Mediating METTL3 variable splicing oligonucleotide and application thereof in preparation of medicine for treating hepatocellular carcinoma

Abstract

The invention discloses oligonucleotide mediating METTL3 variable splicing and application of the oligonucleotide in preparation of a medicine for treating hepatocellular carcinoma. The sequence of the oligonucleotide is any sequence as shown in SEQ ID No.1-7. According to the invention, the expression of the METTL3 splicing isomer is regulated in a targeted manner by using a specific ASO drug, the expression level of the METTL3-A splicing isomer for promoting cancer is reduced, and the expression level of the METTL3-D splicing isomer for inhibiting cancer is increased. The proliferation and clone formation capabilities of hepatoma cells with increased expression of METTL3-D splicing isomers are greatly reduced, and meanwhile, the invasion capability is also remarkably weakened. The expression of the METTL3-D splicing isomer is crucial to the inhibition of liver cancer cells. METTL3 variable splicing regulation provides a new target for clinical treatment of liver cancer, and has a remarkable clinical application prospect. The METTL3-ASO6 and the METTL3-ASO7 can be used for inhibiting the expression of the METTL3-A splicing isomer capable of promoting the cancer in liver cancer cells. The METTL3-ASO6 and the METTL3-ASO7 can be used for inhibiting the proliferation and the clone formation capability of liver cancer cells. The METTL3-ASO6 and the METTL3-ASO7 provided by the invention can be used for efficiently inhibiting the invasion ability of liver cancer cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an oligonucleotide mediating METTL3 variable splicing and an application thereof in preparing a drug for treating hepatocellular carcinoma. Background Art

[0002] Hepatocellular carcinoma (HCC) accounts for 90% of all primary liver cancer cases. Curative treatment including resection, liver transplantation and local ablation is only available for patients with early-stage HCC. However, early detection remains challenging. Chemoembolization therapy is suitable for patients with intermediate-stage HCC, but there is a risk of non-targeted embolism and many side effects. Systemic therapy including four kinase inhibitors, an anti-VEGF antibody and an immune checkpoint inhibitor can be used for patients with advanced HCC. However, the improvement in patient prognosis is still limited. Therefore, new drugs need to be developed to address the unmet needs in HCC treatment.

[0003] RNA plays a central role in gene expression and is an important link in the transmission of genetic information from DNA to protein. The maturation of messenger RNA precursors requires the removal of introns and the joining of exons, a process catalyzed by the spliceosome and known as pre-mRNA splicing. In humans, more than 95% of nascent transcripts are alternatively spliced, generating multiple mRNA isoforms (splice variants) from a single gene. Splice variants differ in their coding sequences and / or untranslated regions (UTRs), of which approximately 60-75% are located in the coding region that provides protein diversity. Tumor biology affected by alternative splicing (AS) includes metabolism, apoptosis, cell cycle control, invasion, and metastasis. AS changes are prevalent in HCC, and many are associated with HCC prognosis; therefore, splice variants are a source of new biomarkers and therapeutic targets for HCC. For example, splice variants of the calcalineurin 1 (RCAN1) regulator, RCAN1, 4, inhibit the proliferation, migration, and invasion activities of HCC cells. CD73S is a short splice variant of CD73 that exhibits functional differences from the canonical isoform CD73L in regulating the proliferation potential of HCC cells.

[0004] Many studies have demonstrated the potential significance of m6A modification in the diagnosis and treatment of liver cancer. METTL3 is upregulated in hepatocellular carcinoma (HCC), and knocking out METTL3 in vivo can significantly inhibit the tumorigenicity of HCC. METTL3 overexpression is associated with poor prognosis in patients with HCC. In addition, patients with high METTL3 expression have poor overall survival and disease-free survival. Although METTL3 has been extensively studied in recent years, the existence and function of its splicing variants are still rarely reported. We compared different splicing isoforms in HepG2 with the reference genome after sequencing. METTL3-A is the most common and full-length spliceosome, with 11 exons consisting of 580 amino acids, a size of 70 kDa, and contains a zinc finger domain and a methyltransferase domain. The variable 3' splicing factor of METTL3-D produces a shorter exon 4 and retains introns 8 and 9; therefore, METTL3-D can be translated into a polypeptide without the terminal catalytic domain MT-A70. The expression of METTL3-A and D in normal tissues is similar, and METTL3-D is even more expressed, but its mRNA and protein levels are significantly reduced in HCC tumor tissues. And compared with the standard full-length METTL3-A, the METTL3-D variant is functionally different. Overexpression of METTL3-D can inhibit the proliferation, migration and invasion of liver cancer cells. METTL3-A and METTL3-D oppositely control the expression of METTL3 downstream target genes. Finally, high levels of METTL3-D are significantly associated with early tumor stage and longer overall survival time in HCC, indicating that METTL3-D is a tumor suppressor in HCC.

