Application of linc02266 and small interfering rna thereof

Abstract

The application discloses application of long-chain non-coding RNA as LINC02266 (RP11-142A22.4) as a biomarker in preparation of products for diagnosing and / or prognosis evaluating gastric cancer diseases, and application of LINC02266 as a target point in preparation of drugs for preventing or treating gastric cancer diseases. The expression of the LINC02266 provided by the application is significantly up-regulated in human gastric cancer tissues and gastric cancer cell lines, and specific detection of the LINC02266 can achieve the purpose of diagnosing and / or prognosis evaluating gastric cancer. The application further provides a small interfering RNA of the LINC02266, which can inhibit the expression of the LINC02266, and can play a role in inhibiting proliferation, invasion and migration of gastric cancer cells, promoting ferroptosis, and further anti-tumor effect. The small interfering RNA of the LINC02266 is used as a drug, and is introduced into a gastric tissue or in vivo, and has a preventive and therapeutic effect on gastric cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of LINC02266 and its small interfering RNA. Background Technology

[0002] lncRNAs are a newly discovered class of endogenous long non-coding RNA molecules. They are a subclass of non-coding RNAs with transcript lengths greater than 200 nucleotides that do not encode proteins. They can interact with biological macromolecules such as DNA, proteins, mRNA, and microRNA, and act as inducing molecules, decoy molecules, and scaffold molecules to regulate gene expression and function at multiple levels, including chromatin remodeling, gene transcription and translation, and protein modification, playing a key regulatory role in life activities.

[0003] Cell proliferation, cell differentiation, and apoptosis are three fundamental activities in the individual development of any multicellular organism. They are interdependent and indispensable. This is an active, highly ordered process controlled by genes and involving a series of enzymes. They play crucial roles in normal embryonic development, maintaining cell populations, and in the progression of malignant diseases, thus ensuring the healthy survival of multicellular organisms. Malignant tumors are characterized by two main features: incessant cell division and malignant differentiation. Inhibiting the malignant proliferation of tumor cells and limiting their metastasis to adjacent or distant tissues and organs is a key issue in cancer treatment.

[0004] Current research has revealed that many lncRNAs participate in cell proliferation, cell differentiation, and the regulation of ferroptosis by modulating the expression of proliferation- or invasion-related target proteins. Some of these lncRNAs promote proliferation or invasion, while others inhibit it. Identifying lncRNAs specifically expressed in gastric cancer tissues that participate in tumor cell proliferation and invasion, and elucidating their mechanisms of action, is of paramount importance and holds significant promise for developing lncRNA-based strategies for the diagnosis and treatment of gastric cancer. Summary of the Invention

[0005] The present invention aims to identify or discover gastric tissue-specific high-expression lncRNAs that regulate the occurrence and development of gastric cancer, determine their key roles in the proliferation, invasion and migration of gastric cancer cells, and apply them to the diagnosis and prevention of gastric cancer.

[0006] The first objective of this invention is to provide the use of LINC02266 in the preparation of products for gastric cancer diagnosis and / or gastric cancer prognostic assessment, wherein the sequence of LINC02266 in this invention is the nucleotide sequence shown in SEQ ID No. 7.

[0007] Furthermore, the expression of LINC02266 was upregulated in patients with gastric cancer.

[0008] Furthermore, the product includes reagents, reagent kits, or chips.

[0009] Furthermore, the product is a kit, which includes primers for specifically amplifying LINC02266 and primers for specifically detecting LINC02266. The primers for specifically amplifying LINC02266 include an upstream primer with the sequence shown in SEQ ID No. 8 and a downstream primer with the sequence shown in SEQ ID No. 9; the primers for specifically detecting LINC02266 include an upstream primer with the sequence shown in SEQ ID No. 10 and a downstream primer with the sequence shown in SEQ ID No. 11.

