CN116622706A - YTHDF2 specific siRNA containing free triphosphate group and application thereof - Google Patents
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- C—CHEMISTRY; METALLURGY
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Abstract
The invention provides YTHDF2 specific siRNA containing free triphosphate groups and application thereof, wherein the siRNA can realize multiple antitumor functions through one molecule: first, it has specific gene silencing function to tumor progression promoting molecule YTHDF 2; second, the double-stranded RNA molecules containing the triphosphate groups can activate RIG-I signaling pathway to promote anti-tumor immune response; third, degradation of RIG-I by YTDDF 2 is also inhibited by siRNA molecules, allowing the body to have sufficient RIG-I receptors to participate in the body's immune response. The YTHDF2 specific siRNA containing the free triphosphate group has very remarkable anti-tumor advantage, and the molecular toxicity of the small molecule is not observed at present. Therefore, the siRNA provided by the invention has good application prospect in preparing a pharmaceutical preparation for tumor treatment, especially in preparing a bladder cancer treatment drug.
Description
Technical Field
The invention relates to the technical field of RNA interference and the technical field of biology and new medical treatment, in particular to YTHDF2 specific siRNA containing a free triphosphate group and application thereof.
Background
Bladder cancer (BLCA) is the most common malignant disease of the urinary system, with incidence ranked 11 and mortality ranked 9 among all cancers worldwide. For BLCA, the currently employed treatments include: (1) Surgery, including transurethral bladder tumor electrotomy, bladder partial excision, and radical bladder total excision; (2) bladder perfusion chemotherapy. However, significant mental and economic stress is placed on the patient due to the extreme recurrence after surgery and the tendency to progress to higher levels of basal invasive bladder cancer after recurrence.
Because of the limited understanding of BLCA occurrence, the treatment methods currently employed can only depend to some extent on reference deductions in the study of other tumors. Therefore, developing an effective BLCA targeted therapeutic drug/regimen for the molecular mechanisms underlying BLCA is an urgent and significant medical need.
Accordingly, the prior art is still in need of improvement and development.
Disclosure of Invention
In view of the shortcomings of the prior art, the invention aims to provide YTHDF2 specific siRNA containing a free triphosphate group and application thereof, and aims to solve the problems that the molecular mechanism aiming at BLCA is unclear and an effective BLCA targeted therapeutic drug is lacking at present.
The technical scheme of the invention is as follows:
a YTHDF 2-specific siRNA comprising a free triphosphate group, wherein the 5' ends of the sense and antisense strands of the YTHDF 2-specific siRNA are modified with a free triphosphate group; the nucleotide sequences of the sense strand and the antisense strand of the YTDDF 2 specific siRNA are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
The YTHDF2 specific siRNA, wherein the siRNA specifically inhibits the expression of YTHDF 2.
The YTDDF 2 specific siRNA, wherein the siRNA causes post-transcriptional silencing of the YTDDF 2 gene.
The YTDDF 2 specific siRNA promotes the mRNA stability of a pattern recognition receptor RIG-I and improves the expression quantity of the pattern recognition receptor RIG-I.
The YTDDF 2 specific siRNA, wherein the siRNA is used as a specific ligand to activate a RIG-I signal path, promote the secretion of cytokines and activate an RIG-I induced anti-tumor immune response.
Use of a YTHDF 2-specific siRNA comprising a free triphosphate as defined in any of the preceding claims for the preparation of a medicament for inhibiting YTHDF2 gene expression in a tumor cell.
Use of a YTHDF 2-specific siRNA comprising a free triphosphate as defined in any of the preceding claims for the preparation of a medicament for activating RIG-I signaling pathway in a tumor cell.
Use of a YTHDF 2-specific siRNA comprising a free triphosphate as defined in any of the preceding claims for the preparation of a medicament for the treatment of a tumor.
The use, wherein the tumor comprises bladder cancer.