[0005] METTL3-D amino acid sequence: MSDTWSSIQAHKKOLDSLRERLQRRRKODSGHLDLRNPEAALSPTFRSDSPVPTAPTSGGPKPSTASAVPELATDPELEKKLLHHLSDLALTLPTDAVSICLAISTPDAPATQDGVESLLQKFAAQELIEVKRGLLQDDAHPTLVTYADHSKLSAMMGAVAEKKGPGEVAGTVTGOKRRAEQ DSTTVAAFASSLVSGLNSSASEPAKEPAKKSRKHAASDVDLEIESLLNQQSTKEQQSKKVGPKCKNSVTMEPRRSA (Alanine A; Phenylalanine F; Cysteine ​​C; Selenocysteine ​​U; Aspartic Acid D; Asparagine N; Glutamic A; Glutamine Q; Glycine G; Histidine H; Leucine L; Isoleucine I; Lysine K; Pyrrolysine O; Methionine M; Proline P; Arginine R; Serine S; Threonine T; Valine V; Tryptophan W; Tyrosine Y)

[0006] As a treatment method, the era of clinical application of ASO drugs has begun since Harvard University scientists first proposed the concept of antisense nucleotides in 1978 and the first antisense nucleotide drug, fomivirsen, was launched in 1998. After 2016, a variety of ASO drugs for rare diseases were launched, and ASO drugs began to enter the historical stage. ASO drugs use Watson & Crick base pairing to target RNA processing and regulate protein expression with high specificity. ASO drugs are usually 15-30 nucleotides in length and contain chemical modifications in sugars and backbones to increase stability (nuclease resistance), prevent immune response and increase binding affinity. The application of single-chain ASO drugs includes downregulating gene expression by triggering RNase H-mediated degradation of target transcripts (gap) and preventing protein binding to RNA (steric blocking). Steric blocking ASO drugs regulate splicing by preventing splicing factors from binding to target pre-mRNA. In turn, this approach can address mutations that cause splicing defects, avoid harmful mutations or change protein isoforms. In studies of beta thalassemia and spinal muscular atrophy, steric blocking ASO drugs rescued splicing defects caused by specific mutations by promoting inclusion of coding exons, resulting in fully functional proteins. In contrast, to avoid specific harmful nonsense or frameshift mutations in mouse models of Duchenne muscular dystrophy, ASO drugs were used to cause skipping of the coding exons where these mutations were located, resulting in partially functional truncated proteins. To date, ASO drugs have only been used to address specific mutations affecting subsets of patients with monogenic diseases. The U.S. Food and Drug Administration (FDA) has approved five ASO drugs: Formivirsen, Mipomersen, Eteplirsen, Nusinersen, and Intertersen.

[0007] ASO drugs have strong and lasting effects, and the pharmaceutical industry is very optimistic about the development of the entire nucleic acid drug field. At this stage, the entire field has entered a period of explosive growth, and in the future, ASO drugs will even surpass the application scenarios of small molecule drugs and antibody drugs.

[0008] Unlike current liver cancer drugs that target vascular development, the immune system, or cell signaling, the ASO drug therapy described here inhibits liver cancer cell growth by increasing the expression of the METTL3-D isoform. This effect is due to increased expression of METTL3-D, while the expression of the allosterically regulated METTL3-A is decreased. In addition, the METTL3-ASO6 and METTL3-ASO7 drugs we discovered by free uptake in two liver cancer cell lines also led to reduced cell proliferation and increased cell cycle arrest, providing important evidence for the clinical application of this drug.

