures 11-13 As shown, the results indicate that overexpression of P311 in pancreatic cancer cell lines CAPAN-1 and BXPC-3 downregulated the protein and mRNA expression levels of the epithelial-like molecular marker E-cadherin, while upregulating the protein and mRNA expression levels of the mesenchymal-like molecular markers N-cadherin and Vimentin (e.g., ...). Figure 11 and Figure 12 When the expression of P311 is interfered with by the siRNA of this invention, the expression of E-cadherin protein is upregulated, while the expression of N-cadherin and Vimentin protein is downregulated (e.g., Figure 13 (As shown).

[0125] The above results show that after the siRNA of the present invention interferes with the expression of P311, it can upregulate the expression of E-cadherin protein and downregulate the expression of N-cadherin and Vimentin protein, thereby inhibiting the epithelial-mesenchymal transition of pancreatic cancer cells.

[0126] Example 4: Application of siRNA in reducing gemcitabine resistance in pancreatic cancer cells

[0127] To further investigate the effects of siRNA on P311-mediated pancreatic cancer cell proliferation and resistance to gemcitabine chemotherapy, this embodiment overexpressed P311 in pancreatic cancer cell lines CAPAN-1 and BXPC-3, respectively. Then, the expression of P311 was interfered with using the siRNA of this invention, and CCK8 cell proliferation assay, crystal violet staining assay, and Annexin V / 7-AAD flow cytometry apoptosis assay were performed.

[0128] CCK8 cell proliferation experiments revealed that P311 overexpression significantly enhanced the proliferation of pancreatic cancer cells; however, siRNA knockdown of P311 expression reversed the increased pancreatic cancer cell proliferation mediated by P311 overexpression (e.g., Figure 14 (As shown). Cells were treated with gemcitabine (20 nM) for 48 hours and then collected. Crystal violet staining was used to detect changes in gemcitabine resistance. It was found that overexpression of P311 significantly enhanced the gemcitabine resistance of pancreatic cancer cells. However, interference with P311 expression using siRNA significantly inhibited P311's gemcitabine resistance (e.g., ...). Figure 15 (As shown). Furthermore, flow cytometry analysis of apoptosis using Annexin V / 7-AAD showed that siRNA knockdown of P311 expression reduced the proliferation of pancreatic cancer cells, specifically as follows: Figure 16 As shown.

[0129] The results above show that siRNA knockdown of P311 expression can reduce the proliferation ability of pancreatic cancer cells and their resistance to gemcitabine chemotherapy, thereby improving the efficacy of gemcitabine.

[0130] Example 5: Application of siRNA in inhibiting the growth of subcutaneous pancreatic cancer tumors

[0131] This embodiment investigates the effect of siRNA on the proliferation of subcutaneous pancreatic cancer tumors in vivo using a subcutaneous tumorigenesis experiment in immunodeficient nude mice. First, a stable P311-overexpressing LUC-transgenic cell line was constructed. The specific construction method is as follows: using pancreatic cancer cell lines CAPAN-1 and BXPC-3 as a background, the pGL4.5[luc2 / CMV / Neo] plasmid was transfected, and neomycin was used for selection to obtain a stable Luc pancreatic cancer cell line. Then, a P311 lentiviral expression vector was constructed using molecular cloning technology. The virus was packaged and transfected into Luc pancreatic cancer cells, and stable lines were selected using drugs (puromycin, Puro), resulting in a stable P311-overexpressing Luc cell line. A subcutaneous tumor model was then constructed, and bioluminescent quantitative methods were used to dynamically monitor subcutaneous tumorigenesis in nude mice before and after siRNA injection. The specific method for in vivo imaging detection (bioluminescent quantitative method) is as follows:

[0132] ① Intraperitoneal injection of luciferase substrate (D-luciferin, dissolved in PBS, concentration 15 mg / ml), based on mouse body weight (10 μL / g);

[0133] ② Fifteen minutes after injection, the mice were anesthetized with isoflurane and placed in the IVIS Spectrum optical imaging system for detection;

[0134] ③ Take a picture and test the fluorescence intensity.

[0135] Test results as follows Figure 17 As shown, the results indicate that pancreatic cancer cells overexpressing P311 have a significantly higher subcutaneous tumorigenicity than the control group, and that interfering with P311 expression using siRNA can significantly inhibit tumor growth.

