Application of combination of target protein circPIAS1-108aa inhibitor and oxaliplatin in preparation of drug synergistic composition for treating cancer

Abstract

The invention discloses application of a target protein circPIAS1-108aa inhibitor combined with oxaliplatin in preparation of a medicine synergistic composition for treating cancers, the specific action mechanism of circPIAS1-108aa in colon cancer treatment is found for the first time, and further research shows that by using siRNA to knock down circPIAS1-108aa, colon cancer cell proliferation can be remarkably inhibited, and the effect of treating the colon cancer can be achieved. Furthermore, the target spot protein circPIAS1-108aa inhibitor can be used for enhancing the treatment effect of the oxaliplatin. Therefore, when the circPIAS1-108aa expression is inhibited in a targeted manner and the oxaliplatin is combined for use, the circPIAS1-108aa can be obviously used for preventing and treating cancers, the dosage and toxic and side effects of the oxaliplatin are reduced, and a new synergistic strategy is provided for tumor treatment.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cancer treatment targets, and relates to an antitumor synergistic drug, in particular to application of an inhibitor of a novel target protein circPIAS1-108aa combined with oxaliplatin (OXA) in preparation of a drug synergistic composition for treating cancer. TECHNICAL BACKGROUND

[0002] Colon cancer is a cancer affecting the colon and is one of the common malignant tumors. Epidemiological data shows that colon cancer has become the third most common and the second most deadly malignant tumor in the world. Due to the extremely high mortality rate, the commonly used treatment means in the clinic are chemotherapy, immunotherapy and targeted therapy. These comprehensive treatment means have achieved certain progress, but the prognosis of tumors is still generally poor, the long-term survival rate is low, and the serious drug resistance of some chemotherapy drugs makes the treatment effect of antitumor drugs decrease significantly, which is an important reason for the failure of antitumor treatment. Oxaliplatin (OXA) as a first-line chemotherapy drug in the treatment of colon cancer occupies an important position in the treatment of colon cancer, however, in clinical practice, its secondary drug resistance will reduce the 5-year survival rate of patients, which is also an important reason for the distant metastasis of colon cancer and the death of patients. Therefore, in clinical treatment, it is urgent to find a target for sensitizing oxaliplatin against colon cancer and improve the treatment effect of oxaliplatin.

[0003] Circular non-coding RNAs (circRNAs) are covalently closed loops formed by reverse splicing. Due to the absence of 5'-3' polarity and polyadenylate tail, it has certain stability in vivo. Many circRNAs play a role by encoding proteins instead of their host genes, and play an important role in cancer treatment. The pathogenesis of colon cancer is relatively complex, involving multiple genes and signaling pathways, affecting tumor cell proliferation, apoptosis, invasion and metastasis. Pyroptosis is a newly discovered programmed cell death, which is regulated by caspase, a unique inflammatory factor that coordinates biological effects, and is an important natural immune response. After cell pyroptosis, the cell continues to swell until the cell membrane ruptures, leading to the release of intracellular components and inflammatory factors, and ultimately leading to cell death. The nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) / cysteine aspartate specific proteinase (caspase1) / gasdermin D (Gasdermin D) signaling pathway is the classical pathway of cell pyroptosis. This pathway is widely involved in the regulation of cell physiological and pathological activities and is closely related to the occurrence and development of colon cancer. The mechanism of action of many circRNAs is related to cell pyroptosis, especially in the field of cancer, for example, CircERC1 promotes chemotherapy resistance by inhibiting pancreatic cancer cell pyroptosis and remodeling the extracellular matrix.

[0004] In our previous study, we found that circPIAS1-108aa from gene PIAS1 located in chr15: 68434283-68439038 (circbase ID: hsa_circ_0008378) can inhibit the proliferation of colon cancer cells by inhibiting the expression of target protein circPIAS1-108aa. However, the specific mechanism of target protein circPIAS1-108aa in colon cancer cells has not been reported, and there is no related report on the synergistic composition of target protein circPIAS1-108aa inhibitor and oxaliplatin for treating colon cancer.

[0005] Based on the current status of the existing technology, a drug synergistic composition for treating colon cancer is provided, which is composed of a target protein circPIAS1-108aa inhibitor and oxaliplatin. The present application will provide a new and potential treatment option for the first-line treatment of colon cancer, further improve the prognosis of patients and improve the quality of life. SUMMARY

[0006] The application aims to provide a drug synergistic composition consisting of a target protein circPIAS1-108aa inhibitor and oxaliplatin and its application in treating cancer, and provide a basis for developing drugs for treating cancer.

