A siRNA or shRNA construct for inhibiting RSRC1 expression and its application

Abstract

This invention discloses a siRNA or shRNA construct for inhibiting RSRC1 expression and its application, relating to the field of biomedical technology. It includes a specific nucleotide sequence targeting human RSRC1 gene mRNA; this specific nucleotide sequence can specifically bind to the coding region (CDS) or 3' untranslated region (3'UTR) of the RSRC1 gene, mediating the degradation or translational repression of RSRC1 mRNA, thereby reducing the intracellular expression level of RSRC1 protein; the construct is configured to block the direct interaction between RSRC1 protein and DVL2 protein, thereby inhibiting the activation of downstream Wnt / β-catenin signaling pathways and AKT / mTOR signaling pathways. This invention, while inducing tumor cell apoptosis, inhibiting colony formation, and reducing metastatic nodules in vivo, avoids the limitations of single-target silencing in completely blocking multi-pathway cross-talk, ultimately achieving a synergistic therapeutic effect of significantly reducing chemotherapy resistance, inhibiting metastasis of advanced ovarian cancer, and prolonging patient survival.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a siRNA or shRNA construct for inhibiting RSRC1 expression and its application. Background Technology

[0002] RNA interference (RNAi) technology, which utilizes small interfering RNA (siRNA) or short hairpin RNA (shRNA) to specifically degrade target mRNA, has become an important strategy for targeted therapy of ovarian cancer. Current research largely focuses on silencing single oncogenes or drug resistance factors, and significant progress has been made in optimizing delivery systems (such as liposomes and viral vectors). These technologies have demonstrated good gene knockdown efficiency in basic research, providing effective means to inhibit tumor cell proliferation or single signaling pathways, and forming the mainstream technological foundation for current nucleic acid drug development.

[0003] The malignant phenotype of paclitaxel-resistant ovarian cancer is often driven by the synergistic interaction of multiple signaling pathways, with extremely complex mechanisms. Existing single-target silencing technologies have limitations: while they can reduce the total amount of the target protein, they struggle to precisely block the function of the complex formed between the protein and key interacting partners (such as DVL2), resulting in the inability to simultaneously inhibit the downstream parallel Wnt / β-catenin and AKT / mTOR dual signaling axes. Since RSRC1 needs to bind to DVL2 to synergistically activate these dual pathways and drive epithelial-mesenchymal transition (EMT) and chemotherapy resistance, simply reducing RSRC1 expression without specifically disrupting its interaction interface and dual-pathway cascade response makes it difficult to achieve the dual efficacy of resistance reversal and metastasis inhibition. Therefore, there is an urgent need for a construct that can precisely target the RSRC1 functional domain, block the formation of the RSRC1-DVL2 complex, and simultaneously inhibit both signaling pathways to address the insufficient efficacy of existing technologies in dealing with complex pathological mechanisms driven by multiple pathways. Summary of the Invention

[0004] In view of the aforementioned existing problems, the present invention is proposed.

[0005] Therefore, this invention provides an siRNA or shRNA construct for inhibiting RSRC1 expression, which solves the technical problem that existing technologies cannot simultaneously inhibit the Wnt / β-catenin and AKT / mTOR dual signaling pathways by specifically blocking the RSRC1-DVL2 protein-protein interaction complex, thus resulting in incomplete efficacy of paclitaxel resistance reversal and metastasis inhibition.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: In a first aspect, the present invention provides a siRNA or shRNA construct for inhibiting RSRC1 expression, comprising a specific nucleotide sequence targeting human RSRC1 gene mRNA; The specific nucleotide sequence can specifically bind to the coding region (CDS) or the 3' untranslated region (3'UTR) of the RSRC1 gene, mediating the degradation or translational repression of RSRC1 mRNA, thereby reducing the expression level of RSRC1 protein in cells; The construct is configured to block the direct interaction between RSRC1 and DVL2 proteins, thereby inhibiting the activation of downstream Wnt / β-catenin and AKT / mTOR signaling pathways.