[0009] ASO drugs have the following advantages: - Strong specificity: They are artificially designed based on target RNA, so the target is clear and the target specificity is strong. Simple design and short R&D cycle: The preclinical R&D of ASO drugs first determines the gene sequence and rationally designs the disease gene to silence the gene target, so it can avoid blind development and save R&D time to a large extent. Rich targets: ASO drugs treat at the post-transcriptional level and can make breakthroughs in special targets that can be effective for some protein targets, and are expected to overcome genetic diseases for which there are no drugs yet. In the future, with the continuous breakthroughs and innovations in the application fields and technical fields of ASO drugs, the update of technology will help the development of ASO drugs. Market demand and market size will continue to expand, and ASO drugs have a wide range of indications, including tumors, rare diseases, viral diseases, kidney diseases, cardiovascular diseases, inflammatory diseases, metabolic diseases, etc. Therefore, ASO drugs have a large base of potential populations. With the development of technology and the maturity of production, the ASO drug market will have a broader development space in the future. Compared with existing small molecule and antibody drugs, ASO drugs have the advantages of fast target screening, high R&D success rate, low resistance, wider therapeutic field and long-term effect, and have great development potential. With the continuous advancement of technology, ASO drugs are expected to form the third wave of modern new drugs after small molecule drugs and antibodies. After special modification, ASO drugs bind to RNA through complementary pairing, and use ASO drugs to regulate RNA splicing. For some genetic diseases, some success has been achieved recently: in clinical trials, they have a certain alleviating effect on Duchenne muscular atrophy (DMD) and spinal muscular atrophy (SMA).

[0010] Unlike other small molecule drugs that are mainly absorbed after oral administration, most ASO drugs need to be injected, which can be divided into intravitreal injection, intrathecal injection, subcutaneous injection (SC), intravenous infusion (IV), etc. Among them, SC or IV administration is the main method, and after IV administration, it is mainly distributed in tissues such as liver and kidney, and the uptake of other tissues is relatively small. Summary of the invention

[0011] The purpose of the present invention is to provide an oligonucleotide mediating METTL3 variable splicing and its application in preparing a drug for treating hepatocellular carcinoma.

[0012] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: an oligonucleotide that mediates the alternative splicing of Mettl3, wherein the sequence of the oligonucleotide is any one of the sequences shown in SEQ ID No. 1-7:

[0013] SEQ ID No.1:AGATCAAATTGGCTCTTAGA;

[0014] SEQ ID No.2:CTTATAGATCAAATTGGCTC;

[0015] SEQ ID No.3:AATGTCTTATAGATCAAATT;

[0016] SEQ ID No.4:TTGGACTTAAAATGTCTTAT;

[0017] SEQ ID No.5:TAGCCTTGGACTTAAAATGT;

[0018] SEQ ID No.6:AGAAGTAGCCTTGGACTTAA;

[0019] SEQ ID No.7:CTTAAAATGTCTTATAGATC.

[0020] The preferred technical solution is: the nucleotides are modified by MOE.

[0021] The preferred technical solution is: the sequence of the oligonucleotide is any one of the sequences shown in SEQ ID No. 1-7:

[0022] SEQ ID No.1:AeGeAeTeCeAAATTGGCTCTeTeAeGeAe;

[0023] SEQ ID No.2:CeTeTeAeTeAGATCAAATTGeGeCeTeCe;

[0024] SEQ ID No.3:AeAeTeGeTeCTTATAGATCAeAeAeTeTe;

[0025] SEQ ID No.4:TeTeGeGeAeCTTAAAATGTCeTeTeAeTe;

[0026] SEQ ID No.5:TeAeGeCeCeTTGGACTTAAAeAeTeGeTe;

[0027] SEQ ID No.6:AeGeAeAeGeTAGCCTTGGACeTeTeAeAe;

[0028] SEQ ID No.7:CeTeTeAeAeAATGTCTTATAeGeAeTeCe;

[0029] e represents 2'-O-MOE modification; 2'-O-MOE modification is as follows:

[0030]

[0031] The preferred technical solution is: the 2'-O-MOE is modified to be cytosine-modified 2'-O-MOE, the structure of which is as follows:

[0032]

[0033] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: use of an oligonucleotide that mediates variable splicing of METTL3 in the preparation of a drug for treating hepatocellular carcinoma, wherein the oligonucleotide is the oligonucleotide described in any one of claims 1-4.