The beneficial effects are that: the invention provides YTHDF2 specific siRNA containing a free triphosphate group and application thereof. The YTHDF2 specific siRNA containing the free triphosphate group provided by the invention can realize multiple antitumor functions through one molecule: (1) The siRNA can specifically inhibit the expression of a tumor progression promoting molecule YTHDF2, thereby inhibiting the progression of tumors; (2) The siRNA can promote the mRNA stability of a pattern recognition receptor RIG-I by inhibiting the expression of YTHDF2, thereby improving the quantity of the pattern recognition receptor RIG-I important in innate immunity; (3) The siRNA is a double-stranded RNA molecule containing a triphosphate group, can be used as a specific ligand to activate a RIG-I signal path and promote secretion of cytokines, thereby improving the killing effect of an organism immune system on tumor cells. Compared with the general chemically synthesized siRNA or the general random sequence double-stranded RNA molecule, the YTHDF2 specific siRNA containing the free triphosphate group has more remarkable anti-tumor advantage, and the molecular toxicity of the small molecule is not observed at present. Therefore, the siRNA provided by the invention has good application prospect in preparing a pharmaceutical preparation for tumor treatment, and particularly has good application potential in preparing a bladder cancer treatment drug.
Drawings
FIG. 1 is a schematic diagram showing the efficiency of detecting ppp-siY 2-specific silencing YTHDF2 by RT-PCR and Western-blot in an embodiment of the invention.
FIG. 2 is a graph showing the effect of ppp-siY 2-specific silencing on activation of RIG-I signaling pathway following YTDDF 2 in an embodiment of the invention.
FIG. 3 is a schematic representation of the tumor inhibition of ppp-siY2 in a mouse bladder tumor model in accordance with an embodiment of the present invention.
FIG. 4 is a graph showing the effect of ppp-siY2 on serum IFN after use in a mouse bladder tumor model in accordance with an embodiment of the present invention.
FIG. 5 is a graph showing the effect of ppp-siY2 on immune cell recruitment in a tumor environment after application to a mouse bladder tumor model in accordance with an embodiment of the present invention.
Detailed Description
The invention provides YTHDF2 specific siRNA containing a free triphosphate group and application thereof, and the YTHDF2 specific siRNA is further described in detail below for making the purposes, technical schemes and effects of the invention clearer and more definite. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Previous studies have shown that YTDDF 2 (YTH N6-Methyladenosine RNA Binding Protein 2, RNA N6-methyladenine modified recognition protein) plays a key oncogenic role in the development of Bladder cancer (BLCA) by promoting the decay of mRNA for the pattern recognition receptor RIG-I. RIG-I acts as a tumor suppressor for BLCA and can promote anti-tumor immune responses. YTDDF 2 is expected to be an ideal target for BLCA treatment based on its important role in BLCA tumor progression.
Based on the above, the embodiment of the invention provides YTHDF2 specific siRNA containing free triphosphates, wherein the 5' ends of the sense strand and the antisense strand of the YTHDF2 specific siRNA are modified with the free triphosphates; the nucleotide sequences of the sense strand and the antisense strand of the YTDDF 2 specific siRNA are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
SEQ ID NO.1：5'-CUAGAGAACAACGAGAAUATT-3'
SEQ ID NO.2：5'-UAUUCUCGUUGUUCUCUAGGC-3'
In some embodiments, the 5 'ends of both the sense and antisense strands of the YTHDF 2-specific siRNA are guanosine, the guanosine having a free triphosphate group modified at the pentose 3' position.
The invention provides a specific siRNA of RNA N6-methyladenosine (m 6A) modified recognition protein YTHDF2 (YTH N6-Methyladenosine RNA Binding Protein 2) containing triphosphate groups, wherein free triphosphate groups are modified on pentose 3 'sites of guanosine at 5' ends of sense strand and antisense strand sequences. The YTHDF2 specific siRNA containing the free triphosphate group has double anti-tumor functions: on the one hand, the gene can specifically silence YTHDF2, namely the oncogene, and also promote the storage capacity of an effective pattern recognition receptor RIG-I in cells; on the other hand, the anti-tumor immune response induced by RIG-I can be effectively activated, and the combination of the anti-tumor immune response and the RIG-I can obviously improve the effect of treating tumors. Compared with the previously disclosed siRNA designed for simply knocking down tumor promotion proteins, the YTHDF2 specific siRNA containing the triphosphate group provided by the invention has more functions, and the obvious effects are as follows: (1) Can specifically inhibit the expression of a tumor progression promoting molecule YTHDF2, thereby inhibiting the progression of tumors; (2) The mRNA stability of the pattern recognition receptor RIG-I can be promoted by inhibiting the expression of YTHDF2, so that the quantity of the pattern recognition receptor RIG-I important in innate immunity is improved; (3) The siRNA is a double-stranded RNA molecule containing a triphosphate group, can be used as a specific ligand to activate a RIG-I signal path and promote secretion of cytokines, thereby improving the killing effect of an organism immune system on tumor cells. The siRNA molecule with the effects has good effect of inhibiting tumor progression, so that the siRNA molecule has great application potential in preparing tumor, especially bladder cancer therapeutic drugs.
