Use of circpias1-108aa and inhibitors thereof in the preparation of antitumor drugs

Abstract

The present application relates to cancer, and particularly to the application of circPIAS1-108aa and its inhibitor in the preparation of antitumor drugs. The application of circPIAS1-108aa as a target in the development or screening or preparation of drugs for preventing or treating tumors is characterized in that the amino acid sequence of the circPIAS1-108aa is SEQ ID NO. 1. The present application finds the application of the inhibitor of the target circPIAS1-108aa in the treatment of cancer through experiments. The inhibitor plays a role in inhibiting proliferation in ten common cancers, including lung cancer, liver cancer, gastric cancer, melanoma, pancreatic cancer, breast cancer, nasopharyngeal carcinoma, colorectal cancer, esophageal cancer and prostate cancer; and the inhibitor of the target circPIAS1-108aa can significantly enhance the efficacy of immune checkpoint inhibitors, providing a new synergistic strategy for tumor immunotherapy.

Description

TECHNICAL FIELD

[0001] The present application relates to cancer, in particular to the application of circPIAS1-108aa and its inhibitor in the preparation of antitumor drugs. BACKGROUND

[0002] Cancer, also known as malignant tumor, is caused by cell malignant proliferation, most of which are invasive and metastatic. Cancer is diverse in types and complex in pathogenesis, and has become the main factor endangering people's life and health in China. The common types of cancer in clinical research in China include lung cancer, liver cancer, gastric cancer, melanoma, pancreatic cancer, breast cancer, nasopharyngeal carcinoma, colorectal cancer, esophageal cancer, prostate cancer, etc. These cancers have rapid onset and are difficult to control by conventional chemotherapy, so immunological checkpoint inhibitors are often used for targeted therapy, but the actual effect is still not good.

[0003] Circular non-coding RNAs (circRNAs) are a class of regulatory non-coding RNAs formed by reverse splicing of pre-mRNA, which have a circular covalent closed structure and can resist degradation by RNase, providing protection for their stable function in vivo. CircRNA plays many important roles in the occurrence and development of diseases, especially in the field of cancer, and plays a very key regulatory role. For example, hsa_circ_102481 is highly expressed in non-small cell lung cancer with EGFR-TKI resistance, which can adsorb miR-30a-50p through the molecular sponge mechanism, adjust the level of ROR1, and promote tumor proliferation and metastasis.

[0004] CircPIAS1 is a circRNA, and its function has not been reported in the literature. SUMMARY

[0005] The present application provides the application of target protein circPIAS1-108aa inhibitor in the treatment of cancer and the enhancement of the efficacy of immunological drug, and provides a basis for the development of drugs for treating cancer.

[0006] Technical scheme: In order to achieve the above-mentioned purpose of the application, the technical scheme of the present application is as follows:

[0007] The application of circPIAS1-108aa as a target in the development or screening or preparation of drugs for preventing or treating tumors, characterized in that the amino acid sequence of circPIAS1-108aa is SEQ ID NO. 1.

[0008] The application of the inhibitor of circPIAS1-108aa in the preparation of antitumor drugs.

[0009] The application, characterized in that the circPIAS1-108aa inhibitor is a substance that inhibits the activity or expression of circPIAS1-108aa.

[0010] The application, characterized in that the circPIAS1-108aa inhibitor includes small-molecule organic substances, small-molecule inorganic substances, anti-circPIAS1-108aa antibodies, nucleic acid fragments or polypeptides that can specifically bind to circPIAS1-108aa and inhibit its activity.

[0011] The application, characterized in that the circPIAS1-108aa inhibitor is selected from artificially synthesized small interfering ribonucleic acids siRNA or antisense oligonucleotides ASOs.

[0012] The application, characterized in that the tumor includes lung cancer, liver cancer, gastric cancer, melanoma, pancreatic cancer, breast cancer, nasopharyngeal cancer, colorectal cancer, esophageal cancer or prostate cancer.

[0013] The application of a pharmaceutical composition in the preparation of an anti-tumor drug, characterized in that the pharmaceutical composition comprises the circPIAS1-108aa inhibitor.

