An antisense oligonucleotide targeting PTBP1 or PKM2 and its application in the preparation of anti-cervical cancer drugs

Abstract

The present invention discloses an antisense oligonucleotide targeting PTBP1 or PKM2 and its application in the preparation of anti-cervical cancer drugs. In cervical cancer and many other solid tumor cancers, the protein levels of PTBP1 and PKM2 are equally up-regulated; PTBP1 and PKM2 are up-regulated in cervical cancer, and PKM2 is a prognostic target. By transfecting ASOs of PTBP1 or PKM2 into HeLa cells respectively, the expression levels of the target proteins are effectively reduced, and the lactic acid production in the cells is also reduced. By intravenous injection of ASOs of PTBP1 or PKM2, the size and weight of the tumors are effectively reduced. The therapeutic value of ASO-PTBP1 / PKM2 is discovered, and an important mechanism related to the regulation of PTBP1 and PKM2 in the regulation of the Warburg effect in cancer is revealed.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and particularly relates to an antisense oligonucleotide targeting PTBP1 or PKM2 and its application in the preparation of anti-cervical cancer drugs. Background Art

[0002] Everyone's DNA is unique. The gene sequences in DNA can encode every kind of protein in the body. If the information stored in genes is to be transmitted outside the cell nucleus, it has to go through mRNA molecules, which hand over the genetic information to ribosomes to produce the specified proteins. However, errors in DNA, such as gene mutations, can produce harmful proteins, leading to diseases. But we can design corresponding antisense oligonucleotide drugs for the mRNA corresponding to the pathogenic proteins. Antisense oligonucleotides (ASOs) are single-stranded DNA or RNA sequences consisting of 15 - 25 nucleotides that pair with the target gene. They can recognize and bind to specific sequences of these mRNAs, and change the protein expression by specifically blocking the transcription or translation process of the target gene, reducing or restoring the protein level to normal. Since a large number of proteins are related to diseases, and antisense oligonucleotides have advantages such as high specificity, high efficiency, and low toxicity and safety, they have great potential in the treatment of a series of diseases, including various genetic rare diseases, cancers, neurological diseases, and autoimmune diseases, showing broad application prospects in the field of gene therapy, etc.

[0003] The specific mechanism of action of ASOs is as follows: Oligonucleotides 15 - 25 nucleotides in length can uniquely bind to a target RNA. After binding to the targeted RNA, RNA cleavage and degradation are promoted through RNase H1 or Argonaute 2 (Ago 2), or a mechanism limited to occupancy, sometimes referred to as a steric hindrance mechanism.

[0004] Therefore, it is necessary to develop an antisense oligonucleotide for the preparation of anti-cervical cancer drugs. Summary of the Invention

[0005] The object of the present invention is to provide an antisense oligonucleotide targeting PTBP1 or PKM2 and its application in the preparation of anti-cervical cancer drugs.

[0006] To solve the above technical problems, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, an antisense oligonucleotide targeting PTBP1 or PKM2 is provided, and the nucleotide sequence of the antisense oligonucleotide targeting PTBP1 is: 5’-GGCAGGAAATTCTGTATTG-3’

[0008] The nucleotide sequence of the antisense oligonucleotide targeting PKM2 is: 5'-TTGGTGAGGACGATTATGGC-3'

[0009] Furthermore, the antisense oligonucleotide targeting PTBP1 or PKM2 further includes: at least one of the antisense oligonucleotides is modified, and the modification includes at least one of phosphorylation, methylation, amination, thiolation, substitution of oxygen with sulfur, substitution of oxygen with selenium, or isotopic labeling.

[0010] Furthermore, at least one of the antisense oligonucleotides is linked to a substance for labeling or treatment; the substance for labeling or treatment includes at least one of a fluorescent label, a radioactive substance, a therapeutic substance, biotin, digoxin, a nano-luminescent material, a small peptide, and siRNA.

[0011] In the second aspect of the present invention, there is provided the use of the antisense oligonucleotide targeting PTBP1 or PKM2 in the preparation of an anti-cervical cancer drug.