[0034] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: a pharmaceutical composition for targeted upregulation of METTL3-D gene expression, comprising an oligonucleotide that mediates METTL3 alternative splicing and a pharmaceutically acceptable material.

[0035] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0036] The present invention utilizes specific ASO drugs to target and regulate the expression of METTL3 splicing isoforms, reduce the expression level of METTL-A splicing isoforms, and increase the expression level of METTL3-D splicing isoforms. The proliferation and cloning ability of liver cancer cells with increased expression of METTL3-D splicing isoforms are greatly reduced, and the invasive ability is also significantly weakened. This shows that the expression of METTL-D splicing isoforms is crucial for liver cancer cells, provides a new target for the clinical treatment of liver cancer, and has significant clinical application prospects. METTL3-ASO6 and METTL3-ASO7 can inhibit the expression of METTL3-A splicing isoforms in liver cancer cells, and METTL3-ASO6 and METTL3-ASO7 can inhibit the proliferation and cloning ability of liver cancer cells. The METTL3-ASO6 and METTL3-ASO7 provided by the present invention can effectively inhibit the invasive ability of liver cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the result diagram of Example 1, which screens possible ASO drugs and verifies them.

[0038] Figure 2 This is the result diagram of Example 2, which proves that the screened ASO drug can produce the application effect of inhibiting the proliferation, migration, invasion and cycle arrest of liver cancer cells.

[0039] Figure 3 Schematic diagram of the modified structure. DETAILED DESCRIPTION

[0040] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in the embodiments.

[0041] See also Figure 1-3 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions under which the present invention can be implemented. Therefore, they have no substantive technical significance, and any structural modification, change in proportional relationship or adjustment of size. The following examples are provided to better understand the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0042] Example 1: Screening and validation of potential ASO drugs.

[0043] Material

[0044] Cells: Human hepatocellular carcinoma cell lines HepG2, MHCC97L and MHCCLM3.

[0045] Reagents: DMEM high-glucose cell culture medium, penicillin, streptomycin, fetal bovine serum, RNA extraction kit, reverse transcription kit Primescript RT reagent kit and real-time fluorescence quantitative PCR SYBR Premix Ex Taq II kit were provided by Shanghai Yisheng Biotechnology Co., Ltd.; qRT-PCR specific primers were provided by Anhui General Biotechnology Co., Ltd.

[0046] method

[0047] Cell culture: Human hepatocellular carcinoma cell lines HepG2, MHCC97L and MHCCLM3 were cultured with complete medium (high-glucose DMEM cell culture medium supplemented with 10% fetal bovine serum and 200 U / mL penicillin-streptomycin double antibody) and cultured in a cell culture incubator at 37°C and 5% CO2.

[0048] Cell transfection

[0049] The human hepatocellular carcinoma cell line MHCC97L was inoculated into a 6-well plate and cultured overnight at 37°C and 5% CO2. When the cell grew to a confluence of 40-50%, a transfection experiment was performed. The transfection method was based on the jet Transfection reagent instructions. The specific steps are as follows: 1. Add 2.5ul ASO drug to 200ul jet prime buffer, vortex for 10s, and shake it in a small centrifuge. 2. Add 4μl JetPRIMEreagent to the above tube, vortex for 10 seconds, and spin at a reduced speed. 3. Incubate at RT for 10 to 15 minutes. 4. Add the transfection mixture dropwise to the cells in the serum-containing culture medium. 5. Gently shake the six-well plate back and forth, shake from side to side, and incubate at 37°C in a 37°C, 5% CO2 incubator; collect cells after transfection, and use qRT-PCR to detect the interference effect of ASO drugs on METTL3, or conduct subsequent functional verification experiments such as cell proliferation and invasion.

[0050] The total RNA extraction kit for hepatocellular carcinoma cell lines was used to extract total RNA from cells. The specific extraction method is as follows: ① Take out the cells cultured in the 6-well plate from the incubator, discard the culture medium, wash three times with phosphate buffered saline (PBS) pre-cooled on ice, and discard the PBS. Add 500mL of lysis reagent to lyse the cells, then add 500mL of anhydrous ethanol and transfer the cell lysate to the centrifuge column. Then centrifuge at 4℃, 4000g for 1min. ③ Add 500mL of Wash Buffer, centrifuge at 4℃, 12000g for 1min, discard the lower layer of centrifuge liquid, centrifuge the empty tube under the same conditions for 1min, and retain the RNA precipitate. Transfer the centrifuge column to an enzyme-free 1.5mL centrifuge tube, open the lid and dry for 2min. ④ Add 20-30μL of Elution Buffer, let stand for 2 minutes, centrifuge at 4℃, 12000g for 1 minute, add the centrifuge solution back into the centrifuge column, let stand for 5 minutes, centrifuge at 4℃, 12000g for 1 minute. Measure the concentration, mark and store in -80℃ ultra-low temperature refrigerator.