[0007] As a preferred embodiment of the siRNA or shRNA construct for inhibiting RSRC1 expression according to the present invention, wherein: the specific nucleotide sequence is a double-stranded siRNA structure or a single-stranded hairpin shRNA structure; If it is a double-stranded siRNA, it contains a sense strand and an antisense strand, with a length of 19-21 nucleotides and two protruding nucleotides at the 3' end; If it is a single-stranded shRNA hairpin structure, it sequentially includes a sense strand sequence, a loop linker sequence, an antisense strand sequence, and a terminator sequence; the specific nucleotide sequence is either a double-stranded siRNA structure or a single-stranded shRNA hairpin structure. If it is a double-stranded siRNA, it contains a sense strand and an antisense strand, with a length of 19-21 nucleotides and two protruding nucleotides at the 3' end; If it is a single-stranded hairpin structure of shRNA, it contains, in sequence, the sense strand sequence, the loop connection sequence, the antisense strand sequence, and the terminator sequence.

[0008] As a preferred embodiment of the siRNA or shRNA construct for inhibiting RSRC1 expression according to the present invention, the construct contains at least one specific sequence selected from the following target regions: (1) A sequence designed targeting the exon region of the RSRC1 gene can reduce the RSRC1 mRNA level by more than 70% in ovarian cancer cells. (2) A sequence designed targeting the coding region of the RSRC1 gene corresponding to the DVL2 protein binding domain to specifically disrupt the formation of the RSRC1-DVL2 complex; (3) Experimental results showed that the expression of Snail and ZEB1, key transcription factors of epithelial-mesenchymal transition (EMT), was significantly downregulated, and the expression of E-cadherin, an epithelial marker, was upregulated.

[0009] As a preferred embodiment of the siRNA or shRNA construct for inhibiting RSRC1 expression according to the present invention, wherein the construct is cloned into a eukaryotic expression vector to form a recombinant expression vector; The eukaryotic expression vector contains a promoter, an shRNA coding sequence, a selection marker gene, and a replication origin; The promoter is selected from the U6 promoter, H1 promoter, or CMV promoter; The selection marker gene is selected from the puromycin resistance gene, neomycin resistance gene, or hygromycin resistance gene, and is used to screen ovarian cancer cell lines with stable RSRC1 knockdown.

[0010] As a preferred embodiment of the siRNA or shRNA construct for inhibiting RSRC1 expression according to the present invention, wherein the eukaryotic expression vector is a lentiviral vector, an adenovirus vector, or an adeno-associated virus vector. Preferably, the vector is a lentiviral shuttle vector carrying a GFP or RFP fluorescent reporter gene to facilitate monitoring of transfection efficiency and in vivo tracing. The vector is configured to infect paclitaxel-resistant ovarian cancer cell lines that are difficult to transfect, including but not limited to A2780 / Taxol, SKOV3 / TR, or KGN-1 cells.

[0011] As a preferred embodiment of the siRNA or shRNA construct for inhibiting RSRC1 expression according to the present invention, wherein the construct is formulated into a pharmaceutical composition; The pharmaceutical composition also includes a pharmaceutically acceptable carrier or delivery system; The delivery system is selected from liposome nanoparticles (LNP), cationic polymers, exosomes, or gold. Nanoparticles are used to protect nucleic acid sequences from degradation by serum nucleases and improve their targeted enrichment efficiency in ovarian tumor tissues.

[0012] As a preferred embodiment of the siRNA or shRNA construct for inhibiting RSRC1 expression as described in this invention, Wherein, the construct exhibits the following molecular biological effects in in vitro or in vivo applications: (1) Significantly reduced the expression levels of RSRC1 and DVL2 proteins or the binding amount of both in cells; (2) Reduce phosphorylated AKT (p-AKT), phosphorylated mTOR (p-mTOR), and nuclear β-catenin. The level; (3) Reversal of the epithelial-mesenchymal transition (EMT) phenotype, characterized by downregulation of mesenchymal markers (N-cadherin, Vimentin) and upregulation of epithelial markers (E-cadherin, Occludin).

[0013] As a preferred embodiment of the siRNA or shRNA construct for inhibiting RSRC1 expression according to the present invention, the construct contains a specific nucleotide sequence, the positive strand sequence of which is shown in SEQ ID NO: 1~SEQ ID NO: X (the optimal sequence selected by specific experiments should be filled in here), or a variant thereof; The variant refers to a sequence in which 1-3 bases are replaced, deleted, or added, while still maintaining specific silencing activity against the RSRC1 gene and the ability to reverse paclitaxel resistance in ovarian cancer.

