Ythdf2-specific sirnas containing free triphosphate groups and uses thereof

Abstract

The application provides YTHDF2-specific siRNA containing a free triphosphate group and application thereof, and the siRNA can realize multiple anti-tumor functions through one molecule: first, the siRNA has specific gene silencing function on tumor progression promoting molecule YTHDF2; second, the double-stranded RNA molecule containing a triphosphate group can activate the RIG-I signal pathway to promote an anti-tumor immune response; third, the degradation of RIG-I by YTHDF2 is also inhibited by the siRNA molecule, so that the body has sufficient RIG-I receptors to participate in the immune response of the body. The YTHDF2-specific siRNA containing a free triphosphate group has very significant anti-tumor advantages, and currently no molecular toxicity of the small molecule has been observed. Therefore, the siRNA provided by the application has good application prospects in the preparation of pharmaceutical preparations for tumor treatment, especially in the preparation of drugs for the treatment of bladder cancer.

Description

Technical Field

[0001] This invention relates to the fields of RNA interference technology and biomedical technology, and particularly to YTHDF2-specific siRNA containing free triphosphate groups and its applications. Background Technology

[0002] Bladder cancer (BLCA) is the most common malignant disease of the urinary system, ranking 11th in incidence and 9th in mortality among all cancers worldwide. Current treatments for BLCA include: (1) surgery, including transurethral resection of bladder tumor (TURP), partial cystectomy, and radical cystectomy; and (2) intravesical chemotherapy. However, the high recurrence rate after surgery and the tendency for recurrence to progress to higher-grade invasive bladder cancer place a significant psychological and financial burden on patients.

[0003] Due to limited understanding of BLCA development, current treatment methods rely to some extent on deductions from studies of other tumors. Therefore, developing effective BLCA-targeted therapies / treatments based on the molecular mechanisms of BLCA development is an urgent and significant medical need.

[0004] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a YTHDF2-specific siRNA containing free triphosphate groups and its application, aiming to solve the problem that the molecular mechanism of BLCA is unclear and there is a lack of effective BLCA-targeted therapies.

[0006] The technical solution of the present invention is as follows:

[0007] A YTHDF2-specific siRNA containing a free triphosphate group, wherein the 5' ends of the sense and antisense strands of the YTHDF2-specific siRNA are modified with free triphosphate groups; the nucleotide sequences of the sense and antisense strands of the YTHDF2-specific siRNA are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0008] The YTHDF2-specific siRNA specifically inhibits the expression of YTHDF2.

[0009] The YTHDF2-specific siRNA, wherein the siRNA induces post-transcriptional silencing of the YTHDF2 gene.

[0010] The YTHDF2-specific siRNA, wherein the siRNA promotes the mRNA stability of pattern recognition receptor RIG-I and increases the expression level of pattern recognition receptor RIG-I.

[0011] The YTHDF2-specific siRNA, wherein the siRNA acts as a specific ligand to activate the RIG-I signaling pathway, promote the secretion of cytokines, and activate RIG-I-induced anti-tumor immune responses.

[0012] The use of a YTHDF2-specific siRNA containing a free triphosphate group as described above in the preparation of a drug for inhibiting YTHDF2 gene expression in tumor cells.

[0013] The use of a YTHDF2-specific siRNA containing a free triphosphate group as described above in the preparation of a drug for activating the RIG-I signaling pathway in tumor cells.

[0014] The application of a YTHDF2-specific siRNA containing a free triphosphate group as described above in the preparation of a drug for treating tumors.

[0015] The application in which the tumor includes bladder cancer.