[0010] The upstream primer for amplifying the full length of LINC02266 is:

[0011] 5′-GAAGTGCTGAACCCTTGACGTGCCT-3′ (SEQ ID No. 8);

[0012] The downstream primers for amplifying the full length of LINC02266 are:

[0013] 5′-TTTTTGTGATTTCAACTTTGTTTTATTTTACAAAATG-3′ (SEQ ID No. 9);

[0014] The upstream primer specifically for detecting the LINC02266 fragment is:

[0015] 5′-TGCCTCTTTAGGCTCTCAAACT-3′(SEQ ID No.10)

[0016] The downstream primer for specific detection of the LINC02266 fragment is:

[0017] 5'-GTGTCCCCACAAACTAAGAGC-3' (SEQ ID No. 11).

[0018] A second objective of this invention is to provide the use of LINC02266 as a target in the preparation of drugs for the prevention and / or treatment of gastric cancer.

[0019] A third objective of this invention is to provide the application of LINC02266 as a target in screening candidate drugs for the prevention or treatment of gastric cancer.

[0020] The fourth objective of this invention is to provide the use of LINC02266 expression inhibitors, knockout agents, or silencing agents in the preparation of drugs for the prevention and / or treatment of gastric cancer.

[0021] Furthermore, the expression inhibitors of LINC02266 include small interfering RNA of LINC02266, at least one of which has a positive strand nucleotide sequence as shown in SEQ ID No. 1-6.

[0022] Another object of the present invention is to provide a pharmaceutical composition for the prevention and / or treatment of gastric cancer, said pharmaceutical composition comprising any of the following substances:

[0023] I. An RNA molecule, a biologically active functional fragment, or a variant of an antisense nucleic acid thereof, having at least one of the sequences shown in SEQ ID No. 1-6;

[0024] II. Recombinant vector encoding the RNA molecule shown in I;

[0025] III. Recombinant viruses encoding the RNA molecule shown in I;

[0026] IV. Recombinant viral vector encoding the RNA molecule shown in I.

[0027] Furthermore, the gastric cancer includes one or more of the following: self-elevating gastric cancer, localized ulcerative gastric cancer, invasive ulcerative gastric cancer, and diffuse invasive gastric cancer.

[0028] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier or excipient.

[0029] Furthermore, the pharmaceutically acceptable carrier is selected from one or more of chitosan, cholesterol, liposomes, and nanoparticles.

[0030] Furthermore, the administration route of the pharmaceutical composition is selected from oral, intravenous, intramuscular, or direct gastric injection.

[0031] The beneficial effects of this invention include at least the following:

[0032] (1) The expression of LINC02266 provided by the present invention is significantly upregulated in human gastric cancer tissues and gastric cancer cell lines. Specific detection of LINC02266 can achieve the purpose of diagnosing and / or prognostic assessment of gastric cancer.

[0033] (2) The present invention also provides six small interfering RNAs of LINC02266, which can inhibit the expression of LINC02266 and play an anti-tumor role by inhibiting the proliferation, invasion and migration of gastric cancer cells; the small interfering RNA of LINC02266 is used as a drug and introduced into gastric tissue or body, which will have a preventive and therapeutic effect on gastric cancer. Attached Figure Description

[0034] Figure 1 The expression level of LINC02266 in gastric cancer cell lines.

[0035] Figure 2 To investigate the inhibitory effect of endogenous LINC02266 on the proliferation of gastric cancer cells BGC823, the MTT assay (A) and EdU staining assay (B) were used to detect the proliferation of BGC823 cells, respectively.

[0036] Figure 3 The effect of small interfering RNA LINC02266 on the invasion and migration ability of gastric cancer cells BGC823.