[0007] The object of the application can be achieved by the following technical solutions.

[0008] In a first aspect, the application claims the use of a circPIAS1-108aa inhibitor combined with oxaliplatin in the preparation of a drug for preventing and / or treating cancer.

[0009] Further, the drug is prepared with the circPIAS1-108aa inhibitor and oxaliplatin as effective components, plus pharmaceutically acceptable auxiliary components. Various clinical dosage forms can be prepared according to clinical needs.

[0010] In a second aspect, the application claims a drug synergistic composition for preventing and / or treating cancer, the effective components of which consist of a circPIAS1-108aa inhibitor and oxaliplatin.

[0011] Further, the drug synergistic composition also contains pharmaceutically acceptable auxiliary components. For example, pharmaceutically acceptable carriers or / and excipients.

[0012] In the technical solution of the application, the circPIAS1-108aa inhibitor is a substance that inhibits the activity or expression of circPIAS1-108aa. Further, the circPIAS1-108aa inhibitor is selected from artificially synthesized small interfering ribonucleic acid (siRNA).

[0013] In a specific embodiment, the circPIAS1-108aa inhibitor is at least one of siRNA#1 and siRNA#2.

[0014] siRNA#1: 5'-GCAGAAATTGGAAGACATC-3'

[0015] siRNA#2: 5'-ATTGGAAGACATCAGACAA-3'.

[0016] In the technical solution of the application, the circPIAS1-108aa inhibitor and oxaliplatin are used in a combined administration or sequential use manner during the treatment of cancer.

[0017] In the technical solution of the application, the cancer is colon cancer.

[0018] The drug synergistic composition of the present application is composed of a target protein circPIAS1-108aa inhibitor and oxaliplatin. The oxaliplatin is a platinum-based anticancer drug, and its main mechanism of action is to form cross-linking with DNA, hinder DNA replication and transcription, and ultimately induce apoptosis. The target protein circPIAS1-108aa inhibitor can inhibit the growth of colon cancer and is beneficial to the prognosis of cancer. The present application first found that the oncogenic factor circPIAS1-108aa can be used as a colon cancer treatment target, and substances that inhibit this target can induce pyroptosis of colon cancer cells for the treatment of colon cancer. The target protein circPIAS1-108aa inhibitor of the present application is an artificially synthesized small interfering ribonucleic acid (siRNA). The target protein circPIAS1-108aa inhibitor itself has no obvious cytotoxicity, and can enhance the therapeutic effect of oxaliplatin by promoting pyroptosis of colon cancer cells, thereby reducing the dosage of oxaliplatin in the treatment of colon cancer and reducing the occurrence of side effects.

[0019] The development process of the technical solution of the present application includes the following steps:

[0020] (1) circPIAS1-108aa is a key factor for promoting cancer progression. The presence and sequence correctness of a special protein (circPIAS1-108aa) encoded by circPIAS1 with 108 amino acids are identified in colon cancer cells using LC-MS / MS technology. The role of circPIAS1 in promoting cancer depends on circPIAS1-108aa by constructing wild-type and ATG mutant overexpression plasmids and verifying through colony formation experiments. According to the loop site of circPIAS1, siRNA targeting inhibitors are designed to specifically down-regulate the level of circPIAS1, thereby significantly inhibiting the growth of colon cancer cells. At least one of the target sequences #1 and #2 of siRNA is designed to be an siRNA targeting inhibitor.

[0021] The target sequence of siRNA is as follows:

[0022] #1: 5'-GCAGAAATTGGAAGACATC-3' (SEQ ID NO: 3)

[0023] #2: 5'-ATTGGAAGACATCAGACAA-3' (SEQ ID NO: 4).

[0024] The ORF sequence (such as SEQ ID NO: 2) encoding the circPIAS1-108aa protein was inserted into the PGMLV-CMV-MCS-3xFlag-EF1-ZsGreen1-T2A-Puro vector to construct a linear-ORF-108aa plasmid for high expression of circPIAS1-108aa. The linear-ORF-108aa plasmid can be ordered from Jieman Biotechnology Co., Ltd. (or directly purchased from Jieman Biotechnology Co., Ltd., PGMLV-CMV-MCS-3xFlag-EF1-ZsGreen1-T2A-Puro vector with the product number PGMLV-6751, and linear-ORF-108aa plasmid ID: 81629). The construction of the plasmid is based on the directional cloning of the vector multiple cloning site (MCS) region, and all constructions are verified by DNA sequencing. The sequencing results are shown in SEQ ID NO: 2. The construction and sequencing verification process are completed by Jieman Biotechnology Co., Ltd.