[0016] Beneficial Effects: This invention provides a YTHDF2-specific siRNA containing free triphosphate groups and its applications. The YTHDF2-specific siRNA containing free triphosphate groups provided by this invention can achieve multiple anti-tumor functions through a single molecule: (1) The siRNA can specifically inhibit the expression of the YTHDF2 molecule, thereby inhibiting tumor progression; (2) The siRNA can promote the mRNA stability of the pattern recognition receptor RIG-I by inhibiting the expression of YTHDF2, thereby increasing the amount of the important pattern recognition receptor RIG-I in innate immunity; (3) The siRNA is a double-stranded RNA molecule containing triphosphate groups, which can act as a specific ligand to activate the RIG-I signaling pathway, promote the secretion of cytokines, and thus enhance the killing effect of the body's immune system on tumor cells. Compared with general chemically synthesized siRNA or ordinary random sequence double-stranded RNA molecules, the YTHDF2-specific siRNA containing free triphosphate groups of this invention has more significant anti-tumor advantages, and no molecular toxicity of this small molecule has been observed so far. Therefore, the siRNA provided by the present invention has good application prospects in the preparation of pharmaceutical formulations for tumor treatment, especially in the preparation of drugs for bladder cancer treatment. Attached Figure Description

[0017] Figure 1This is a schematic diagram illustrating the efficiency of RT-PCR and Western blot detection of ppp-siY2-specific silencing of YTHDF2 in embodiments of the present invention.

[0018] Figure 2 This is a schematic diagram illustrating the effect of ppp-siY2-specific silencing of YTHDF2 on the activation of the RIG-I signaling pathway in an embodiment of the present invention.

[0019] Figure 3 This is a schematic diagram illustrating the inhibitory effect of ppp-siY2 on tumors in a mouse bladder tumor model according to an embodiment of the present invention.

[0020] Figure 4 This is a schematic diagram showing the effect of ppp-siY2 on serum IFN in a mouse bladder tumor model, as described in this embodiment of the invention.

[0021] Figure 5 This is a schematic diagram illustrating the effect of ppp-siY2 on the recruitment of immune cells in the tumor environment after being used in a mouse bladder tumor model in an embodiment of the present invention. Detailed Implementation

[0022] This invention provides a YTHDF2-specific siRNA containing free triphosphate groups and its applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0023] Previous studies have shown that YTHDF2 (YTH N6-Methyladenosine RNA Binding Protein 2) plays a crucial oncogenic role in bladder cancer (BLCA) development by promoting the mRNA decay of pattern recognition receptor RIG-I. RIG-I, as a tumor suppressor in BLCA, can promote anti-tumor immune responses. Based on the important role of the YTHDF2-RIG-I axis in BLCA tumor progression, YTHDF2 shows promise as an ideal target for BLCA therapy.

[0024] Based on this, embodiments of the present invention provide a YTHDF2-specific siRNA containing a free triphosphate group, wherein the 5' ends of the sense and antisense strands of the YTHDF2-specific siRNA are modified with free triphosphate groups; the nucleotide sequences of the sense and antisense strands of the YTHDF2-specific siRNA are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0025] SEQ ID NO.1: 5'-CUAGAGAACAACGAGAAUATT-3'

[0026] SEQ ID NO.2: 5'-UAUUCUCGUUGUUCUCUAGGC-3'

[0027] In some embodiments, the 5' ends of both the sense and antisense strands of the YTHDF2-specific siRNA are guanine nucleoside, and the 3' site of the pentose of the guanine nucleoside is modified with a free triphosphate group.

[0028] The present invention provides a specific siRNA containing a triphosphate group, specifically targeting the N6-methyladenosine (m6A)-modified recognition protein YTHDF2 (YTH N6-Methyladenosine RNA Binding Protein 2). The 3' site of the pentose guanine nucleotide at the 5' end of both the sense and antisense strands is modified with a free triphosphate group. This YTHDF2-specific siRNA containing a free triphosphate group possesses dual anti-tumor functions: on the one hand, it can specifically silence the oncogene YTHDF2 and also promote the storage of the effective pattern recognition receptor RIG-I in cells; on the other hand, it can effectively activate the RIG-I-induced anti-tumor immune response. The combination of these two aspects can significantly improve the therapeutic effect on tumors. The YTHDF2-specific siRNA containing triphosphate groups provided by this invention has multiple functions compared with previously disclosed siRNAs designed to knock down pro-tumor proteins. Its significant effects are: (1) it can specifically inhibit the expression of the tumor progression-promoting molecule YTHDF2, thereby inhibiting tumor progression; (2) it can promote the mRNA stability of the pattern recognition receptor RIG-I by inhibiting the expression of YTHDF2, thereby increasing the amount of RIG-I, an important pattern recognition receptor in innate immunity; (3) the siRNA is a double-stranded RNA molecule containing triphosphate groups, which can act as a specific ligand to activate the RIG-I signaling pathway, promote the secretion of cytokines, and thus enhance the killing effect of the body's immune system on tumor cells. The siRNA molecule of this invention, combining the above effects, has a good effect in inhibiting tumor progression and therefore has great potential for application in the preparation of drugs for the treatment of tumors, especially bladder cancer.