[0136] Furthermore, after euthanizing the mice, the control group, the P311 overexpression group, and the group with added siRNA to interfere with P311 were photographed and compared. The results are as follows: Figure 18 and Figure 19 As shown, the subcutaneous tumors of mice in the P311 overexpression group were larger and heavier than those in the control group. However, after interfering with P311 expression using siRNA, the subcutaneous tumors of mice in the control group were smaller than those in the P311 overexpression group.

[0137] The above results show that using the siRNA of the present invention to interfere with P311 expression can significantly inhibit P311-mediated subcutaneous tumor growth of pancreatic cancer.

[0138] In summary, this invention provides an siRNA that inhibits the P311 gene, a composition thereof, and its application. This siRNA can specifically knock down the expression of the P311 gene in human tumor cells, and can be used as an adjuvant to enhance gemcitabine drug efficacy, improve the drug sensitivity of pancreatic cancer patients to gemcitabine, reduce gemcitabine resistance in patients with advanced pancreatic cancer, improve the prognosis of pancreatic cancer patients, and prolong the survival of patients with advanced pancreatic cancer, which is of great significance for the treatment of pancreatic cancer.

[0139] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Use of siRNA or composition for inhibiting P311 gene in the preparation of a product for reducing the resistance of pancreatic cancer cells to gemcitabine, the siRNA consisting of a sense strand and an antisense strand, wherein, the nucleotide sequence of the siRNA sense strand is: 5'-UGAUAAAUAGCAUGAUUGCAC-3', the nucleotide sequence of the siRNA antisense strand is: 5'-GCAAUCAUGCUAUUUAUCAAA-3'; the composition comprises: the siRNA, or an expression system capable of expressing the siRNA. The P311 gene is selected from a human P311 gene. The composition further comprises: a pharmaceutically acceptable carrier and / or adjuvant; and other active ingredients for preventing or treating tumors. The pharmaceutically acceptable carrier and / or adjuvant includes, but is not limited to, buffers, emulsifiers, suspending agents, preservatives, physiological saline, excipients, fillers, coagulants, harmonizing agents, diffusing agents, antifoaming agents.

2. Use according to claim 1, characterized in that, The other active ingredients for preventing or treating tumors include: chemotherapeutic agents, radiotherapeutic agents, or antibody drugs.

3. Use according to claim 1, characterized in that, The form of the composition can be selected from any of solid, liquid, semi-liquid, and aerosol.

4. Use according to claim 3, characterized in that, 2. The siRNA for use in the preparation of a product for reducing the resistance of pancreatic cancer cells to gemcitabine, the siRNA consisting of a sense strand and an antisense strand, wherein, the nucleotide sequence of the siRNA sense strand is: 5'-UGAUAAAUAGCAUGAUUGCAC-3', the nucleotide sequence of the siRNA antisense strand is: 5'-GCAAUCAUGCUAUUUAUCAAA-3'.

5. Use according to claim 3, characterized in that, The P311 gene is selected from a human P311 gene.

6. Use according to any one of claims 3 to 5, characterized in that, The composition further comprises: a pharmaceutically acceptable carrier and / or adjuvant; and other active ingredients for preventing or treating tumors. The pharmaceutically acceptable carrier and / or adjuvant includes, but is not limited to, buffers, emulsifiers, suspending agents, preservatives, physiological saline, excipients, fillers, coagulants, harmonizing agents, diffusing agents, antifoaming agents. The other active ingredients for preventing or treating tumors include: chemotherapeutic agents, radiotherapeutic agents, or antibody drugs. The form of the composition can be selected from any of solid, liquid, semi-liquid, and aerosol.

3. The siRNA for use in the preparation of a product for reducing the resistance of pancreatic cancer cells to gemcitabine, the siRNA consisting of a sense strand and an antisense strand, wherein, the nucleotide sequence of the siRNA sense strand is: 5'-UGAUAAAUAGCAUGAUUGCAC-3', the nucleotide sequence of the siRNA antisense strand is: 5'-GCAAUCAUGCUAUUUAUCAAA-3'. The P311 gene is selected from a human P311 gene. The composition further comprises: a pharmaceutically acceptable carrier and / or adjuvant;

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