In some embodiments, the siRNA specifically inhibits expression of YTHDF 2.
In particular, the siRNA is capable of causing post-transcriptional silencing of the YTHDF2 gene.
The YTHDF2 specific siRNA containing the free triphosphate group provided by the invention has a specific gene silencing function on a tumor progression promoting molecule YTHDF2 by causing the post-transcriptional silencing of YTHDF2 genes.
In some embodiments, the siRNA is capable of promoting the stability of mRNA of the pattern recognition receptor RIG-I and increasing the amount of expression of the pattern recognition receptor RIG-I. This is because by inhibiting the expression of YTDDF 2, the degradation of RIG-I mRNA by YTDDF 2 is also inhibited by the siRNA molecule, which allows the body to have sufficient RIG-I receptor to participate in the immune response of the body.
In some embodiments, the siRNA activates a RIG-I signaling pathway as a specific ligand, promotes secretion of cytokines, and activates a RIG-I induced anti-tumor immune response. The YTHDF2 specific siRNA containing the free triphosphate group provided by the invention is a double-stranded RNA molecule containing the triphosphate group, can activate RIG-I signal channels to promote anti-tumor immune response, and well plays a role in inhibiting tumor progression.
The YTHDF2 specific siRNA (named ppp-siY) containing the free triphosphate group provided by the invention can realize multiple anti-tumor functions through one molecule, and comprises the following components: (1) The ppp-siY2 can specifically inhibit the expression of a tumor progression promoting molecule YTHDF2, thereby inhibiting the progression of tumors; (2) The ppp-siY2 can promote the mRNA stability of the pattern recognition receptor RIG-I by inhibiting the expression of YTHDF2, thereby improving the quantity of the pattern recognition receptor RIG-I important in innate immunity; (3) The ppp-siY molecule is a double-stranded RNA molecule containing a triphosphate group, can be used as a specific ligand to activate a RIG-I signal path and promote secretion of cytokines, thereby improving the killing effect of an organism immune system on tumor cells. The key point is that ppp-siY2 molecules have more remarkable anti-tumor advantages with common chemically synthesized siRNA (the molecular end is an-OH bond instead of a triphosphate group), or common random sequence double-stranded RNA molecules, and the molecular toxicity of the small molecules is not observed at present. Therefore, the ppp-siY2 molecule of the invention can be applied to tumor treatment schemes, especially bladder cancer treatment schemes, and has good application potential for developing and preparing medicines for treating tumors, especially bladder cancer.
In some embodiments, free triphosphate group-containing siRNA (ppp-siRNA) molecules specific for YTHDF2, derivatives of ppp-siRNA, or similar triphosphate group-containing siRNA designed for YTHDF2 may also be designed for other sequence targets of YTHDF2 molecules. The above-mentioned modification scheme is similar to the design thought of the invention, and belongs to the protection scope of the invention.
The embodiment of the invention also provides application of the YTDDF 2 specific siRNA containing the free triphosphate group in preparation of drugs for inhibiting YTDDF 2 gene expression in tumor cells.
The embodiment of the invention also provides application of the YTDDF 2 specific siRNA containing the free triphosphate group in preparing a medicament for activating RIG-I signal paths in tumor cells.
The embodiment of the invention also provides application of the YTDDF 2 specific siRNA containing the free triphosphate group in preparing a medicine for treating tumors.