[0029] In some implementations, the siRNA specifically inhibits the expression of YTHDF2.

[0030] Specifically, the siRNA can induce post-transcriptional silencing of the YTHDF2 gene.

[0031] The YTHDF2-specific siRNA containing free triphosphate groups provided by this invention has the function of specifically silencing the tumor progression-promoting molecule YTHDF2 by inducing posttranscriptional silencing of the YTHDF2 gene.

[0032] In some embodiments, the siRNA can promote the stability of pattern recognition receptor RIG-I mRNA and increase the expression level of pattern recognition receptor RIG-I. This is because, by inhibiting the expression of YTHDF2, the degradation of RIG-I mRNA by YTHDF2 is also inhibited by the siRNA molecule, which ensures that the body has sufficient RIG-I receptors to participate in the body's immune response.

[0033] In some embodiments, the siRNA acts as a specific ligand to activate the RIG-I signaling pathway, promote cytokine secretion, and activate RIG-I-induced anti-tumor immune responses. The YTHDF2-specific siRNA containing free triphosphate groups provided by this invention is a double-stranded RNA molecule containing triphosphate groups, which can activate the RIG-I signaling pathway to promote anti-tumor immune responses and effectively inhibit tumor progression.

[0034] The YTHDF2-specific siRNA (named ppp-siY2) containing free triphosphate groups provided by this invention can achieve multiple anti-tumor functions through a single molecule, including: (1) ppp-siY2 of this invention can specifically inhibit the expression of YTHDF2, thereby inhibiting tumor progression; (2) ppp-siY2 of this invention can promote the mRNA stability of pattern recognition receptor RIG-I by inhibiting the expression of YTHDF2, thereby increasing the amount of RIG-I, an important pattern recognition receptor in innate immunity; (3) ppp-siY2 molecule of this invention is a double-stranded RNA molecule containing triphosphate groups, which can act as a specific ligand to activate the RIG-I signaling pathway, promote the secretion of cytokines, thereby enhancing the killing effect of the body's immune system on tumor cells. The above key points make ppp-siY2 molecule more significantly effective against tumors than general chemically synthesized siRNA (with -OH bonds at the molecular end instead of triphosphate groups) or ordinary random sequence double-stranded RNA molecules, and no molecular toxicity of this small molecule has been observed so far. Therefore, the ppp-siY2 molecule of this invention can be applied in tumor treatment programs, especially bladder cancer treatment programs, and has great potential for developing and preparing drugs for the treatment of tumors, especially bladder cancer.

[0035] In some embodiments, siRNA molecules containing free triphosphate groups specific to YTHDF2 (ppp-siRNA), derivatives of ppp-siRNA, or similar triphosphate-based siRNAs designed for other sequence targets of the YTHDF2 molecule can also be designed. These variations are similar to the design concept of this invention and are all within the scope of protection of this invention.

[0036] This invention also provides the application of the above-mentioned YTHDF2-specific siRNA containing free triphosphate groups in the preparation of a drug for inhibiting YTHDF2 gene expression in tumor cells.

[0037] This invention also provides the application of the above-mentioned YTHDF2-specific siRNA containing free triphosphate groups in the preparation of a drug for activating the RIG-I signaling pathway in tumor cells.

[0038] This invention also provides the application of the above-mentioned YTHDF2-specific siRNA containing free triphosphate groups in the preparation of drugs for treating tumors.

[0039] Specifically, the tumors include bladder cancer.