[0025] Circ-control (overexpression plasmid), C-PIAS1 (overexpression of RNA and protein levels), C-PIAS1-ATG-mut (only overexpression of RNA level), the ORF sequence (such as SEQ ID NO: 2) encoding the circPIAS1-108aa protein was inserted into the GPLVX-Laccase2-MCS-GFP-Puro vector to construct the C-PIAS1 plasmid. The C-PIAS1 plasmid can be entrusted to GenScript Biotech Co., Ltd. to construct (or directly purchased from GenScript, GPLVX-Laccase2-MCS-GFP-Puro vector item number is GM-9457, C-PIAS1 plasmid ID: 81625); the ATG mutant sequence (such as SEQ ID NO: 4) was inserted into the GPLVX-Laccase2-MCS-GFP-Puro vector to construct the C-PIAS1-ATG-mut plasmid. The C-PIAS1-ATG-mut plasmid was entrusted to GenScript Biotech Co., Ltd. to construct (or directly purchased from GenScript, GPLVX-Laccase2-MCS-GFP-Puro vector item number is GM-9457, C-PIAS1-ATG-mut plasmid ID: 81627). The construction of C-PIAS1 plasmid and C-PIAS1-ATG-mut plasmid is based on the directional cloning of vector multiple cloning site (MCS) region, all constructions are verified by DNA sequencing, and the construction and sequencing verification process are completed by GenScript Biotech Co., Ltd. The C-PIAS1-ATG-mut mutation method is to mutate both ATG sites of circPIAS1 (ID: hsa_circ_0008378, SEQ ID NO: 3) to ACG (hsa_circ_0008378-ATG-MUT, SEQ ID NO: 4).

[0026] The method for testing C-PIAS1-ATG-mut mutation is to detect by RT-qPCR and Western Blot, and the detection results show that the RNA level of circPIAS1 is increased, and the protein level of circPIAS1-108aa is decreased, which indicates that the construction is successful.

[0027] (2) The use of target protein circPIAS1-108aa inhibitor can significantly induce pyroptosis of colon cancer cells to play an anti-tumor effect. The use of overexpression plasmid of circPIAS1-108aa can significantly promote the proliferation of colon cancer cells and inhibit the pyroptosis of tumor cells.

[0028] (3) Target protein circPIAS1-108aa inhibitor combined with oxaliplatin for anti-tumor can significantly improve the anti-tumor effect of oxaliplatin. Through cell in vitro experiment, it is verified that the anti-tumor effect of oxaliplatin is significantly improved when circPIAS1-108aa level is down-regulated and oxaliplatin is given at the same time.

[0029] The beneficial effects of the present application are:

[0030] The present application finds that the target protein circPIAS1-108aa inhibitor plays a specific mechanism of anti-tumor in the treatment of colon cancer. And the target protein circPIAS1-108aa inhibitor can significantly enhance the therapeutic effect of oxaliplatin, providing a new synergistic strategy for tumor treatment. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 It is circPIAS1 coding protein prediction based on circPrimer2.0 and mass spectrometry verification.

[0032] Among them, A is the circPrimer2.0 predicted protein sequence coded by circPIAS1; B is the mass spectrometry detection of circPIAS1-108aa, which shows that the target protein circPIAS1-108aa really exists in colon cancer cells and is consistent with the predicted amino acid sequence.

[0033] Figure 2 It is the carcinogenic function of circPIAS1 which depends on its coded protein circPIAS1-108aa.

[0034] Among them, A is the clonal formation experiment result graph; B is the relative quantification, which shows that the target circPIAS1 plays a carcinogenic role mainly depending on its coded new protein, not the function of RNA level.

[0035] Figure 3 It is the influence of down-regulating circPIAS1 or over-expressing circPIAS1-108aa on colon cancer cell proliferation.