[0037] Figure 4 The effect of small interfering RNA LINC02266 on ferroptosis in gastric cancer cells BGC-823 is shown in Example 4; where A represents MDA level and B represents GSH level. Detailed Implementation

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0039] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0040] The inventors of this invention discovered that the small interfering RNA of LINC02266 exerts an anti-tumor effect by inhibiting the proliferation, invasion, and migration of gastric cancer cells. The expression of LINC02266 is significantly upregulated in gastric cancer cell lines, and this upregulation may induce gastric cancer development. Inhibiting endogenous LINC02266 within cell lines can suppress the proliferation, invasion, and migration of gastric cancer cells and promote ferroptosis. Combining the small interfering RNA of LINC02266 with a suitable vector to form a drug, and introducing it into gastric tissue or the body, could have both preventative and therapeutic effects against gastric cancer. It is also possible to consider combining the small interfering RNA of LINC02266 with chitosan, cholesterol, liposomes, nanoparticles, etc., to form drug molecules for the prevention and treatment of gastric cancer via oral, intravenous, intramuscular, or direct gastric injection.

[0041] Application of the small interfering RNA LINC02266 (siRNA-LINC02266) in the preparation of drugs for the prevention and / or treatment of gastric cancer.

[0042] The nucleotide sequence of the small interfering RNA (siRNA-LINC02266) synthesized in this invention is shown in SEQ ID No. 1-6;

[0043] 5'-AAGATCCCAGATGGAAGATG-3' (SEQ ID NO. 1);

[0044] 5'-CCTGAATTCCTGCCCACAAA-3' (SEQ ID NO. 2);

[0045] 5'-TGCAGTATTCCTTCAGGGAA-3' (SEQ ID NO. 3);

[0046] 5'-CCAATTGGGACTGCAGTAT-3' (SEQ ID NO.4);

[0047] 5'-GGCTCTCAAACTCCATAGA-3' (SEQ ID NO.5);

[0048] 5'-CACTCAAACAGCCAATGGA-3'(SEQ ID NO.6)

[0049] A pharmaceutical composition for the prevention and / or treatment of gastric cancer, the pharmaceutical composition comprising any one of the following substances:

[0050] I. RNA molecules, biologically active functional fragments, or variants shown in SEQ ID Nos. 1-6;

[0051] II. Recombinant vector encoding the RNA molecule shown in I;

[0052] III. Recombinant viruses encoding the RNA molecule shown in I;

[0053] IV. Recombinant viral vector encoding the RNA molecule shown in I;

[0054] The aforementioned gastric cancers include one or more of the following: self-elevating gastric cancer, localized ulcerative gastric cancer, invasive ulcerative gastric cancer, and diffuse invasive gastric cancer;

[0055] The pharmaceutical composition also includes a pharmaceutically acceptable carrier or excipient.

[0056] In a preferred embodiment, the pharmaceutically acceptable carrier is selected from one or more of chitosan, cholesterol, liposomes, and nanoparticles.

[0057] In another preferred embodiment, the route of administration of the drug is selected from oral, intravenous, intramuscular, or direct gastric injection.

[0058] Application of LINC02266 in the preparation of kits for the diagnosis and / or prognostic assessment of gastric cancer.

[0059] The nucleotide sequence of LINC02266 is shown in SEQ ID No. 7.

[0060] A kit for the diagnosis and / or prognostic assessment of gastric cancer, the kit containing an RNA molecule as shown in SEQ ID No. 7;

[0061] The aforementioned gastric cancers include one or more of the following: self-elevating gastric cancer, localized ulcerative gastric cancer, invasive ulcerative gastric cancer, and diffuse invasive gastric cancer.

[0062] Unless otherwise specified, the cell line used in the following examples is the gastric cancer cell line BGC-823.

[0063] Unless otherwise specified, the test methods used in the following examples are all conventional methods used in the art.

[0064] Unless otherwise specified, all reagents used in the following examples are analytical grade reagents and are available from legitimate commercial sources.

[0065] The following specific embodiments illustrate the solution proposed in this invention:

[0066] Example 1: Detection of LINC02266 expression in gastric cancer cell lines

[0067] In this embodiment, both the normal gastric mucosal epithelial cells and the gastric cancer cell lines used were cultured and cryopreserved under normal culture conditions.

[0068] Total RNA was extracted from normal gastric mucosal epithelial cells and gastric cancer cells using the Trizol reagent method, and the expression level of LINC02266 was detected using real-time quantitative PCR. LINC02266 was amplified by PCR, and the full-length nucleotide sequence was 1153 bp, as shown in SEQ ID No. 7.