[0040] siRNA (small interfering RNA) drugs are among the most strategically promising biopharmaceutical technologies, and their approval and market launch have accelerated in recent years, with more siRNA drugs expected to enter the market in the coming years. siRNA drugs can block the expression of pathogenic proteins at the mRNA level, offering advantages such as high efficiency, good specificity, and long-lasting effects. However, currently, there are no small molecule siRNA drugs that utilize RNA interference (RNAi) technology to inhibit YTHDF2 expression and treat bladder cancer. Based on this, this invention also provides the application of YTHDF2-specific siRNA containing free triphosphate groups in the preparation of drugs for treating tumors. This siRNA has a specific gene silencing function against the tumor progression-promoting molecule YTHDF2; furthermore, the double-stranded RNA molecule containing triphosphate groups can activate the RIG-I signaling pathway to promote anti-tumor immune responses. Simultaneously, the degradation of RIG-I by YTHDF2 is inhibited by the siRNA molecule, ensuring sufficient RIG-I receptors in the body to participate in the immune response. The siRNA molecules designed based on these three characteristics can be used to develop siRNA drugs that inhibit tumor progression and have promising applications in the preparation of drug formulations for tumor treatment.

[0041] The following specific embodiments further illustrate the YTHDF2-specific siRNA containing free triphosphate groups and its applications according to the present invention:

[0042] Unless otherwise specified, the reagents, methods and equipment used in this invention are all conventional reagents, methods and equipment in the field.

[0043] Example 1: Synthesis of siRNA molecules that specifically silence YTHDF2

[0044] 1. Synthesis of YTHDF2-specific siRNA

[0045] Based on the cDNA sequences of YTHDF2 from humans and mice in the database, and considering the design principles of siRNA, a universal sequence for both humans and mice was selected as the target sequence. An siRNA molecule sequence for silencing YTHDF2 was designed (SEQ ID NO. 1-2). A commercial company synthesized a common siRNA molecule without triphosphate groups through chemical synthesis (after preliminary screening, the sequence with the best silencing efficiency was finally selected for this invention and named siY2, with forward and reverse strand sequences of SEQ ID NO. 1-2, respectively). At the same time, a control siRNA molecule without gene silencing function was synthesized with reference to the control siRNA molecule sequence in the literature (named siNC, with forward and reverse strand sequences of SEQ ID NO. 3-4, respectively).

[0046] SEQ ID NO.1:5'-CUAGAGAACAACGAGAAUATT-3'

[0047] SEQ ID NO.2:5'-UAUUCUCGUUGUUCUCUAGGC-3'

[0048] SEQ ID NO.3:5'-UUCUCCGAACGUGUCACGUTT-3'

[0049] SEQ ID NO.4:5'-ACGUGACACGUUCGGAGAATT-3'

[0050] 2. Synthesis and purification of YTHDF2-specific triphosphate siRNA (ppp-siRNA): Based on the above siRNA sequences, sense and antisense strand templates bearing the T7 polymerase promoter sequence (TAATACGACTCACTATA) were synthesized to guide in vitro transcription synthesis. For the synthesis of each double-stranded siRNA, four template primers were designed, and then annealed in pairs to form template 1 and template 2. Transcription was then performed to form the forward and reverse strands of RNA, followed by annealing to form double-stranded RNA. DNA templates were synthesized and purified commercially. The template for synthesizing YTHDF2-specific ppp-siRNA (named ppp-siY2) is sequence SEQ ID NO. 5-8, and the corresponding control template (named ppp-siNC) is sequence SEQ ID NO. 9-12. The in vitro synthesis and purification using the T7 polymerase promoter were performed according to the instructions for use of the Vazyme-T7 RNAi Transcription kit_TR102-V21.1. The final purified siRNA was analyzed for concentration and purity using electrophoresis and NanoDrop.