[0036] Among them, A is the clonal formation experiment result graph; B is the relative quantification; C is the CCK8 experiment result graph, which shows the change of colon cancer cell proliferation ability after down-regulating circPIAS1 by siRNA inhibitor or over-expressing circPIAS1-108aa target protein by circPIAS1-108aa over-expression plasmid.

[0037] Figure 4 It is the influence of circPIAS1-108aa on related protein expression and lactate dehydrogenase (LDH) release.

[0038] Wherein, A is the change level of each protein in colon cancer cells after administration of circPIAS1-108aa inhibitor or overexpression plasmid of circPIAS1-108aa; B is the quantification chart of each protein; C is the change of LDH in colon cancer cells after administration of circPIAS1-108aa inhibitor or overexpression plasmid of circPIAS1-108aa.

[0039] Figure 5 The synergistic anti-tumor effect of circPIAS1-108aa inhibitor and oxaliplatin.

[0040] Wherein, A is the 24 h half inhibitory concentration (IC50) of oxaliplatin on colon cancer cells; B is the change of LDH in colon cancer cells after administration of circPIAS1-108aa inhibitor combined with oxaliplatin; C is the change of cell survival rate of colon cancer cells after administration of circPIAS1-108aa inhibitor combined with oxaliplatin. DETAILED DESCRIPTION

[0041] The present application is further described below in conjunction with examples. The following description is merely intended to be a best mode of the present application and is not intended to limit the present application in other forms. Any skilled person in the art can modify the above-mentioned technical content to obtain equivalent embodiments. Any modification or equivalent change to the following examples without departing from the technical essence of the present application falls within the scope of the present application.

[0042] The HCT-116 (colon cancer) and HT-29 (colon cancer) cell lines used in the examples were provided by the Chinese Academy of Sciences Stem Cell Bank (Shanghai, China). The target protein circPIAS1-108aa amino acid sequence used in the study was predicted by circPrimer2.0 and verified by LC-MS / MS. The siRNA and circPIAS1-108aa overexpression plasmid used in the experiment were designed and synthesized by the research group.

[0043] Example 1 circPIAS1 is confirmed to encode a novel target protein of 108 amino acids long

[0044] Predicted by circPrimer 2.0 software, circPIAS1 (ID: hsa_circ_0008378) encodes a specific polypeptide of 108 amino acids, with the protein sequence: MDISGTKCDFTVQVQLRFCLSETSCPQEDHFPPNLCVKVNTKPCSLPGYLPPTKNGVEPKRPSRPINITSLVRLSTTVPNTIVVSWTAEIGRLQTTASAFGKPVLHLP (SEQ ID NO: 1). The presence of circPIAS1-108aa was successfully verified by LC-MS / MS technology.

[0045] The results are as follows Figure 1 As shown, circPIAS1-108aa is actually present in colon cancer cells and matches the predicted amino acid sequence.

[0046] Example 2: circPIAS1-108aa is a key oncogene.

[0047] In the clonogenic assay, HCT 116 or HT-29 cells were used at a concentration of 3 × 10⁻⁶. 3 The cells were laid at a density of cells per well in a 6-well plate to reduce the cell density so that cell colonies could form from individual cells.

[0048] Three overexpression plasmids were used: Circ-control (empty vector), C-PIAS1 (RNA and protein overexpression), and C-PIAS1-ATG-mut (RNA-only overexpression). The ORF sequence encoding the circPIAS1-108aa protein (e.g., SEQ ID NO:2) was inserted into the GPLLVX-Laccase2-MCS-GFP-Puro vector to construct the C-PIAS1 plasmid. The C-PIAS1 plasmid can be constructed by Jiman Biotechnology Co., Ltd. (or purchased directly from Jiman Biotechnology; the GPLLVX-Laccase2-MCS-GFP-Puro vector catalog number is GM-9457, and the C-PIAS1 plasmid ID is 81625). The ATG mutant sequence (e.g., SEQ ID NO:4) was inserted into the GPLLVX-Laccase2-MCS-GFP-Puro vector to construct the C-PIAS1-ATG-mut plasmid. The C-PIAS1-ATG-mut plasmid can be constructed by Jimon Biotechnology Co., Ltd. (or purchased directly from Jimon Biotechnology; the GPLVX-Laccase2-MCS-GFP-Puro vector catalog number is GM-9457, and the C-PIAS1-ATG-mut plasmid ID is 81627). For both the C-PIAS1 and C-PIAS1-ATG-mut plasmids, construction is based on directed cloning within the vector's multiple cloning site (MCS) region. All constructions are verified by DNA sequencing, and both the construction and sequencing verification processes are performed by Jimon Biotechnology Co., Ltd. The C-PIAS1-ATG-mut mutation is detected using RT-qPCR and Western Blot. Successful construction is indicated by an increased RNA level of circPIAS1 and a decreased protein level of circPIAS1-108aa. The mutation method involved mutating both ATG sites of circPIAS1 (ID: hsa_circ_0008378, SEQ ID NO:3) to ACG (hsa_circ_0008378-ATG-MUT, SEQ ID NO:4).