[0014] The application, characterized in that the pharmaceutical composition comprises a circPIAS1-108aa inhibitor and a pharmaceutically acceptable carrier.

[0015] Specifically:

[0016] The present application first discovers that the carcinogenic factor circPIAS1-108aa can be used as a cancer treatment target, therefore, substances that inhibit this target can be used for the treatment of cancer, and the target circPIAS1-108aa d inhibitor is a substance that inhibits the activity or expression of circPIAS1-108aa.

[0017] The target circPIAS1-108aa d inhibitor of the present application can inhibit the expression of circPIAS1-108aa protein or inhibit its activity / function, thereby being used for the prevention and / or treatment of cancer:

[0018] siRNA and / or ASOs that target circPIAS1 to promote its degradation;

[0019] Knock out circPIAS1 by gene knockout technology;

[0020] Blockers, antagonists or inhibitors that target circPIAS1-108aa protein and inhibit its function;

[0021] Inhibitors that inhibit the expression of the circPIAS1 gene or protein;

[0022] Neutralizing antibodies, blocking antibodies or blocking peptides of circPIAS1-108aa, but not limited to the above means.

[0023] As a further choice:

[0024] The target protein circPIAS1-108aa inhibitor includes, but is not limited to, small-molecule organic matter, small-molecule inorganic matter, anti-circPIAS1-108aa antibody, nucleic acid fragments or polypeptides that can specifically bind to circPIAS1-108aa and inhibit its activity.

[0025] The target protein circPIAS1-108aa inhibitor also includes an inhibitor precursor.

[0026] For example, the circPIAS1-108aa inhibitor can be selected from artificially synthesized small interfering ribonucleic acid (siRNA) or antisense oligonucleotide (ASOs).

[0027] The cancer includes all kinds of cancers, such as lung cancer, liver cancer, gastric cancer, melanoma, pancreatic cancer, breast cancer, nasopharyngeal carcinoma, colorectal cancer, esophageal cancer, prostate cancer, etc.

[0028] The target protein circPIAS1-108aa inhibitor can inhibit the growth of cancer and enhance the efficacy of immunotherapy, thereby preventing, improving or treating cancer.

[0029] Beneficial effects

[0030] Recent studies have shown that even if circRNA is defined as non-coding RNA, a small part of them still has the ability to encode proteins, and the polypeptides encoded by them have been confirmed to be key factors affecting tumor progression. Some studies have found that Circ-E-cadherin can promote the progression of glioblastoma, but interestingly it does not work through the miRNA molecular sponge mechanism, but through the ribosome to produce a 254-amino acid long polypeptide through multiple rounds of translation, to bind the CR2 domain of EGFR, thereby reactivating the EGFR downstream pathway, limiting the efficacy of EGFR inhibitors. circPIAS1 is also a non-coding RNA with the ability to encode proteins.

[0031] The function of circPIAS1 as a circRNA has not been reported in the literature. The present application unexpectedly found that circPIAS1 itself has no effect on promoting tumor proliferation, but the polypeptide circPIAS1-108aa expressed by it is a cancer-promoting factor, which is a novel target protein encoded by circPIAS1 with 108 amino acids. There is no report on inhibiting circPIAS1-108aa for treating cancer.

[0032] Specifically:

[0033] (1) circPIAS1-108aa is a key factor for promoting cancer progression. The presence and sequence correctness of the special protein (circPIAS1-108aa) encoded by circPIAS1 with 108 amino acids are identified by 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 by colony formation experiment. The key is to design siRNA or ASOs targeting inhibitors according to the loop site of circPIAS1 to specifically down-regulate the level of circPIAS1, thereby significantly inhibiting the growth of cancer cells.

[0034] The target sequence of the ASOs is as follows:

[0035] #1: 5'-GCAGAAATTGGAAGACATC-3', SEQ ID NO. 2

[0036] #2: 5'-ATTGGAAGACATCAGACAA-3', SEQ ID NO., 3

[0037] #3: 5'-GAAGACATCAGACAACAGT-3', SEQ ID NO., 4

[0038] #4: 5'-GACTGCAGAAATTGGAAGAC-3', SEQ ID NO., 5

[0039] #5: 5'-AGACATCAGACAACAGTCAG-3', SEQ ID NO., 6.