In particular, the neoplasm includes bladder cancer.
siRNA (small interfering RNA ) drugs are one of the most strategically promising biopharmaceutical technologies, and in recent years the rate of mass market arrival has been accelerating, and more siRNA drugs are expected to enter the market in the next few years. The siRNA medicine can prevent the expression of pathogenic protein from mRNA level, and has the advantages of high efficiency, good specificity, long acting and the like. However, there is no small-molecule siRNA drug for inhibiting YTHDF2 expression and treating bladder cancer using RNA interference (RNAi) technology. Based on the above, the embodiment of the invention also provides an application of YTHDF2 specific siRNA containing a free triphosphate group in preparing a medicine for treating tumors. The siRNA has a specific gene silencing function on the tumor progress promoting molecule YTHDF2, and on the other hand, double-stranded RNA molecules containing triphosphate groups can activate RIG-I signal paths to promote anti-tumor immune response, and meanwhile, the degradation of RIG-I by YTHDF2 is inhibited by the siRNA molecules so that the organism has enough RIG-I receptors to participate in the immune response of the organism. The siRNA molecule designed by combining the three characteristics can be applied to the development of siRNA drugs for inhibiting tumor progression, and has good application prospect in the aspect of preparing pharmaceutical preparations for tumor treatment.
The following is a further explanation of YTDDF 2 specific siRNA containing free triphosphate group and its application according to the present invention by way of specific examples:
the reagents, methods and apparatus employed in the present invention are those conventional in the art unless otherwise indicated.
EXAMPLE 1 Synthesis of siRNA molecules specifically silencing YTHDF2
1. Synthesis of YTHDF 2-specific siRNA
According to cDNA sequences of YTHDF2 of human and mice in a database, taking siRNA design principles into consideration, selecting a sequence common to human and mice as a target sequence, designing an siRNA molecule sequence (SEQ ID NO. 1-2) for silencing YTHDF2, synthesizing a common siRNA molecule without a triphosphate group by a commercial company through a chemical synthesis mode (through early screening, finally selecting a sequence with the best silencing efficiency for the invention, namely siY2, wherein forward and reverse strand sequences are SEQ ID NO.1-2 respectively), and simultaneously synthesizing a control siRNA molecule (namely SiNC, wherein forward and reverse strand sequences are SEQ ID NO. 3-4) without a gene silencing function by referring to a sequence of a control siRNA molecule in literature.
SEQ ID NO.1:5'-CUAGAGAACAACGAGAAUATT-3'
SEQ ID NO.2:5'-UAUUCUCGUUGUUCUCUAGGC-3'
SEQ ID NO.3:5'-UUCUCCGAACGUGUCACGUTT-3'
SEQ ID NO.4:5'-ACGUGACACGUUCGGAGAATT-3'
2. Synthesis and purification of YTHDF 2-specific triphosphate group siRNA (ppp-siRNA) based on the above siRNA sequences, sense and antisense templates with T7 polymerase promoter sequence (TAATACGACTCACTATA) were synthesized to direct in vitro transcription synthesis. Wherein, for the synthesis of each double-stranded siRNA, 4 template primers are designed first, and then two by two annealing is carried out to form a template 1 and a template 2. Then, transcription is carried out to form the positive strand and the negative strand of RNA, and annealing is carried out to form double-stranded RNA. DNA templates were synthesized and purified by commercial companies. The template for synthesizing YTHDF2 specific ppp-siRNA (named ppp-siY 2) is the sequence SEQ ID NO.5-8, and the corresponding control (named ppp-siNC) template is the sequence SEQ ID NO.9-12. The method of synthesis and purification outside the promoter using T7 polymerase was performed according to the kit Vazyme-T RNAi Transcription kit _TR 102-instructions for use-V21.1. The purified siRNA finally obtained was subjected to concentration and purity determination using electrophoresis and NanoDrop.