[0049] The specific procedure is as follows: After transfecting tumor cells with the plasmid, the culture medium is replaced with fresh medium, and the cells are cultured for another 7 days. Three replicates are set up for each group to ensure accuracy. When cell colonies are clearly visible, the culture medium is discarded, and the cells are washed with PBS. Cells are fixed with 4% paraformaldehyde for 15 min, washed again with PBS, and stained with crystal violet dye for 10-15 min. Shaking may be necessary depending on the tightness of cell adhesion. Finally, after washing thoroughly with PBS, images are taken using a camera or scanner.

[0050] The results are as follows Figure 2As shown, this indicates that circPIAS1 exerts its oncogenic effect mainly through its encoded novel protein rather than RNA-level function, and also demonstrates that circPIAS1-108aa has high target potential.

[0051] Example 3: The proliferation of tumor cells was significantly inhibited after using a specific inhibitor of circPIAS1-108aa.

[0052] (1) Based on the circPIAS1 cyclic site, siRNA targeting inhibitors were designed to specifically downregulate the level of circPIAS1, thereby significantly inhibiting the growth of colon cancer cells. Two targeting sequences were used to design siRNA targeting inhibitors.

[0053] The target sequence for siRNA is either #1 or #2 as follows:

[0054] #1: 5'-GCAGAAATTGGAAGACATC-3' (SEQ ID NO: 3)

[0055] #2: 5'-ATTGGAAGACATCAGACAA-3' (SEQ ID NO: 4).

[0056] (2) By constructing wild-type and circPIAS1-108aa overexpression plasmids, and verifying by clone formation experiments that the cancer-promoting effect of circPIAS1 depends on circPIAS1-108aa.

[0057] The ORF sequence encoding the circPIAS1-108aa protein (as shown in SEQ ID NO:2) was inserted into the PGMLV-CMV-MCS-3×Flag-EF1-ZsGreen1-T2A-Puro vector to construct the linear-ORF-108aa plasmid for high expression of circPIAS1-108aa. The linear-ORF-108aa plasmid was constructed by Jimon Biotechnology Co., Ltd. (or purchased directly from Jimon Biotechnology Co., Ltd., the catalog number of the PGMLV-CMV-MCS-3×Flag-EF1-ZsGreen1-T2A-Puro vector is PGMLV-6751, and the linear-ORF-108aa plasmid ID is 81629). The plasmid was constructed by directional cloning based on the multiple cloning site (MCS) region of the vector. All constructions were verified by DNA sequencing, and the sequencing results are shown in SEQ ID NO:2. The construction and sequencing verification processes were all completed by Jimon Biotechnology Co., Ltd.

[0058] In the clonogenic assay, HCT 116 or HT-29 cells were used at a concentration of 3 × 10⁻⁶. 3Cells were seeded at a density of 1 cell per well in 6-well plates to reduce cell density and allow for cell colony formation from single cells. Two inhibitors of circPIAS1-108aa, siRNA#1 and siRNA#2 (siRNA inhibition group), were used. The siRNAs were synthesized by Qingke Biotechnology and were unmodified. Lipo3000 liposome transfection reagent was used in this study. In sterile centrifuge tubes, 5 µM of siRNA and Lipo3000 were diluted separately with serum-free / low-serum medium and incubated at room temperature (25±5℃) for 5 min. After gentle mixing, they were incubated for another 15 min to allow the liposomes to form a stable complex with the nucleic acid. This complex was then added to the seeded cells, and the cells were cultured for 6–8 h, then replaced with normal medium and cultured for an additional time. Alternatively, after transfection with linear-control (empty vector control group) or linear-ORF-108aa (circPIAS1-108aa overexpression group, only overexpressing circPIAS1-108aa protein level), the culture medium was replaced with fresh medium 8 hours later, and the cells were cultured for another 7 days. Three replicates were set for each group to ensure accuracy. When cell colonies were clearly visible, the culture medium was discarded and the cells were washed with PBS. Cells were fixed with 4% paraformaldehyde for 15 min, washed again with PBS, and stained with crystal violet dye for 10-15 min. Shaking was optional depending on the tightness of cell adhesion. Finally, the cells were washed thoroughly with PBS and photographed using a camera or scanner.