[0012] In the third aspect of the present invention, there is provided an anti-cervical cancer drug, which includes the antisense oligonucleotide targeting PTBP1 or PKM2 as claimed in claims 1-3.

[0013] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0014] The present invention provides an antisense oligonucleotide targeting PTBP1 or PKM2 and its use in the preparation of an anti-cervical cancer drug. The ASO-PTBP1 of the present invention reduces the protein level of PTBP1 and appropriately reduces the protein level of PKM2; ASO-PKM2 reduces the protein level of PKM2, and at the same time ASO-PKM2 effectively inhibits cell proliferation, resulting in cell cycle arrest in the S phase; both ASO-PTBP1 and ASO-PKM2 can effectively reduce the production of lactic acid in HeLa cells ( Figure 1 D), indicating that the antisense oligonucleotide targeting PTBP1 or PKM2 has the potential to prepare an anti-cervical cancer drug.

[0015] The roles of PTBP1 and PKM2 ASOs in drug delivery and efficacy were evaluated for the first time, filling the gap in previous related research:

[0016] Meanwhile, this ASO has demonstrated the possible mechanism of action (by inhibiting the expression of PTBP1 and PKM2 proteins through ASOs, thereby reducing the production of lactic acid, inhibiting the metabolic process of "aerobic glycolysis" in tumor cells, blocking the cell cycle in the S phase, inhibiting cell proliferation, and ultimately achieving the inhibition of tumor growth and anti-cancer effects) and the effectiveness of inhibiting tumor growth in mouse tumor treatment through in vitro experiments and intravenous injection into mice. Both ASO-PKM2 and ASO-PTBP1 showed significant effects on inhibiting tumor growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following-described drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0018] Figure 1 Blocking PTBP1 and PKM2 inhibits the proliferation and lactic acid production of HeLa cells in Example 1 of the present invention; wherein Figure 1 A shows that Western Blot analysis indicates that the antisense oligonucleotides of PKM2 and PTBP1 can effectively reduce the protein levels of the targets in HeLa cells. Figure 1 B shows the effects of blocking PTBP1 and PKM2 on the proliferation of HeLa cells. Figure 1 C shows the effects of blocking PTBP1 and PKM2 on the cell cycle of HeLa cells. Figure 1 D shows the effects of blocking PTBP1 and PKM2 on the lactic acid production of HeLa cells. Figure 1 E and Figure 1 F shows Figure 1 The quantification results of A; error bars are SEM, *, 0.01 < p < 0.05, **, p < 0.01.

[0019] Figure 2 Blocking PTBP1 and PKM2 inhibits mouse tumor growth; wherein Figure 2 A shows the distribution after intravenous injection of ASO-PKM2 into nude mice bearing HeLa cell tumors. Figure 2 B shows the volumes of xenograft tumors during the treatment with ASO-PTBP1 and ASO-PKM2. Figure 2 C shows the weights of xenograft tumors on the 19th day of treatment with ASO-PTBP1 and ASO-PKM2. Figure 2 D shows the relative protein levels of PKM2 in tumor tissues; error bars are SEM, *, 0.01 < p < 0.05, **, p < 0.01

[0020] Figure 3Changes in the body weight of mice during ASO-PTBP1 and ASO-PKM2 treatments. Detailed implementation manners

[0021] The present invention will be specifically described below in combination with the detailed implementation manners and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation manners and examples are used to illustrate the present invention, rather than limiting the present invention.

[0022] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention pertains. In case of conflict, this specification shall prevail.

[0023] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchases or by existing methods.

[0024] The antisense oligonucleotides targeting PTBP1 or PKM2 of the present application will be described in detail below in combination with examples and experimental data.