[0051] SEQ ID NO.5:5'-GATCACTAATACGACTCACTATAGGGCtAGAGAACAACGAGAAtATT-3'

[0052] SEQ ID NO.6:5'-AATaTTCTCGTTGTTCTCTaGCCCTATAGTGAGTCGTATTAGTGATC-3'

[0053] SEQ ID NO.7:5'-GATCACTAATACGACTCACTATAGGGtAttCtCGttGttCtCtAGTT-3'

[0054] SEQ ID NO.8:5'-AACTaGaGaaCaaCGaGaaTaCCCTATAGTGAGTCGTATTAGTGATC-3'

[0055] SEQ ID NO.9:5'-GATCACTAATACGACTCACTATAGGGtTCTCCGAACGTGTCACGTTT-3'

[0056] SEQ ID NO.10:5'-AAACGTGACACGTTCGGAGAaCCCTATAGTGAGTCGTATTAGTGATC-3'

[0057] SEQ ID NO.11:5'-GATCACTAATACGACTCACTATAGGGACGtGACACGttCGGAGAATT-3'

[0058] SEQ ID NO.12:5'-AATTCTCCGaaCGTGTCaCGTCCCTATAGTGAGTCGTATTAGTGATC-3'

[0059] Example 2: Detection of the efficiency of ppp-siY2-specific silencing of YTHDF2 by RT-PCR and Western blot

[0060] Each siRNA molecule was transfected into the human bladder cancer cell line SW780. Transfection protocol: cells were infected at a concentration of 10... 5 Spread siRNA evenly in each well of a 12-well plate and incubate overnight. Transfect each well with 3 μl of Lipofectamine RNAiMAX reagent containing 1 μg siRNA, following the RNAiMAX instructions. After 36 h, extract total RNA or total protein from the cells and detect the efficiency of ppp-siY2-specific silencing of YTHDF2 using qRT-PCR and Western blot, respectively.

[0061] Figure 1 The figure shows the efficiency of RT-PCR and Western blot in detecting ppp-siY2-specific silencing of YTHDF2. Figure 1 A represents the relative quantification of YTHDF2 mRNA in SW780 cells after ppp-siY2 transfection by RT-PCR. Figure 1 B represents the amount of YTHDF2 protein detected by Western blot after ppp-siY2 transfection into SW780 cells. The results show that both ppp-siY2 and siY2 can effectively reduce the amount of YTHDF2, and as expected by the design principle, the expression of the pattern recognition receptor RIG-I (gene DDX58) is also upregulated.

[0062] Example 3: Activation of the RIG-I signaling pathway after specific silencing of YTHDF2 by ppp-siY2

[0063] Each siRNA molecule was transfected into the human bladder cancer cell line SW780. Transfection protocol: cells were infected at a concentration of 10... 5Seeds were evenly distributed in 12-well plates and incubated overnight. 1 μg of siRNA was coated in 3 μl of Lipofectamine RNAiMAX reagent in each well and transfected according to the RNAiMAX instructions. After 36 h, total RNA or total protein was extracted from the cells. qRT-PCR was used to detect the relative mRNA levels of ISG15, IFNβ, and CXCL10, downstream molecules of the RIG-I signaling pathway activated after ppp-siY2-specific silencing of YTHDF2. Western blot was used to determine the knockdown status of the siRNA molecules.

[0064] Figure 2 The diagram shows the effect of ppp-siY2-specific silencing of YTHDF2 on the activation of the RIG-I signaling pathway. Figure 2 A represents the effect of ppp-siY2 transfection on ISG15, IFNβ, and CXCL10 in SW780 cells as detected by RT-PCR. Figure 2 B represents the amount of YTHDF2 protein in SW780 cells after transfection with ppp-siY2, as determined by Western blot. The results show that both ppp-siY2 and siY2 can effectively reduce the amount of YTHDF2 and promote the activation of the RIG-I signaling pathway. Furthermore, the activation effect of ppp-siY2 is more pronounced.

[0065] Example 4: Inhibitory effect of ppp-siY2 on tumors in a mouse bladder tumor model

[0066] 100 μl of MBT-2 cells (1×10⁻⁶) 5 (one) and 15% matrix adhesive (

[0067] After thorough mixing, the siRNA was inoculated into C57BL / 6J mice. On days 3, 6, and 9 post-inoculation, 50 μg of the above siRNA molecules were coated with in vivo-JetPEI reagent and dissolved in 5% glucose solution, and then injected intravenously into each mouse. On day 11, tumor cells were photographed and their size and weight were measured.