[0059] In the CCK8 assay, HCT 116 or HT-29 cells were transfected with circPIAS1-108aa inhibitor siRNA, linear-control (empty vector), or linear-ORF-108aa (overexpressing only circPIAS1-108aa protein level) for 8 hours. After digestion with 0.5 mg / mL trypsin, centrifugation, and dilution with complete culture medium, the cells were counted using a cell counter. The transfected HCT 116 or HT-29 cells were then cultured at a rate of 3 × 10⁻⁶ cells / year. 3 Cells were seeded at a density of 100 μL / well in 96-well plates and cultured under suitable conditions to allow cell adhesion. Four replicates were set up for each group to ensure accuracy. After incubation, 10 μL of CCK8 reagent was added to every 100 μL of medium, and the plates were incubated for another 2 h. Finally, the absorbance (OD) value at 450 nm was measured using a microplate reader. Cell viability and inhibition rate were calculated based on the OD values ​​of the control group and each experimental group.

[0060] The results are as follows Figure 3As shown, downregulation of circPIAS1 by siRNA inhibitors significantly reduced the number of tumor cell clones (by approximately 50%, P<0.01) and cell survival rate, indicating that the inhibitor of circPIAS1 can significantly suppress tumor growth. Overexpression of circPIAS1-108aa protein by linear-ORF-108aa significantly increased the number of tumor cell clones and improved cell survival rate, indicating that circPIAS1 is an oncogene that can significantly promote tumor growth.

[0061] Example 4: Inhibitors of the target protein circPIAS1-108aa can significantly promote pyroptosis in colon cancer cells.

[0062] HCT116 or HT-29 cells were transfected for 48 h using siRNA#1 and siRNA#2 (siRNA inhibition group) or linear-control (empty vector) and linear-ORF-108aa (overexpression of circPIAS1-108aa protein only), followed by cell collection and lysis. The collected cells were thoroughly mixed with cell lysis buffer, and then centrifuged at 12000 g for 10 min to ensure efficient protein collection. Next, protein quantification was performed using a BCA assay kit. After detection, 50 μg of protein was accurately loaded onto each sample and added to a 10% SDS-PAGE gel for electrophoresis separation. Under an electric field, proteins of different molecular weights migrated at different speeds in the gel, thus achieving separation. After electrophoresis, the separated protein bands were transferred to a PVDF membrane in preparation for subsequent antibody incubation. After the transfer was completed, the PVDF membrane was blocked with 5% skim milk for 2 hours to reduce non-specific binding and improve experimental accuracy.

[0063] After sealing, the PVDF membrane was placed in the corresponding primary antibody solution and incubated overnight at 4°C. After primary antibody incubation, the membrane was washed with 1×TBST and then incubated for 2 hours with horseradish peroxidase-labeled secondary antibodies (goat anti-mouse IgG, catalog number SE131; goat anti-rabbit IgG, catalog number SE134, Beijing Solarbio Science & Technology Co., Ltd.). Finally, the membrane was developed using a chemiluminescent reagent under a Bio-Rad imaging system.

[0064] Primary antibody information is shown in Table 1:

[0065] Table 1 Primary Antibody Information

[0066] Antibody Name Source Cat. No. GAPDH Proteintech 60004-1-Ig NLRP3 Proteintech 19771-1-AP Caspase1 / P20 / p10 Proteintech 22915-1-AP GSDMD Proteintech 20770-1-AP GSDMD-N Abcam ab215203 GSDMD-N CST 36425T Cleaved-IL-1β CST 83186T Cleaved-IL-18 Proteintech 60070-1-Ig

[0067] HCT 116 or HT-29 cells were transfected for 48 h using siRNA#1 and siRNA#2 (inhibitors of circPIAS1-108aa) or linear-control (empty vector) and linear-ORF-108aa (overexpressing only circPIAS1-108aa protein). LDH release levels were measured using the CytoTox 96 LDH release reagent. The procedure was as follows: After cell transfection, cell supernatant was collected. 50 µl of the cell supernatant to be analyzed was transferred to the corresponding measurement well. Then, 50 µl of CytoTox 96® reaction working solution was added to each well, and the cells were incubated at room temperature for 30 min. After incubation, 50 µl of stop solution was added to each well of the 96-well plate. The absorbance was recorded at 490 nm or 492 nm within 1 hour after the addition of the stop solution.