[0025] Example 1. Antisense oligonucleotides targeting PTBP1 or PKM2

[0026] I. Design of ASOs targeting PTBP1 and PKM2

[0027] The nucleotide sequence of the random antisense oligonucleotide is: 5'-AACATCGCCATCTAATTCA-3' (SEQ ID NO.3);

[0028] The nucleotide sequence of the antisense oligonucleotide targeting PTBP1 is: 5'-GGCAGGAAATTCTGTATTG-3' (SEQ ID NO.1);

[0029] The nucleotide sequence of the antisense oligonucleotide targeting PKM2 is: 5'-TTGGTGAGGACGATTATGGC-3' (SEQ ID NO.2);

[0030] II. Validation of effectiveness

[0031] (I) Transfecting ASOs of PTBP1 and PKM2 into HeLa cells respectively effectively reduced the protein levels of their target genes

[0032] 1. Cell culture

[0033] HeLa cells were cultured in MEM basal medium + 10% fetal bovine serum + 1% penicillin and streptomycin at 100 U / mL at 37 °C and 5% CO 2 under saturated humidity conditions. All cells used in the experiments were in the logarithmic growth phase.

[0034] 2. Cell seeding

[0035] Take normally cultured cells, aspirate the original culture medium, add PBS to wash, then add trypsin to digest for 1 - 3 min and terminate digestion. Pipette to break into single cells. Take 15 μL of the cell suspension for counting. Centrifuge the remaining cell suspension at 1000 rpm for 5 min, resuspend with the medium, and seed at 2×10 5 cells / well in 12-well plates, with 3 replicates in each group, and culture overnight at 37 °C in 5% CO 2 .

[0036] 3. ASO transfection

[0037] After the cells were seeded and cultured overnight, ASO transfection was performed. The pre-designed scramble oligonucleotides and antisense oligonucleotides targeting PTB / PKM2 were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). The scramble oligonucleotide sequence was AACATCGCCATCTAATTCA. The PTB antisense oligonucleotide 5'-GGCAGGAAATTCTGTATTG-3' specifically targeted exon 10 of PTBP1. The PKM2 antisense oligonucleotide 5'-TTGGTGAGGACGATTATGGC-3' specifically targeted exon 10 of PKM. The specific method was as follows:

[0038] 1) According to the instructions of Lipofectamine 2000, add ASO to the corresponding volume of serum-free medium and incubate for 5 minutes;

[0039] 2) Meanwhile, take Lipofectamine 2000 Transfection Reagent, add it to serum-free medium and incubate for 5 minutes;

[0040] 3) Add the mixture from step (1) to step (2) and incubate for 20 minutes;

[0041] 4) Add the above mixture evenly to the cell culture medium, place it in an incubator at 37 °C with 5% CO 2 for 6 h, then replace it with the corresponding fresh complete medium, and continue to culture in an incubator at 37 °C with 5% CO 2 until 48 h. Collect the samples for subsequent operations.

[0042] 4. WB detection

[0043] (1) Sample denaturation

[0044] Take an appropriate loading volume from the lysate, add SDS loading buffer to it, and boil it in boiling water for 5 min or treat it at 99 °C on a PCR instrument for 5 min to denature the protein.

[0045] (2) Electrophoresis

[0046] Prepare separating gel and stacking gel with appropriate concentrations according to the size of the target protein, prepare electrophoresis buffer, load protein ladder and denatured protein samples in the expected loading order, first run the stacking gel at 60 - 80 V electrophoresis, and when the samples are concentrated into a line, adjust the voltage to 80 - 120 V and run until the target protein size position is separated.

[0047] (3) Blotting

[0048] Prepare the transfer buffer, soak the cut filter paper in the transfer buffer for 5 min, cut out the parts of the target protein and internal reference, cut a suitable PVDF membrane according to the size of the gel, soak it in methanol for 15 s, stack them neatly in the order of filter paper, PVDF membrane, gel, filter paper from bottom to top, use a roller to drive out the bubbles, pour a little transfer buffer, put it into the transfer instrument and transfer with a constant current, and rinse the membrane with RO water after the transfer is completed.

[0049] (4) Blocking and antibody incubation

[0050] Block with 5% skim milk at room temperature for 1 h, and then incubate the membrane overnight at 4 °C in the antibody dilution solution (diluted with 5% milk).