[0051] Reverse transcription

[0052] Carry out reverse transcription reaction, the system is as follows:

[0053] The total amount of RNA is 2000 ng, RNA volume X = 2000 / concentration c, ⅡBuffer 5μL, ddH2O=20-5-XμL, total volume 20μL, placed in PCR instrument at 25℃, 5min; 55℃, 15min; incubate at 85℃ for 5min to inactivate reverse transcriptase and obtain cDNA.

[0054] qRT-PCR

[0055] Using the SYBR Premix Ex Taq II kit from Yisheng Company, the reaction system is as follows:

[0056] qPCR SYBR Green Master Mix 10 μL, forward primer 0.5 μL, reverse primer 0.5 μL, cDNA 0.6 μL, ddH2O 7.4 μL, total volume 20 μL.

[0057] The reaction conditions were as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, and at 60°C for 34 s.

[0058] Annealing and extension, 34 cycles. The internal reference Gapdh gene was used as a reference.

[0059] Figure 1 A, B, and C are schematic diagrams for screening ASO drugs. Using METTL3 intron 3 as a template, Western blotting experiments showed that the expression level of METTL3-D was significantly upregulated in the groups transfected with METTL3-ASO6 and METTL3-ASO7. "*" indicates a statistically significant difference compared with the control group, P<0.5 "**" indicates a statistically significant difference compared with the control group, P<0.05.

[0060] METTL3 intron 3:

[0061]

[0062] ASO drugs-abc

[0063] METTL3-ASO drug-a:CTGTGACAGAAGTAGCCTTG;

[0064] METTL3-ASO drug-b:GCTCTTAGACCAACTCCGCA;

[0065] METTL3-ASO drug-c (ASO drug-7): CTTAAAATGTCTTATAGATC;

[0066] METTL3-ASO Drugs-1-6:

[0067] METTL3-ASO drug-1:AGATCAAATTGGCTCTTAGA;

[0068] METTL3-ASO drug-2:CTTATAGATCAAATTGGCTC;

[0069] METTL3-ASO drug-3:AATGTCTTATAGATCAAATT;

[0070] METTL3-ASO drug-4:TTGGACTTAAAATGTCTTAT;

[0071] METTL3-ASO drug-5:TAGCCTTGGACTTAAAATGT;

[0072] METTL3-ASO Drug-6:AGAAGTAGCCTTGGACTTAA;

[0073] 2'-O-MOE modification; no PS modification.

[0074] 2'-O-MOE modified structure

[0075]

[0076] Such as the structure of cytosine:

[0077] Purification method: Suzhou Hongxun Biotechnology Co., Ltd. uses HPLC purification, which uses the principle of high performance liquid chromatography to purify antisense oligonucleotides. This method can achieve high purity and sensitivity when used for separation, purification or analysis.

[0078] METTL3-A, D

[0079] METTL3-A: F-CTAAACCTAAGAATTTATAGAAG;

[0080] METTL3-A: R-CCAACTACAATACAAATGTTTAT;

[0081] METTL3-D: F-CAACAGAGCAAGAAGGTCGGGCC;

[0082] METTL3-D: R-CCATGGCCCTGCCTGTGACCAG.

[0083] Example 2: It is demonstrated that the screened ASO drug can produce the application effect of inhibiting the proliferation, migration, invasion and cycle arrest of liver cancer cells.