[0068] The results are as follows Figure 4 As shown, the use of the target protein circPIAS1-108aa inhibitor significantly promoted pyroptosis in colon cancer cells and significantly increased LDH release (by approximately 8%, P<0.01). Overexpression of circPIAS1-108aa protein level via linear-ORF-108aa significantly inhibited pyroptosis in colon cancer cells and significantly decreased LDH release (by approximately 7%, P<0.01). All data are expressed as mean ± standard deviation, and statistical analysis was performed using one-way ANOVA. Example 5: The target protein circPIAS1-108aa inhibitor significantly improved the therapeutic effect of oxaliplatin.

[0069] HCT 116 or HT-29 cells in logarithmic growth phase were digested with 0.5 mg / mL trypsin, centrifuged, diluted with complete culture medium, counted using a cell counter, and then cultured at a rate of 3 × 10⁻⁶ cells / mL. 3 Cells were seeded at a density of 100 μL / well in 96-well plates and cultured overnight. Oxaliplatin was then added, and the plates were cultured for another 24 hours. Four replicates were performed for each group to ensure accuracy. After culture, 10 μL of CCK8 reagent was added to every 100 μL of medium, and the plates were incubated for another 2 hours. Finally, the absorbance (OD) at 450 nm was measured using a microplate reader. The IC50 value of the cells was calculated based on the OD values ​​of the control group and each experimental group.

[0070] HCT 116 or HT-29 cells were transfected with circPIAS1-108aa inhibitor siRNA#1 and inhibitor siRNA#2 for 24 h, followed by oxaliplatin treatment for 24 h. Cell supernatant was collected, and LDH release levels were measured using the CytoTox 96 LDH release reagent. Specifically, 50 µl of the cell supernatant to be analyzed was transferred to the corresponding measurement well. Then, 50 µl of CytoTox 96® reaction working solution was added to each well, and the cells were incubated at room temperature for 30 min. After incubation, 50 µl of stop solution was added to each well of the 96-well plate. The absorbance was recorded at 490 nm or 492 nm within one hour after the addition of the stop solution.

[0071] In the CCK8 assay, HCT 116 or HT-29 cells were transfected with circPIAS1-108aa inhibitor siRNA for 8 hours. After trypsin digestion with 0.5 mg / mL, centrifugation, and dilution with complete culture medium, the cells were counted using a cell counter. The transfected HCT 116 or HT-29 cells were then cultured at a rate of 3 × 10⁻⁶ cells / year. 3 Cells were seeded at a density of 100 μL / well in 96-well plates, maintaining a culture medium volume of 100 μL. The plates were then incubated overnight, followed by incubation with oxaliplatin for 24 h. Four replicates were performed for each group to ensure accuracy. After incubation, 10 μL of CCK8 reagent was added to every 100 μL of culture medium, and the plates were incubated for another 2 h. Finally, the absorbance (OD) at 450 nm was measured using a microplate reader. Cell viability and inhibition rate were calculated based on the OD values ​​of the control group and each experimental group.

[0072] The results are as follows Figure 5 As shown, the IC50 values ​​of HCT 116 and HT-29 cells were 45.91 µM and 15.71 µM, respectively, 24 h after oxaliplatin administration. Combining the circPIAS1-108aa inhibitor siRNA with oxaliplatin enhanced LDH release and simultaneously reduced the survival rate of colon cancer cells.