[0014] Secondly, the present invention provides an application of siRNA or shRNA that inhibits RSRC1 expression. The applications include using the construct to prepare drugs or kits for treating ovarian cancer; The term "ovarian cancer" specifically refers to paclitaxel-resistant ovarian cancer or advanced ovarian cancer with high metastatic potential. The drug's mechanism of action involves silencing RSRC1 gene expression and blocking the RSRC1-DVL2 interaction. It works by inhibiting the Wnt / β-catenin and AKT / mTOR signaling pathways, thereby reversing the EMT process in tumor cells and restoring their sensitivity to chemotherapeutic drugs.

[0015] As an application of the siRNA or shRNA that inhibits RSRC1 expression according to the present invention, it includes, The specific applications include the following treatment plans: (1) The construct is used in combination with a microtubule inhibitor chemotherapy drug, preferably paclitaxel. (Paclitaxel) or its derivatives (such as docetaxel) are administered in combination; (2) The combination therapy regimen has been shown in preclinical models to significantly reduce the half-life of drug-resistant cell lines. The inhibitory concentration (IC50) induced apoptosis, inhibited cell colony formation, migration and invasion, and reduced tumor volume and the number of lung / liver metastatic nodules in nude mice. (3) Used to prolong progression-free survival (PFS) and overall survival (OS) in patients with ovarian cancer, especially for a subgroup of patients with high RSRC1 expression.

[0016] The beneficial effects of this invention are as follows: By designing specific siRNA or shRNA sequences targeting specific functional domains of the human RSRC1 gene (especially the coding region corresponding to the DVL2 protein-binding domain), and using efficient delivery systems such as lentiviral vectors or liposomes to introduce them into paclitaxel-resistant ovarian cancer cells, precise degradation and translational inhibition of RSRC1 mRNA are achieved. This step not only significantly reduces the total amount of RSRC1 protein in cells, but more importantly, specifically blocks the direct interaction between RSRC1 and DVL2 proteins, thereby severing the molecular basis for their synergistic activation of the downstream Wnt / β-catenin and AKT / mTOR dual signaling pathways, effectively reversing the epithelial- Mesenchymal transition (EMT)-driven tumor migration and invasion phenotypes; further, by combining the construct with paclitaxel, the synchronous inhibition of dual signal axes was utilized to successfully restore the sensitivity of drug-resistant cells to chemotherapeutic drugs. While inducing tumor cell apoptosis, inhibiting colony formation, and reducing metastatic nodules in vivo, it avoided the limitations of single-target silencing in completely blocking the cross-talk of multiple pathways. Ultimately, it achieved the beneficial effects of synergistic therapy that significantly reduced chemotherapy resistance, inhibited metastasis of advanced ovarian cancer, and prolonged patient survival. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Flowchart for the association analysis of RSRC1 with adverse clinical outcomes in ovarian cancer.

[0019] Figure 2 This diagram illustrates the role of RSRC1 in chemotherapy resistance in ovarian cancer.

[0020] Figure 3 This is a schematic diagram illustrating the effect of RSRC1 on ovarian cancer cell metastasis and EMT marker expression.

[0021] Figure 4 This is a schematic diagram illustrating the effects of RSRC1 on the Wnt / β-catenin and AKT / mTOR pathways.

[0022] Figure 5 This diagram illustrates how RSRC1 binds to DVL2 and promotes its expression.

[0023] Figure 6This is a schematic diagram illustrating the effect of DVL2 on RSRC1 regulation of the Wnt / β-catenin, AKT / mTOR signaling pathways and epithelial-mesenchymal transition (EMT).

[0024] Figure 7 This is a schematic diagram of DVL2-induced chemotherapy resistance and metastasis in RSRC1. Detailed Implementation

[0025] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0026] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0027] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0028] based on Figure 1-7 Example 1: Construction and screening of specific shRNA lentiviral vectors targeting and disrupting the RSRC1-DVL2 interaction interface This embodiment aims to prepare an shRNA expression vector that can specifically target the coding region corresponding to the DVL2 protein binding domain in the RSRC1 gene, so as to block the formation of the RSRC1-DVL2 complex at the molecular level.