[0084] Experimental methods

[0085] Cell proliferation assay

[0086] The CCK-8 experiment and detection of the effect of targeted ASO drug expression on cell proliferation activity. The specific operation method of the CCK-8 experiment: collect MHCC97L liver cancer cells in the experimental group and the control group after siRNA transfection for 24 hours, add complete medium to resuspend, count the cells, and inoculate them in a 96-well plate at a density of 5000 cells / well. Set 5 rows for the experimental group and the control group, 5 duplicate wells in each row, and culture them in a 37°C, 5% CO2 incubator. Set 24h, 48h, and 72h three time points. When testing, add 10μL CCK-8 reagent to each well and incubate in a 37°C incubator for 2 hours. Then, use an enzyme reader to detect the optical density (OD) of each well at 450nm. Add complete medium to the cell-free wells as zero wells.

[0087] Cell invasion assay

[0088] MHCC97L liver cancer cells from the experimental and control groups were collected 24 hours after transfection with ASO drugs, resuspended in complete culture medium, and the cells were counted. 5 / mL cells. Add 100μL of cell suspension (DMEM high-glucose cell culture medium containing 1% fetal bovine serum) to the Transwell chamber (pre-coated with Matrigel), place the chamber in a 24-well cell culture plate, add 600μL of DMEM high-glucose cell culture medium containing 20% ​​fetal bovine serum to the lower layer, and culture in a 37°C, 5% CO2 incubator for 48h. Take out the chamber, discard the culture medium in the 24-well plate, add 500μL of 90% ethanol to fix for 10min, gently wipe the residual ethanol and cells inside the chamber with a sterile cotton swab, add 500μL of 0.1% crystal violet staining solution after air drying, and stain for 10min. Under an inverted microscope at low power, randomly select 5-8 fields of view from each chamber for observation, photography, and counting.

[0089] Cell scratch assay

[0090] 1. Marking: First, use a marker pen to draw horizontal lines evenly on the back of the 6-well plate with a ruler, about every 0.5-1 cm, and cross the holes horizontally, with at least 5 lines in each hole; 2. Plate: Cells in the logarithmic growth phase are digested with trypsin into a single cell suspension and inoculated into a 6-well culture plate; 3. 24 hours after transfection with ASO drugs, use a 200μL gun tip to measure the 6-well plate cover or ruler, parallel or perpendicular to the horizontal lines on the back, and the gun tip should be vertical and not tilted; 4. Washing: Rinse the cells with PBS 3 times, remove the scratched cells, and add serum-free culture medium; 5. Take pictures: Take pictures under a 4x microscope to ensure that the scratches are centered and vertical, pay attention to the consistency of the background, add the sample to be tested, and take samples at 0, 24, 48, and 72 hours, and take pictures.

[0091] Flow cytometry to detect apoptosis

[0092] MHCC97L liver cancer cells from the experimental and control groups were collected 24 hours after transfection with ASO drugs. 1. Collect cells: Be sure to combine the supernatant and digested cells (Note: suspended cells can be directly centrifuged); 2. Wash cells: Wash cells twice with pre-cooled PBS; 3. Grouping: Each experiment is divided into an unstained group, a single-stained Annexin V group, a single-stained PI group, and a double-stained PI and Annexin V group. The treatment groups are double-stained from low to high; 4. Staining: Dilute 4×binding buffer to 1× buffer with PBS, aspirate the residual PBS in the centrifuge tube, add 100μL of 1× binding buffer to each tube, blow the cells with a pipette to fully resuspend the cells, and add the dye under light-proof conditions. No addition was made to the unstained group, 5 μL of Annexin V or PI was added to the single-stained group, and 5 μL of Annexin V and PI were added to the double-stained group, and gently mixed with a pipette; 5. Detection on the flow cytometer: After incubation at room temperature in the dark for 15 minutes, 300 μL of 1× binding buffer was added and mixed, and the cell suspension was transferred to a 5 mL flow tube in the dark, and detected on the flow cytometer within 1 hour.

[0093] Detection of cell cycle: Add 1 ml DNA staining solution and 10 μL Permeabilization solution to the cells in step 1, vortex for 5-10 seconds to mix. Incubate at room temperature in the dark for 30 minutes.

[0094] Figure 2 A verified the inhibitory effect of ASO drugs on cell proliferation. The CCK8 experiment showed that transfection of METTL3-ASO6 and METTL3-ASO7 reduced the cell density of 97L and HepG2 cells, and the growth of cells in the two cell lines was significantly slowed down.