[0051] (5) Secondary antibody incubation and ECL exposure

[0052] Take out the membrane incubated with the primary antibody from the refrigerator, recover the antibody, write the antibody name, source (mouse or rabbit), dilution ratio, date on the tube, wash the membrane 5 times with TBST for 5 min each time, incubate the membrane in the secondary antibody dilution solution (diluted with TBST) at room temperature for 45 min, wash the membrane 5 times with TBST for 5 min each time, prepare the ECL luminescent solution at a ratio of 1:1, and expose it with an exposure instrument.

[0053] The experimental results are as Figure 1 shown in A. The ASOs of PTBP1 and PKM2 effectively reduced the protein levels of their target genes. Consistent with the role of PTBP1 in promoting PKM2 production, as Figure 1 shown in A, the ASO-PTBP1 targeting PTBP1 could effectively reduce the PTBP1 protein level and simultaneously reduce the PKM2 protein level, while the ASO-PKM2 targeting PKM2 could effectively reduce the PKM2 protein level and simultaneously reduce the PTBP1 protein level.

[0054] The quantification results are as follows Figure 1 E and Figure 1 F. In the figure, ASO-scr represents random antisense oligonucleotides, indicating that both of the two antisense oligonucleotides of the present invention can simultaneously reduce the protein levels of PKM2 and PTBP1.

[0055] (II) Results

[0056] The results are as follows Figure 1 A, indicating that ASO-PKM2 and ASO-PTBP1 can effectively reduce the protein levels of the corresponding targets in HeLa cells.

[0057] Example 2: ASO-PKM2 effectively inhibits cell proliferation and arrests the cell cycle in the S phase

[0058] I. Cell proliferation detection

[0059] (I) Method

[0060] 1. For the cell samples transfected in 96-well plates, incubate them at 37°C in 5% CO 2 for the corresponding time;

[0061] 2. Add 10 μl of CCK8 solution to each well and incubate in a 5% CO 2 incubator at 37°C for 4 h;

[0062] 3. Immediately mix well and measure OD450 with an enzyme-labeled instrument;

[0063] (II) Results

[0064] The results are as follows Figure 1 B, indicating that ASO-PKM2 effectively inhibits cell proliferation.

[0065] II. Cell cycle detection

[0066] (I) Method

[0067] 1. For the cell samples 48 h after transfection, aspirate the supernatant in the culture dish or plate.

[0068] 2. Wash the cells twice with PBS.

[0069] 3. Digest the cells with 0.25% trypsin (without EDTA) at 37°C for 2 - 3 min.

[0070] 4. Transfer the cell suspension to a centrifuge tube with an appropriate amount of medium or PBS.

[0071] 5. Centrifuge at 1000 rpm for 5 min and discard the supernatant.

[0072] 6. After adding PBS, centrifuge at 1000 rpm for 5 min and discard the supernatant. Repeat this step once.

[0073] 7. Resuspend the cell pellet with 50 - 100 μl of PBS, and control the cell count at 5×10^5 - 10^6 cells.

[0074] 8. Slowly add the cell pellet into the pre-prepared pre-cooled 70% ethanol, mix well, and fix overnight at 4°C.

[0075] 9. Centrifuge at 1500 rpm for 10 min and discard the supernatant.

[0076] 10. After adding PBS, centrifuge at 1500 rpm for 10 min and discard the supernatant. Repeat this step once.

[0077] 11. Add 300 μl of the pre-prepared DNA staining solution, incubate at room temperature in the dark for 15 min. Detect by machine as soon as possible.

[0078] (II) Results

[0079] The results are as Figure 1 shown in C, indicating that the cell cycle is arrested in the S phase.

[0080] Example 3: Both ASO-PTBP1 and ASO-PKM2 can effectively reduce the production of lactic acid in HeLa cells

[0081] (I) Detection of lactic acid production

[0082] For the cell samples 48 h after transfection, immediately collect the cell culture supernatant, and use a lactic acid detection kit (Nanjing Jiancheng Bioengineering Institute) to measure the lactic acid level according to the instructions.