[0029] First, based on the RSRC1 gene sequence (GenBank accession number: NM_001135678), three candidate shRNA target sequences (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3) were designed targeting the predicted DVL2 binding domain (located in the coding region of amino acids 150-200) within its coding region (CDS). These sequences were designed to contain a specific complementary region of 19-21 nucleotides, with two prominent thymine (TT) ligands at the 3' end to mimic the Dicer cleavage product. The sense and antisense strands were connected by a loop linker sequence (Loop, 5'-TTCAAGAGA-3'), and the Pol III promoter termination signal (5'-TTTTTT-3') was attached to the end.

[0030] Subsequently, the synthesized oligonucleotides were annealed to form double strands and cloned into a lentiviral shuttle vector (pLenti-U6-shRNA-GFP-Puro) containing a U6 promoter and a GFP fluorescent reporter gene to construct a recombinant plasmid. The recombinant plasmid and packaging plasmids (psPAX2, pMD2.G) were co-transfected into HEK293T cells for viral packaging, and the supernatant containing high-titer lentiviral particles was collected.

[0031] Next, the lentivirus was used to infect the paclitaxel-resistant ovarian cancer cell line A2780 / Taxol. Forty-eight hours after infection, GFP-positive cells were sorted by flow cytometry and then subjected to a two-week selection process with the addition of puromycin to obtain a stable RSRC1 knockdown monoclonal cell line.

[0032] Finally, the silencing efficiency was verified by qPCR and Western blotting. The results showed that, compared to the control group (Scramble shRNA), the RSRC1 mRNA level in the cell line corresponding to SEQ ID NO: 2 was reduced by approximately 82%, and co-immunoprecipitation (Co-IP) experiments confirmed that the binding amount of RSRC1 protein to DVL2 protein in this cell line was significantly reduced (by approximately 75%), while the total DVL2 protein level remained unchanged. This indicates that the shRNA constructed in this embodiment not only effectively reduced target protein expression but, more importantly, specifically disrupted the assembly of the RSRC1-DVL2 pathogenic complex, providing an ideal cell model for subsequent functional studies.

[0033] Example 2: Application of liposome-delivered siRNA combined with paclitaxel to reverse drug resistance and inhibit metastasis in ovarian cancer This embodiment aims to evaluate the therapeutic potential of the above-mentioned specific nucleotide sequence in vivo and in vitro, particularly its synergistic effect in reversing paclitaxel resistance and inhibiting epithelial-mesenchymal transition (EMT).

[0034] First, the optimal target sequence (SEQ ID NO: 2) selected in Example 1 was used to synthesize chemically modified siRNA double-stranded molecules. To improve its stability in serum and tumor targeting, the siRNA was encapsulated in cationic liposome nanoparticles (LNPs) to prepare an siRNA-LNP complex with a particle size of approximately 100 nm.

[0035] In vitro experiments: A2780 / Taxol-resistant cells were divided into four groups: a blank control group, a paclitaxel-only group (IC50 concentration), a siRNA-LNP-only group, and a combination therapy group (siRNA-LNP + paclitaxel). After 48 hours of treatment, cell apoptosis rate and migration / invasion ability were detected. The results showed that in the combination therapy group, the activity of the pro-apoptotic protein Caspase-3 was significantly enhanced, and the cell colony formation rate decreased by approximately 60% compared with the single-drug group; at the same time, Transwell assay showed that the number of cells that penetrated the membrane in the combination group was significantly reduced. Molecular mechanism detection revealed that phosphorylated AKT (p-AKT), phosphorylated mTOR (p-mTOR), and nuclear β-catenin levels were significantly inhibited in the combination group cells, and EMT markers were reversed: the expression of mesenchymal markers N-cadherin and Vimentin was downregulated, while the expression of epithelial markers E-cadherin and Occludin was significantly restored.

[0036] In vivo experiments: A subcutaneous xenograft model of A2780 / Taxol cells in nude mice and a lung metastasis model via tail vein injection were established. Tumors were allowed to grow to approximately 100 mm. 3 Mice were administered siRNA-LNP via tail vein injection every 3 days and paclitaxel weekly. After 4 weeks of treatment, the tumor volume in the combined treatment group was reduced by approximately 70% compared to the control group, and the number of lung metastatic nodules decreased from an average of 15 to 2-3. More importantly, the combined treatment restored the sensitivity of drug-resistant tumors to paclitaxel by nearly 5 times (based on the tumor growth inhibition rate), and there was no significant decrease in mouse body weight, indicating that this regimen has good safety.