SEQ ID NO.5:5'-GATCACTAATACGACTCACTATAGGGCtAGAGAACAACGAGAAtATT-3'
SEQ ID NO.6:5'-AATaTTCTCGTTGTTCTCTaGCCCTATAGTGAGTCGTATTAGTGATC-3'
SEQ ID NO.7:5'-GATCACTAATACGACTCACTATAGGGtAttCtCGttGttCtCtAGTT-3'
SEQ ID NO.8:5'-AACTaGaGaaCaaCGaGaaTaCCCTATAGTGAGTCGTATTAGTGATC-3'
SEQ ID NO.9:5'-GATCACTAATACGACTCACTATAGGGtTCTCCGAACGTGTCACGTTT-3'
SEQ ID NO.10:5'-AAACGTGACACGTTCGGAGAaCCCTATAGTGAGTCGTATTAGTGATC-3'
SEQ ID NO.11:5'-GATCACTAATACGACTCACTATAGGGACGtGACACGttCGGAGAATT-3'
SEQ ID NO.12:5'-AATTCTCCGaaCGTGTCaCGTCCCTATAGTGAGTCGTATTAGTGATC-3'
EXAMPLE 2RT-PCR and Western blot detection of efficiency of ppp-siY2 specific silencing YTHDF2
Each siRNA molecule was transfected into human bladder cancer cell line SW780, respectively, transfection protocol: cell press 10 5 The cells/wells were spread evenly in 12-well plates overnight. Mu.l of Lipofectamine RNAiMAX reagent per well was used to encapsulate 1. Mu.g siRNA and transfected according to RNAiMAX instructions. After 36h, total RNA or total protein of the cells is extracted, and the efficiency of specific silencing YTHDF2 of ppp-siY2 is detected by qRT-PCR and Western-blot respectively.
FIG. 1 shows the efficiency of RT-PCR and Western-blot detection of ppp-siY2 specific silencing YTHDF 2. FIG. 1A shows the relative quantification of YTHDF2 mRNA after transfection of SW780 cells with ppp-siY2 by RT-PCR; FIG. 1B shows the protein amount of YTHDF2 after SW780 cells were transfected with ppp-siY by Western-blot detection. From the results, it can be seen that: both ppp-siY2 and siY2 are effective in reducing the amount of YTHDF2 and, as expected from the design principle, the expression of the pattern recognition receptor RIG-I (gene DDX 58) is also up-regulated.
Example 3ppp-siY2 specific silencing of YTHDF2 post RIG-I Signaling activation
Each siRNA molecule was transfected into human bladder cancer cell line SW780, respectively, transfection protocol: cell press 10 5 The cells/wells were spread evenly in 12-well plates overnight. Mu.l of Lipofectamine RNAiMAX reagent per well was used to encapsulate 1. Mu.g siRNA and transfected according to RNAiMAX instructions. After 36h, total RNA or total protein of the cells is extracted, qRT-PCR is used for respectively detecting mRNA relative quantification of downstream molecules ISG15, IFN beta and CXCL10 activated by RIG-I signal paths after ppp-siY2 specific silencing YTHDF2, and Western-blot is used for determining the knocking-down condition of siRNA molecules.
FIG. 2 is a graph showing the effect of ppp-siY 2-specific silencing on activation of the RIG-I signaling pathway following YTHDF 2. FIG. 2A shows the effect of RT-PCR on ISG15, IFN beta and CXCL10 after transfection of ppp-siY2 into SW780 cells; FIG. 2B shows the protein amount of YTHDF2 after SW780 cells were transfected with PPp-siY by Western-blot detection. From the results, it can be seen that: both ppp-siY2 and siY2 are effective in reducing the amount of YTDDF 2 and promoting RIG-I signaling pathway activation. Moreover, the ppp-siY2 molecule promotes activation to a more pronounced extent.
Example 4 inhibition of tumors by ppp-siY2 in a mouse bladder tumor model
Mu.l of MBT-2 cells (1X 10 5 And 15% matrigel%
) After thorough mixing, the mixture was inoculated into C57BL/6J mice, and 50. Mu.g of the siRNA molecules described above were coated with in vivo-JetPEI reagent and dissolved in 5% glucose solution at days 3, 6 and 9 after inoculation, respectively, and each mouse was intravenously injected. Mice tumor cells were photographed on day 11 and measured for size and body weight.
FIG. 3 shows the inhibition of tumors by ppp-siY2 in a mouse bladder tumor model. Wherein FIG. 3A is a dosing strategy of ppp-siY2 for a mouse subcutaneous bladder tumor model; FIG. 3B is a diagram showing tumor sizes for each group; FIG. 3C is a statistical plot of tumor sizes; fig. 3D is tumor weight. From the results, it can be seen that: both ppp-siY2 and siY2 are effective in reducing tumor size and weight, and ppp-siY2 shows a stronger therapeutic effect.