[0069] The PCR amplification reaction volume is 50 μL. 5×PS Buffer 10.0 μL, dNTP Mix (2.5 mM each) 4.0 μL, upstream amplification primer (10 μM) 1.0 μL, downstream amplification primer (10 μM)

[0070] 1.0 μL of template (10 ng / μL), 1.0 μL of PrimeSTAR HSDNA polymerase

[0071] 0.5 μL of double-distilled water was added to bring the volume to 50 μL. The PCR conditions were as follows: 95℃, 5 min, one cycle; 95℃, 30 sec; 56℃, 30 sec; 72℃, 1 min, for a total of 30 cycles; and a final extension at 72℃ for 7 min. The PCR primers were designed as follows: the upstream primer for full-length amplification was 5'-TGCCTCTTTAGGCTCTCAAACT' (SEQ ID No. 8); the downstream primer for full-length amplification was 5'-GTGTCCCCACAAACTAAGAGC' (SEQ ID No. 9).

[0072] The expression level of LINC02266 was detected by real-time quantitative PCR. The PCR reaction system was 20 μL and consisted of: SYBR Green Real-time PCR MasterMix 10.0 μL; Primer F 1.0 μL; Primer R 1.0 μL; cDNA 2.0 μL; triple-distilled water to make up to 20.0 μL.

[0073] The qPCR conditions were as follows: 95℃, 30 sec, one cycle; 95℃, 5 sec; 60℃, 30 sec; 72℃, 1 min, for a total of 39 cycles; and a final extension at 72℃ for 10 min. The PCR primers were designed as follows: the upstream primer for specific detection of the LINC02266 fragment was 5′-GATCCTAGAGGAAAGTGGCAAG-3′ (SEQ ID No. 10); the downstream primer for specific detection of the LINC02266 fragment was 5′-AGGGTGTACAGCAGTGAACAA-3′ (SEQ ID No. 11).

[0074] In this embodiment, the expression level of LINC02266 was detected in different gastric cancer cell lines using real-time quantitative PCR (qPCR reaction and conditions were the same as above). The detection results are as follows: Figure 1 As shown, Figure 1 The vertical axis represents the expression level of LINC02266 in gastric cancer cell lines SGC-7901, MGC-803, BGC-823, and AGS, using the normal gastric mucosal epithelial cell line GES-1 as a baseline. Figure 1 This indicates that, compared to gastric mucosal epithelial cells GES-1, the expression level of LINC02266 was significantly increased in gastric cancer cells BGC-823, AGS, and SGC-7901.

[0075] Example 2: Experiment on the inhibition of gastric cancer cell proliferation by small interfering RNA of LINC02266

[0076] The sense strand sequence in the synthetic small interfering RNA that inhibits LINC02266 is as follows:

[0077] 5'-AAGATCCCAGATGGAAGATG-3' (SEQ ID NO. 1);

[0078] 5'-CCTGAATTCCTGCCCACAAA-3' (SEQ ID NO. 2);

[0079] 5'-TGCAGTATTCCTTCAGGGAA-3' (SEQ ID NO. 3);

[0080] 5'-CCAATTGGGACTGCAGTAT-3' (SEQ ID NO.4);

[0081] 5'-GGCTCTCAAACTCCATAGA-3' (SEQ ID NO.5);

[0082] 5'-CACTCAAACAGCCAATGGA-3' (SEQ ID NO. 6).

[0083] Transfection was performed using lipofectamine 2000, with separate control and small interfering RNA mix experimental groups. Transfection was performed according to the kit instructions. Lipofectamine 2000 was purchased from Invitrogen.