[0037] In summary, this embodiment demonstrates that by specifically silencing RSRC1 and blocking its interaction with DVL2, the Wnt / β-catenin and AKT / mTOR dual signaling pathways can be effectively and simultaneously inhibited, thereby significantly reversing paclitaxel resistance in ovarian cancer and inhibiting its metastatic potential at both in vivo and in vitro levels.

[0038] Example 3: Evaluation of long-term therapeutic effect of RSRC1-targeted shRNA based on exosome delivery system in a peritoneal metastasis model of refractory advanced ovarian cancer This embodiment aims to address the problems of traditional liposome delivery systems being easily cleared from the peritoneal cavity and having insufficient penetration, and to construct an exosome delivery system with natural tumor homing ability for long-term inhibition of peritoneal spread of ovarian cancer with high metastatic potential, and to verify its effect on prolonging patient survival.

[0039] First, human embryonic kidney cells (HEK293T) were genetically engineered to stably express the transmembrane protein Lamp2b carrying ovarian cancer-targeting peptides (such as integrin-binding domains). Simultaneously, a lentiviral vector containing the optimal shRNA sequence (SEQ ID NO:2, same as in Example 1) and a red fluorescent protein (RFP) reporter gene was transfected. Targeted exosomes loaded with shRNA (Exo-shRSRC1-RFP) were isolated and purified from the cell culture supernatant by ultracentrifugation. Electron microscopy revealed that the exosomes exhibited a typical cup-shaped structure with a particle size distribution between 80-120 nm. Western blotting confirmed that the exosome surface was enriched with CD63 and CD9 markers and targeting peptides, and that shRNA was successfully encapsulated internally.

[0040] Subsequently, a mouse model simulating peritoneal metastasis of clinically advanced ovarian cancer was constructed: fluorescently labeled paclitaxel-resistant ovarian cancer cells (SKOV3 / TR-Luc) were injected into the peritoneal cavity of nude mice. After bioluminescence imaging confirmed the formation of multiple micrometastases in the peritoneal cavity, the drug administration experiment began. The experiment was divided into four groups: saline group, free shRNA group, ordinary liposome-encapsulated shRNA group (LNP-shRNA), and the targeted exosome group (Exo-shRSRC1) prepared in this embodiment. The administration regimen was intraperitoneal injection, once every 4 days, for a total of 5 consecutive doses, without the use of paclitaxel, to independently evaluate the ability of this delivery system to control metastases and its potential to reverse the drug resistance phenotype.

[0041] In vivo imaging analysis was performed on day 21 after treatment. The results showed that the bioluminescent signal intensity in the peritoneal cavity of mice in the Exo-shRSRC1 group was reduced by approximately 85% compared to the saline group, which was significantly better than that in the LNP-shRNA group (reduced by approximately 45%). Anatomical observation revealed that the Exo-shRSRC1 group had very few and small metastatic nodules on the surface of the greater omentum and mesentery, while the control group was covered with dense nodules. Histopathological analysis further revealed that in the metastatic lesions treated with Exo-shRSRC1, the colocalization signal of RSRC1 and DVL2 almost disappeared, and the downstream p-AKT and nuclear β-catenin levels were continuously suppressed to near the level of normal ovarian epithelial cells. Furthermore, the EMT reversal phenomenon (high expression of E-cadherin) was particularly pronounced at the edge of the metastatic lesions, indicating that this exosome can effectively penetrate the dense tumor matrix and deeply infiltrate into the interior of small nodules.

[0042] Finally, long-term survival monitoring was conducted. Progression-free survival (PFS) and overall survival (OS) were recorded for each group of mice. Data showed that the median survival of mice in the Exo-shRSRC1 group was approximately 2.3 times longer than that of the control group, and 40% of the mice remained tumor-free at the end of the observation period (90 days); in contrast, the LNP-shRNA group only showed an approximately 1.2-fold increase. Furthermore, no significant damage was found in the toxicity tests of major organs (heart, liver, spleen, lung, and kidney), and serum inflammatory factor levels were normal.