Example 5 effect of ppp-siY2 on serum IFN in a mouse bladder tumor model
Mice were treated as described in example 4 and serum was isolated on day 11 after blood was taken from the heart and the amount of serum IFN was determined as indicated by ELISA Kit.
FIG. 4 shows the effect of ppp-siY2 on serum IFN after use in a mouse bladder tumor model. From the results, it can be seen that: both ppp-siY2 and siY2 are effective in promoting serum IFN and ppp-siY2 shows a stronger promoting effect.
Example 6 effect of ppp-siY2 on immune cell recruitment in the tumor environment in a mouse bladder tumor model.
Mouse tumor sections obtained as described in example 4 were labeled with Multiplex Immunofluorescence (MIHC) starting, YTHDF2, CD8, CD11b and DAPI, respectively.
FIG. 5 shows the effect of ppp-siY2 on immune cell recruitment in the tumor environment after use in a mouse bladder tumor model. From the results, it can be seen that: both ppp-siY2 and siY2 inhibit YTHDF2 protein levels and are effective in promoting CD8 + ,CD11b + Recruitment of cells.
In summary, the invention provides YTHDF2 specific siRNA containing free triphosphate groups and application thereof. The YTHDF2 specific siRNA containing the free triphosphate group provided by the invention can realize multiple antitumor functions through one molecule: (1) The siRNA can specifically inhibit the expression of a tumor progression promoting molecule YTHDF2, thereby inhibiting the progression of tumors; (2) The siRNA can promote the mRNA stability of a pattern recognition receptor RIG-I by inhibiting the expression of YTHDF2, thereby improving the quantity of the pattern recognition receptor RIG-I important in innate immunity; (3) The siRNA is a double-stranded RNA molecule containing a triphosphate group, can be used as a specific ligand to activate a RIG-I signal path and promote secretion of cytokines, thereby improving the killing effect of an organism immune system on tumor cells. Compared with the general chemically synthesized siRNA or the general random sequence double-stranded RNA molecule, the YTHDF2 specific siRNA containing the free triphosphate group has more remarkable anti-tumor advantage, and the molecular toxicity of the small molecule is not observed at present. Therefore, the siRNA provided by the invention has good application prospect in preparing a pharmaceutical preparation for tumor treatment, and particularly has good application potential in preparing a bladder cancer treatment drug.
It is to be understood that the invention is not limited in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.
Claims (9)
1. A YTHDF 2-specific siRNA comprising a free triphosphate group, wherein the 5' ends of the sense strand and the antisense strand of the YTHDF 2-specific siRNA are modified with the free triphosphate group; the nucleotide sequences of the sense strand and the antisense strand of the YTDDF 2 specific siRNA are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
2. The YTHDF 2-specific siRNA of claim 1, wherein the siRNA specifically inhibits expression of YTHDF 2.
3. The YTHDF 2-specific siRNA of claim 2, wherein the siRNA causes post-transcriptional silencing of a YTHDF2 gene.
4. The YTHDF 2-specific siRNA according to claim 1, wherein the siRNA promotes mRNA stability of pattern recognition receptor RIG-I and increases expression level of pattern recognition receptor RIG-I.
5. The YTHDF 2-specific siRNA of claim 1, wherein the siRNA activates RIG-I signaling pathway as a specific ligand, promotes secretion of cytokines, activates RIG-I-induced anti-tumor immune response.
6. Use of a free triphosphate group-containing YTHDF 2-specific siRNA according to any one of claims 1-5 for the manufacture of a medicament for inhibiting YTHDF2 gene expression in a tumor cell.
7. Use of a YTHDF 2-specific siRNA comprising a free triphosphate according to any one of claims 1-5 for the manufacture of a medicament for activating RIG-I signaling pathway in tumor cells.
8. Use of a YTHDF 2-specific siRNA comprising a free triphosphate according to any one of claims 1-5 for the manufacture of a medicament for the treatment of a tumor.
9. The use of claim 8, wherein the tumor comprises bladder cancer.
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