[0084] Using the gastric cancer cell line BGC-823 as a cell model, gastric cancer cells BGC-823 (with a cell mass of approximately 1×10⁻⁶) were subjected to treatment. 6 The cells were transfected with small interfering RNA (MIRNA) from LINC02266. After 4 hours of transfection, the medium was replaced with normal medium and cultured for 1-5 days. After incubation for different times in an incubator, 100 μL of medium was discarded, and 10 μL MTT (final concentration 500 μg / mL) was added. After 4 hours of culture, 150 μL LDMSO was added to dissolve the formazan particles. The OD 490 nm light absorbance was measured using a microplate reader.

[0085] Experimental results are as follows Figure 2 As shown in Figure A, the results indicate that inhibiting endogenous LINC02266 can significantly suppress the proliferation of gastric cancer cells BGC-823.

[0086] The efficacy of LINC02266 small interfering RNA in inhibiting the proliferation of gastric cancer cells was detected using EdU staining. Gastric cancer cells BGC-823 (approximately 1 × 10⁻⁶ cells) were used in this study. 6The cells were randomly divided into a control group and an experimental group. The experimental group underwent transfection with LINC02266 small interfering RNA. Four hours after transfection, the medium was replaced with normal medium, and the cells were cultured for another 20 hours. The control group underwent the same treatment as the experimental group, except that the LINC02266 interfering RNA was replaced with a negative control RNA containing a meaningless sequence. EdU solution (purchased from Guangzhou Ruibo) was diluted 1000:1 with cell culture medium to prepare an appropriate amount of 50 μM EdU medium. 100 μL of 50 μM EdU medium was added to each well and incubated for 2 hours, then the medium was discarded. Cells were washed 1-2 times with PBS for 5 minutes each time. 100 μL of cell fixation solution (PBS buffer containing 4% paraformaldehyde) was added to each well and incubated at room temperature for 30 minutes. The fixation solution was discarded. 2 mg / mL glycine was added to each well, and the cells were incubated on a shaker for 5 minutes to decolorize. The glycine solution was discarded. 100 μL of PBS was added to each well, and the cells were washed on a shaker for 5 minutes to decolorize. The PBS was discarded. 100 μL of PBS was added to each well. Incubate 1×Apollo staining reaction solution at room temperature, protected from light, on a destaining shaker for 30 minutes; discard the staining reaction solution, add 100 μL of osmotic buffer (0.5% Triton X-100 in PBS buffer) to each well and wash on a destaining shaker for 10 minutes, 2-3 times; dilute 100×Hoechst 33342 stock solution with deionized water at a ratio of 100:1 to prepare an appropriate amount of 1×Hoechst 33342 reaction solution, and store in the dark; add 100 μL of 1×Hoechst 33342 reaction solution to each well, incubate at room temperature, protected from light, on a destaining shaker for 30 minutes, then discard the staining reaction solution; wash each well with 100 μL of PBS 1-3 times each time; add 100 μL of PBS to each well and store for later use. Finally, perform image acquisition and analysis. When adjusting the instrument, adjust the exposure time to approximately 30 milliseconds, not exceeding 1 second.

[0087] Experimental results are as follows Figure 2 As shown in Figure B, the results indicate that inhibiting endogenous LINC02266 can significantly suppress the proliferation of gastric cancer cells.

[0088] Example 3: LINC02266's small interfering RNA inhibits the invasion and migration of gastric cancer cells.

[0089] The gastric cancer cell line BGC-823, treated with the small interfering RNA of LINC02266 in Example 2, was used as a cell model. The invasive ability of the cells was detected using the Transwell assay, the specific procedure of which is as follows:

[0090] The upper surface of the bottom membrane of the Transwell chamber was coated with 50 mg / L Atrigel at a 1:10 dilution and incubated at 37°C. After treatment with LINC02266 small interfering RNA, the cells were digested 12 hours later, washed once with PBS and once with serum-free medium, resuspended in serum-free medium, counted, and the concentration adjusted to 3 × 10⁻⁶ cells / mL.5 / mL; Add 600-800μL of culture medium containing 20% ​​serum to the lower chamber of the Transwell plate (i.e., the bottom of the 24-well plate), and add 100-150μL of cell suspension to the upper chamber. Continue incubation for 24-48 hours. Carefully remove the upper chamber with forceps, aspirate the liquid in the upper chamber, and transfer it to wells pre-filled with about 800μL of methanol. Fix at room temperature for 60 minutes. Remove the upper chamber, aspirate the fixative solution in the upper chamber, and transfer it to wells pre-filled with about 800μL of crystal violet staining solution. Stain at room temperature for 15-30 minutes. Gently rinse and soak several times with water, remove the upper chamber, aspirate the liquid in the upper chamber, and carefully wipe away the cells on the membrane surface at the bottom of the upper chamber with a moistened cotton swab. Wash each well 1-3 times with 100μL of PBS each time. Count the cells in 5 random fields under a microscope and statistically analyze the results.

[0091] Experimental results are as follows Figure 3 As shown, the results indicate that inhibiting endogenous LINC02266 can significantly suppress the migration and invasion of gastric cancer cells.

[0092] Example 4

[0093] The small interfering RNA of LINC02266 promotes ferroptosis in gastric cancer cells.

[0094] Using the gastric cancer cell line BGC-823 treated with small interfering RNA (MIRNA) from LINC02266 in Example 2 as a cell model, the levels of ferroptosis-related indicators, lipid peroxidation (MDA) and glutathione (GSH), were detected. The specific procedures are as follows:

[0095] (1) Detection of lipid oxidation (MDA)

[0096] Cells were first washed with PBS, then digested with trypsin and centrifuged. After lysing the cells with Western lysis buffer, they were centrifuged at 11000g-13000g for 20 minutes, and the supernatant was collected. After preparing the cell samples, protein concentration was determined using a BCA protein assay kit. Appropriate amounts of standards were diluted with distilled water to concentrations of 1, 2, 5, 10, 20, and 50 μM for subsequent standard curve preparation. The reaction system was set up according to the manufacturer's instructions.

[0097] After mixing, heat in a 100°C water bath for 15 minutes. Cool to room temperature under water bath conditions and centrifuge at 1100g for 15 minutes at room temperature. Add 200 μL of supernatant to a 96-well plate and measure the absorbance at A532 nm using a microplate reader. For cell samples, calculate the MDA content in the solution and express the initial sample content as protein content per unit weight.

[0098] (2) GSH detection

[0099] Dilute the 10 mM GSSH stock solution with protein removal reagent M to prepare a 15 μM GSSH solution. Then, dilute the solution sequentially to 10, 5, 2, 1, and 0.5 μM GSSH solutions to plot a standard curve at six points.

[0100] Wash cells once with PBS, centrifuge to collect cells, and aspirate the supernatant. Add three times the volume of protein removal reagent M solution to the cell pellet; for example, if the cell pellet volume is 10 μL, add 30 μL of protein removal reagent M solution and vortex thoroughly. Then, perform two rapid freeze-thaw cycles in liquid nitrogen and a 37°C water bath. Incubate at 4°C or on ice for 5 minutes. Centrifuge at 10000g for 10 minutes at 4°C. Use the supernatant for GSH determination; the sample should be temporarily stored at 4°C. Take the prepared sample and add it and the standard to a 96-well plate according to the table below, and mix well. Add 150 μL of GSH detection working solution, mix well, and incubate at room temperature for 5 minutes. Add NADPH solution, mix well, and let stand for 25 minutes. Immediately measure A412 using a microplate reader.

[0101]

[0102] Experimental results are as follows Figure 4 As shown, inhibiting endogenous LINC02266 can significantly increase lipid oxidation (MDA) levels and decrease glutathione (GSH) levels in gastric cancer cells.

[0103] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0104] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0105] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The application of an inhibitor of LINC02266 expression in the preparation of a drug for treating gastric cancer, characterized in that, The expression inhibitor of LINC02266 is a small interfering RNA of LINC02266, and the positive strand nucleotide sequence of the small interfering RNA of LINC02266 is at least one of the following shown in SEQ ID No. 1-6.

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