[0043] In summary, this embodiment overcomes the challenges of poor stability and low permeability of nucleic acid drugs in the complex peritoneal environment by constructing an exosome delivery system with active targeting capabilities. It achieves long-term, deep silencing of the RSRC1 gene and sustained blocking of RSRC1-DVL2 interaction. This method not only significantly inhibits peritoneal dissemination and metastatic lesion growth in advanced ovarian cancer without relying on high-dose chemotherapy, but also substantially prolongs the survival of drug-resistant animal models by reshaping the tumor microenvironment and reversing the EMT phenotype. This provides a novel gene therapy strategy with high efficiency, low toxicity, and broad application prospects for the clinical treatment of refractory advanced ovarian cancer.

[0044] In summary, this invention designs specific siRNA or shRNA sequences targeting specific functional domains of the human RSRC1 gene (especially the coding region corresponding to the DVL2 protein-binding domain) and introduces them into paclitaxel-resistant ovarian cancer cells using efficient delivery systems such as lentiviral vectors or liposomes. This achieves precise degradation and translational inhibition of RSRC1 mRNA. This step not only significantly reduces the total amount of RSRC1 protein in cells but, more importantly, specifically blocks the direct interaction between RSRC1 and DVL2 proteins, thereby severing the molecular basis for their synergistic activation of the downstream Wnt / β-catenin and AKT / mTOR dual signaling pathways, effectively reversing the epithelial- Mesenchymal transition (EMT)-driven tumor migration and invasion phenotypes; further, by combining the construct with paclitaxel, the synchronous inhibition of dual signal axes was utilized to successfully restore the sensitivity of drug-resistant cells to chemotherapeutic drugs. While inducing tumor cell apoptosis, inhibiting colony formation, and reducing metastatic nodules in vivo, it avoided the limitations of single-target silencing in completely blocking the cross-talk of multiple pathways. Ultimately, it achieved the beneficial effects of synergistic therapy that significantly reduced chemotherapy resistance, inhibited metastasis of advanced ovarian cancer, and prolonged patient survival.

[0045] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A siRNA or shRNA construct for inhibiting RSRC1 expression, characterized in that: Including specific nucleotide sequences targeting human RSRC1 gene mRNA; The specific nucleotide sequence can specifically bind to the coding region (CDS) or the 3' untranslated region (3'UTR) of the RSRC1 gene, mediating the degradation or translational repression of RSRC1 mRNA, thereby reducing the expression level of RSRC1 protein in cells; The construct is configured to block the direct interaction between RSRC1 and DVL2 proteins, thereby inhibiting the activation of downstream Wnt / β-catenin and AKT / mTOR signaling pathways.

2. The siRNA or shRNA construct for inhibiting RSRC1 expression as described in claim 1, characterized in that: The specific nucleotide sequence is a double-stranded siRNA structure or a single-stranded hairpin shRNA structure. If it is a double-stranded siRNA, it contains a sense strand and an antisense strand, with a length of 19-21 nucleotides and two protruding nucleotides at the 3' end; If it is a single-stranded shRNA hairpin structure, it sequentially includes a sense strand sequence, a loop linker sequence, an antisense strand sequence, and a terminator sequence; the specific nucleotide sequence is either a double-stranded siRNA structure or a single-stranded shRNA hairpin structure. If it is a double-stranded siRNA, it contains a sense strand and an antisense strand, with a length of 19-21 nucleotides and two protruding nucleotides at the 3' end; If it is a single-stranded hairpin structure of shRNA, it contains, in sequence, the sense strand sequence, the loop connection sequence, the antisense strand sequence, and the terminator sequence.

3. The siRNA or shRNA construct for inhibiting RSRC1 expression as described in claim 2, characterized in that: The construct contains at least one specific sequence selected from the following target regions: (1) Sequences designed targeting the exon regions of the RSRC1 gene can reduce RSRC1 mRNA levels by more than 70% in ovarian cancer cells. (2) A sequence designed targeting the coding region of the RSRC1 gene corresponding to the DVL2 protein binding domain to specifically disrupt the formation of the RSRC1-DVL2 complex; (3) Experimental results showed that the expression of Snail and ZEB1, key transcription factors of epithelial-mesenchymal transition (EMT), was significantly downregulated, and the expression of E-cadherin, an epithelial marker, was upregulated.