[0095] Figure 2 The BCDE of ASO drugs was used to verify the arrest of the cell cycle and explore the reasons for the decrease in cell proliferation. The cell cycle analysis of liver cancer cells treated with ASO drugs was performed. For 97L and HepG2 cells, we observed a significant decrease in the number of G2 / M cells, while the number of cells in the S phase increased. There were slight changes in apoptotic cells in liver cancer cells treated with METTL3-ASO6 and METTL3-ASO7, but there was no statistical significance. "**" indicates a statistical difference compared with the control group, P<0.05, "***" indicates a statistical difference compared with the control group, P<0.005, and "****" indicates a statistical difference compared with the control group, P<0.0005.

[0096] Figure 2FGHI verified the inhibition of cell migration and invasion by ASO drugs. The scratch test showed that transfection with ASO drugs 6 and 7 significantly reduced the migration ability of MHCC97L and HepG2 cells. The Transwell experiment showed that the invasion ability of these two liver cancer cells was inhibited by METTL3-ASO6 and METTL3-ASO7.

[0097] Example 3: Demonstrate the application effect of the screened ASO drug on the proliferation, migration, invasion and cycle inhibition of liver cancer cells in nude mice.

[0098] Nude mouse tumorigenesis experiment

[0099] Four-week-old BALB / c-nu nude mice were selected and housed in ventilated cages and cultured indoors for one week. HepG2 subcutaneous tumors were established as described: 2×106HepG2 cells (40,000 cells / μl in Hank's balanced salt solution) were injected subcutaneously into the right flank of nude mice. After the injection of tumor cells, tumor formation was waited for, and the tumor volume was measured once every three days after the treatment, and the tumor volume weight was measured after sacrifice. The tumor was allowed to grow 10 days before the first ASO treatment. ASOs were injected subcutaneously at a dose of 2 mg / kg / week for 5 consecutive days, followed by a 2-day rest for a total of 3 weeks.

[0100] The above description is only used to explain the preferred embodiments of the present invention, and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. An oligonucleotide that mediates alternative splicing of METTL3, characterized in that: The sequence of the oligonucleotide is any one of the sequences shown in SEQ ID No. 1-7: SEQ ID No.1:AGATCAAATTGGCTCTTAGA; SEQ ID No.2:CTTATAGATCAAATTGGCTC; SEQ ID No.3:AATGTCTTATAGATCAAATT; SEQ ID No.4:TTGGACTTAAAATGTCTTAT; SEQ ID No.5:TAGCCTTGGACTTAAAATGT; SEQ ID No.6:AGAAGTAGCCTTGGACTTAA; SEQ ID No.7:CTTAAAATGTCTTATAGATC.

2. The oligonucleotide that mediates alternative splicing of METTL3 according to claim 1, characterized in that: The nucleotides were modified by MOE.

3. The oligonucleotide that mediates alternative splicing of METTL3 according to claim 1, characterized in that: The sequence of the oligonucleotide is any one of the sequences shown in SEQ ID No. 1-7: SEQ ID No.1:AeGeAeTeCeAAATTGGCTCTeTeAeGeAe; SEQ ID No.2:CeTeTeAeTeAGATCAAATTGeGeCeTeCe; SEQ ID No.3:AeAeTeGeTeCTTATAGATCAeAeAeTeTe; SEQ ID No.4:TeTeGeGeAeCTTAAAATGTCeTeTeAeTe; SEQ ID No.5:TeAeGeCeCeTTGGACTTAAAeAeTeGeTe; SEQ ID No.6:AeGeAeAeGeTAGCCTTGGACeTeTeAeAe; SEQ ID No.7:CeTeTeAeAeAATGTCTTATAeGeAeTeCe; e represents 2'-O-MOE modification; 2'-O-MOE modification is as follows:

4. The oligonucleotide that mediates alternative splicing of METTL3 according to claim 3, characterized in that: The 2'-O-MOE modification is cytosine-modified 2'-O-MOE, and the structure is as follows:

5. Use of an oligonucleotide that mediates alternative splicing of METTL3 in the preparation of a drug for treating hepatocellular carcinoma, characterized in that: The oligonucleotide is the oligonucleotide according to any one of claims 1 to 4.

6. A pharmaceutical composition for targeting upregulation of METTL3-D gene expression, characterized in that: The method comprises the oligonucleotide mediating METTL3 alternative splicing according to claims 1 to 4 and a pharmaceutically acceptable material.

Images