[0068] Figure 3 The diagram shows the inhibitory effect of ppp-siY2 on tumors in a mouse bladder tumor model. Figure 3 A represents the administration strategy of ppp-siY2 for a mouse subcutaneous bladder tumor model; Figure 3 B shows the tumor size display for each group; Figure 3 C represents a statistical chart of tumor size; Figure 3 D represents tumor weight. The results show that both ppp-siY2 and siY2 can effectively reduce tumor size and weight, with ppp-siY2 exhibiting a stronger therapeutic effect.

[0069] Example 5: Effect of ppp-siY2 on serum IFN in a mouse bladder tumor model

[0070] Mice were treated according to the method described in Example 4, and serum was separated after blood was collected from the heart on day 11. The amount of serum IFN was measured according to the instructions of the ELISA Kit.

[0071] Figure 4 The image shows the effect of ppp-siY2 on serum IFN in a mouse bladder tumor model. The results indicate that both ppp-siY2 and siY2 can effectively promote serum IFN, with ppp-siY2 showing a stronger promoting effect.

[0072] Example 6: Effect of ppp-siY2 on immune cell recruitment in a mouse bladder tumor model.

[0073] Mouse tumor sections obtained according to the method described in Example 4 were labeled with YTHDF2, CD8, CD11b and DAPI respectively using multiple immunofluorescence (MIHC) staining.

[0074] Figure 5 The image shows the effect of ppp-siY2 on immune cell recruitment in the tumor environment after application in a mouse bladder tumor model. The results indicate that both ppp-siY2 and siY2 inhibit YTHDF2 protein levels and effectively promote CD8+. + CD11b + Cell recruitment.

[0075] In summary, this invention provides a YTHDF2-specific siRNA containing free triphosphate groups and its applications. The YTHDF2-specific siRNA containing free triphosphate groups provided by this invention can achieve multiple anti-tumor functions through a single molecule: (1) the siRNA can specifically inhibit tumor progression and promote the expression of the YTHDF2 molecule, thereby inhibiting tumor progression; (2) the siRNA can promote the mRNA stability of the pattern recognition receptor RIG-I by inhibiting the expression of YTHDF2, thereby increasing the amount of the important pattern recognition receptor RIG-I in innate immunity; (3) the siRNA is a double-stranded RNA molecule containing triphosphate groups, which can act as a specific ligand to activate the RIG-I signaling pathway, promote the secretion of cytokines, and thus enhance the killing effect of the body's immune system on tumor cells. Compared with general chemically synthesized siRNA or ordinary random sequence double-stranded RNA molecules, the YTHDF2-specific siRNA containing free triphosphate groups of this invention has a more significant anti-tumor advantage, and no molecular toxicity of this small molecule has been observed so far. Therefore, the siRNA provided by the present invention has good application prospects in the preparation of pharmaceutical formulations for tumor treatment, especially in the preparation of drugs for bladder cancer treatment.

[0076] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A YTHDF2-specific siRNA containing a free triphosphate group, characterized in that, The 5' ends of the sense and antisense strands of the YTHDF2-specific siRNA are modified with free triphosphate groups; the nucleotide sequences of the sense and antisense strands of the YTHDF2-specific siRNA are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

2. The YTHDF2-specific siRNA according to claim 1, characterized in that, The siRNA specifically inhibits the expression of YTHDF2.

3. The YTHDF2-specific siRNA according to claim 2, characterized in that, The siRNA caused post-transcriptional silencing of the YTHDF2 gene.

4. The YTHDF2-specific siRNA according to claim 1, characterized in that, The siRNA promotes the mRNA stability of pattern recognition receptor RIG-I in bladder cancer cells and increases the expression level of pattern recognition receptor RIG-I.

5. The YTHDF2-specific siRNA according to claim 1, characterized in that, The siRNA acts as a specific ligand to activate the RIG-I signaling pathway in bladder cancer cells, promote the secretion of cytokines, and activate RIG-I-induced anti-tumor immune responses.

6. The use of a YTHDF2-specific siRNA containing a free triphosphate group as described in any one of claims 1-5 in the preparation of a medicament for treating bladder cancer.

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