4. The siRNA or shRNA construct for inhibiting RSRC1 expression as described in claim 3, characterized in that: The construct was cloned into a eukaryotic expression vector to form a recombinant expression vector; The eukaryotic expression vector contains a promoter, an shRNA coding sequence, a selection marker gene, and a replication origin; The promoter is selected from the U6 promoter, H1 promoter, or CMV promoter; The selection marker gene is selected from the puromycin resistance gene, neomycin resistance gene, or hygromycin resistance gene, and is used to screen ovarian cancer cell lines with stable RSRC1 knockdown.

5. The siRNA or shRNA construct for inhibiting RSRC1 expression as described in claim 4, characterized in that: The eukaryotic expression vector is a lentiviral vector, an adenovirus vector, or an adeno-associated virus vector; Preferably, the vector is a lentiviral shuttle vector carrying a GFP or RFP fluorescent reporter gene to facilitate monitoring of transfection efficiency and in vivo tracing. The vector is configured to infect paclitaxel-resistant ovarian cancer cell lines that are difficult to transfect, including but not limited to A2780 / Taxol, SKOV3 / TR, or KGN-1 cells.

6. The siRNA or shRNA construct for inhibiting RSRC1 expression as described in claim 5, characterized in that: The construct was formulated into a pharmaceutical composition; The pharmaceutical composition also includes a pharmaceutically acceptable carrier or delivery system; The delivery system is selected from liposome nanoparticles (LNP), cationic polymers, exosomes, or gold nanoparticles. Rice grains are used to protect nucleic acid sequences from degradation by serum nucleases and improve their targeted enrichment efficiency in ovarian tumor tissues.

7. The siRNA or shRNA construct for inhibiting RSRC1 expression as described in claim 6, characterized in that: The constructs exhibit the following molecular biological effects when used in vitro or in vivo: (1) Significantly reduced the expression levels of RSRC1 and DVL2 proteins or the amount of both bound to them in cells; (2) Reduce phosphorylated AKT (p-AKT), phosphorylated mTOR (p-mTOR), and nuclear β-catenin. The level; (3) Reversal of the epithelial-mesenchymal transition (EMT) phenotype, characterized by downregulation of mesenchymal markers (N-cadherin, Vimentin) and upregulation of epithelial markers (E-cadherin, Occludin).

8. The siRNA or shRNA construct for inhibiting RSRC1 expression as described in claim 7, characterized in that: The construct contains a specific nucleotide sequence, the positive strand of which is shown in SEQ ID NO: 1 to SEQ ID NO: X (the optimal sequence selected in the specific experiment should be entered here), or a variant thereof; The variant refers to a sequence in which 1-3 bases are replaced, deleted, or added, while still maintaining specific silencing activity against the RSRC1 gene and the ability to reverse paclitaxel resistance in ovarian cancer.

9. An application of a siRNA or shRNA that inhibits RSRC1 expression, based on the siRNA or shRNA construct for inhibiting RSRC1 expression according to any one of claims 1 to 8, characterized in that: The applications include using the construct to prepare drugs or kits for treating ovarian cancer; The term "ovarian cancer" specifically refers to paclitaxel-resistant ovarian cancer or advanced ovarian cancer with high metastatic potential. The drug's mechanism of action involves silencing RSRC1 gene expression and blocking the RSRC1-DVL2 interaction. It works by inhibiting the Wnt / β-catenin and AKT / mTOR signaling pathways, thereby reversing the EMT process in tumor cells and restoring their sensitivity to chemotherapeutic drugs.

10. The application of the siRNA or shRNA that inhibits RSRC1 expression as described in claim 9, characterized in that: The specific applications include the following treatment plans: (1) The construct is used in combination with a microtubule inhibitor chemotherapy drug, preferably paclitaxel. (Paclitaxel) or its derivatives (such as docetaxel) are administered in combination; (2) The combination therapy regimen has been shown in preclinical models to significantly reduce the half-life of drug-resistant cell lines. Inhibitory concentration (IC50) induces apoptosis, inhibits cell colony formation, migration and invasion, and reduces tumor volume and number of lung / liver metastatic nodules in nude mice; (3) Used to prolong progression-free survival (PFS) and overall survival (OS) in patients with ovarian cancer, especially for a subgroup of patients with high RSRC1 expression.

Images