[0073] sequence list

[0074] The amino acid sequence of the target protein circPIAS1-108aa (SEQ ID NO: 1):

[0075] MDISGTKCDFTVQVQLRFCLSETSCPQEDHFPPNLCVKVNTKPCSLPGYLPPTKNGVEPKRPSRPINITSLVRLSTTVPNTIVVSWTAEIGRLQTTASAFGKPVLHLP

[0076] The ORF sequence encoding the circPIAS1-108aa protein (SEQ ID NO:2):

[0077] GCCACCATGGATATTTCTGGGACCAAATGTGACTTCACAGTACAGGTCCAGTTAAGGTTTTGTTTATCAGAAACCAGTTGTCCACAAGAAGATCACTTCCCACCCAATCTTTGTGTGAAAGTGAATACAAAACCTTGCAGCCTTCCAGGTTACCTTCCACCTACAAAAAATGGCGTGGAACCAAAGCGACCCAGCCGACCAATTAATATCACCTCACTTGTCCGACTGTCCACAACAGTACCAAACACGATTGTTGTTTCTTGGACTGCAGAAATTGGAAGACATCAGACAACAGTCAGCGCTTTCGAGAAACCTGTTTTGCATTTGCCT

[0078] >hsa_circ_0008378 (SEQ ID NO:3)

[0079] CATCAGACAACAGTCAGCGCTTTCGAGAAACCTGTTTTGCATTTGCCTTGACACCACAACAAGTGCAGCAAATCAGTAGTTCCATGGATATTTCTGGGACCAAATGTGACTTCACAGTACAGGTCCAGTTAAGGTTTTGTTTATCAGAAACCAGTTGTCCACAAGAAGATCACTTCCCACCCAATCTTTGTGTGAAAGTGAATACAAAACCTTGCAGCCTTCCAGGTTACCTTCCACCTACAAAAAATGGCGTGGAACCAAAGCGACCCAGCCGACCAATTAATATCACCTCACTTGTCCGACTGTCCACAACAGTACCAAACACGATTGTTGTTTCTTGGACTGCAGAAATTGGAAGA

[0080] >hsa_circ_0008378-ATG-MUT (SEQ ID NO:4)

[0081] CATCAGACAACAGTCAGCGCTTTCGAGAAACCTGTTTTGCATTTGCCTTGACACCACAACAAGTGCAGCAAATCAGTAGTTCCACGGATATTTCTGGGACCAAATGTGACTTCACAGTACAGGTCCAGTTAAGGTTTTGTTTATCAGAAACCAGTTGTCCACAAGAAGATCACTTCCCACCCAATCTTTGTGTGAAAGTGAATACAAAACCTTGCAGCCTTCCAGGTTACCTTCCACCTACAAAAAACGGCGTGGAACCAAAGCGACCCAGCCGACCAATTAATATCACCTCACTTGTCCGACTGTCCACAACAGTACCAAACACGATTGTTGTTTCTTGGACTGCAGAAATTGGAAGA

[0082] Inhibitor siRNA#1 (SEQ ID NO:5): 5’-GCAGAAATTGGAAGACATC-3’

[0083] Inhibitor siRNA#2 (SEQ ID NO:6): 5’-ATTGGAAGACATCAGACAA-3’.

Claims

1. Use of a circPIAS1-108aa inhibitor in combination with oxaliplatin in the preparation of a drug for preventing and / or treating cancer.

2. Use according to claim 1, characterized in that, The circPIAS1-108aa inhibitor is a substance that inhibits the activity or expression of circPIAS1-108aa.

3. Use according to claim 2, characterized in that, The circPIAS1-108aa inhibitor is selected from artificially synthesized small interfering ribonucleic acid.

4. Use according to claim 1, characterized in that, The cancer is colon cancer.

5. The use according to claim 1, characterized in that, The circPIAS1-108aa inhibitor and oxaliplatin are used in a combined administration or sequential use manner.

6. Use according to any one of claims 1 to 5, characterized in that, The drug is prepared by taking the circPIAS1-108aa inhibitor and oxaliplatin as effective components, and adding pharmaceutically acceptable auxiliary components.

7. A pharmaceutical synergistic composition for preventing and / or treating cancer, characterized by, The effective components of the drug synergistic composition consist of the circPIAS1-108aa inhibitor and oxaliplatin.

8. The pharmaceutical synergistic composition according to claim 7, wherein The drug synergistic composition further comprises pharmaceutically acceptable auxiliary components.

9. The pharmaceutical synergistic composition according to claim 7, wherein The circPIAS1-108aa inhibitor is a substance that inhibits the activity or expression of circPIAS1-108aa; preferably, the circPIAS1-108aa inhibitor is selected from artificially synthesized small interfering ribonucleic acid.

10. The pharmaceutical synergistic composition according to claim 7, wherein The cancer is colon cancer.

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