[0083] (II) Results

[0084] The results are as Figure 1 shown in D, indicating that both ASO-PTBP1 and ASO-PKM2 can effectively reduce the production of lactic acid in HeLa cells.

[0085] Example 4: Application of ASO treatment

[0086] (I) Experimental method:

[0087] 1. Male BALB / c nude mice (6 weeks old) were purchased from Jilin University and raised under sterile conditions. HeLa-GFP cells were digested with trypsin and collected, and washed and resuspended with 1×10 7 cells / ml PBS.

[0088] 2. Then inject 0.2 ml of the cell suspension subcutaneously into the male nude mice without thymus.

[0089] 3. Four days later, the animals were divided into four groups. Group 1 was the vehicle control group (mice were intravenously injected with 200 μl of normal saline); Group 2 was the scramble control group (mice were intravenously injected with 15 mg / kg of scramble oligonucleotide, added to 200 μl of 0.9% normal saline); Group 3 was the anti-PTB group (mice were intravenously injected with 15 mg / kg of anti-ptb antisense oligonucleotide, added to 200 μl of 0.9% normal saline); Group 4 was the anti-PKM2 animal group (mice were intravenously injected with 15 mg / kg of anti-pkm2 antisense oligonucleotide, added to 200 μl of 0.9% normal saline). There were 10 mice per group in each experiment.

[0090] 4. After anesthesia injection, a NightOwl LB983 in vivo fluorescence imaging system (Berthold, Germany) was used to image the mice at different time points. Meanwhile, the tumors were measured two-dimensionally in vitro with calipers every day to determine the tumor volume (in mm 3 ), and then calculated according to the following formula: V = L × W2 × 0.5, where L is the length of the xenograft and W is the width of the graft.

[0091] 5. After the last imaging, the mice were immediately sacrificed by cervical dislocation, the xenografts were excised and weighed. Then, part of the xenograft tumors were homogenized with lysis buffer [137 mM NaCl, 20 mM TRIS, 1% NP40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 μg / ml aprotinin, 1 μg / ml albumin, 0.5 mM sodium vanadate]. The homogenate was centrifuged at 10000 × g for 20 min, and the supernatant was collected. The preparation of cell extracts and Western blotting was as described previously. The animal experiments were conducted in accordance with the established guidelines. The animal experiments were approved by the Animal Protection and Utilization Committee of the Chinese Academy of Sciences.

[0092] (II) Experimental results

[0093] The results were as Figure 3 described, indicating that during the ASO treatment, the body weight of the mice did not change ( Figure 3 ); as Figure 2 shown in B and C, both ASO-PTBP1 or ASO-PKM2 treatment significantly reduced the size and weight of the tumors.

[0094] The results were as Figure 2 shown in D. We also detected the protein levels of PKM2 and PTBP1 in different tumor groups. GAPDH protein was used as an internal reference in the WB detection. The results showed that ASO-PKM2 was significantly reduced and ASO-PTBP1 was moderately reduced, supporting that the reduction of tumor growth by the two ASO treatments may be at least partially attributed to the reduction of the Warburg effect related to the PKM2 expression level.

[0095] Finally, it should also be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article or apparatus comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or apparatus.

[0096] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0097] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations. Sequence Listing <110> Wuhan Ruixing Biotechnology Co., Ltd. <120> Antisense Oligonucleotides Targeting PTBP1 or PKM2 and Their Application in the Preparation of Anti-Cervical Cancer Drugs <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <400> 1 ggcaggaaat tctgtattg 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 ttggtgagga cgattatggc 20 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <400> 3 aacatcgcca tctaattca 19

Claims

1. Use of antisense oligonucleotides targeting PTBP1 in the preparation of anti-cervical cancer drugs, characterized in that, the nucleotide sequence of the antisense oligonucleotides targeting PTBP1 is: 5'-GGCAGGAAATTCTGTATTG-3', and the antisense oligonucleotides targeting PTBP1 are modified, and the modification includes at least one of phosphorylation, methylation, amination, thiolation, substitution of oxygen with sulfur, substitution of oxygen with selenium or isotopic labeling.

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