[0040] (2) The use of inhibitors targeting circPIAS1-108aa can significantly improve the anti-tumor effect of immune drugs. The anti-tumor effect of immune drugs is significantly improved when down-regulating the level of circPIAS1-108aa and giving immune drugs at the same time, compared with single administration of immune drugs.

[0041] The present application finds through experiments that the inhibitor of the target point circPIAS1-108aa has an application in cancer treatment. The inhibitor plays a role in inhibiting proliferation in ten common cancers, including lung cancer, liver cancer, gastric cancer, melanoma, pancreatic cancer, breast cancer, nasopharyngeal carcinoma, colorectal cancer, esophageal cancer and prostate cancer. The inhibitor of the target point circPIAS1-108aa can significantly enhance the efficacy of immune checkpoint inhibitors, providing a new synergistic strategy for tumor immunotherapy. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 circPIAS1 encodes circPIAS1-108aa verification results, wherein a is the circPrimer2.0 predicted circPIAS1 encoded protein sequence, b is the mass spectrometry detection of circPIAS1-108aa, showing that the target point circPIAS1-108aa really exists in tumor cells and is consistent with the predicted amino acid sequence.

[0043] Figure 2 The screening results of circPIAS1-108aa as a carcinogenic factor and a drug target point, wherein: a is the clonal formation experiment result graph, b is the relative quantification, showing that the target point circPIAS1 plays a role in promoting cancer mainly depending on the novel protein circPIAS1-108aa it encodes, rather than the function at the RNA level.

[0044] Figure 3 The in vitro efficacy results of the inhibitor of circPIAS1-108aa, wherein: a is the clonal formation experiment result graph, b is the relative quantification statistical result, showing the change of tumor cell proliferation ability after down-regulation of circPIAS1 by ASOs inhibitor.

[0045] Figure 4 The in vivo efficacy results of the inhibitor of circPIAS1-108aa, wherein: a is the schematic diagram of animal in vivo experiment method, b is the body weight change of mice after different treatments, c is the tumor tissue of different treatment groups, d is the relative quantification of tumor tissue size of different groups, showing that the inhibitor of the target point circPIAS1-108aa can significantly enhance the inhibitory effect of immune drugs on tumors, while not affecting the normal growth of mice; *P<0.05, **P<0.01. DETAILED DESCRIPTION

[0046] The present application will be described in detail below in combination with specific embodiments

[0047] H1975 (lung cancer), HepG2 (liver cancer), SGC-7901 (gastric cancer), A375 (melanoma), AsPC-1 (pancreatic cancer), T-47D (breast cancer), CNE-1 (nasopharyngeal carcinoma), HCT-116 (colorectal cancer), SKGT-4 (esophageal cancer), Du 145 (prostate cancer) cell lines used in the examples were provided by the Chinese Academy of Sciences Cell Library (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 ASOs used in the experiment were designed by the research group and synthesized by Guangzhou Ribo Biological Technology Co., Ltd.

[0048] Example 1 circPIAS1 was confirmed to encode a novel target protein of 108 amino acids

[0049] According to the prediction of circPrimer2.0 software, circPIAS1 (ID: hsa_circ_0008378) can encode a special polypeptide of 108 amino acids, and the protein sequence is:

[0050] MDISGTKCDFTVQVQLRFCLSETSCPQEDHFPPNLCVKVNTKPCSLPGYLPPTKNGVEPKRPSRPINITSLVRLSTTVPNTIVVSWTAEIGRLQTTASAFGKPVLHLP, see SEQ ID NO. 1.

[0051] The existence of circPIAS1-108aa was successfully verified by LC-MS / MS technology.

[0052] As shown in the results, Figure 1 circPIAS1-108aa truly exists in tumor cells and is consistent with the predicted amino acid sequence.

[0053] Example 2 circPIAS1-108aa is a key carcinogenic factor and drug target

[0054] In the colony formation experiment, ten kinds of tumor cells were inoculated into 6-well plates at a density of 2x103 cells per well. Vector (empty vector), circPIAS1-108aa (overexpression of RNA and protein levels), circPIAS1-ATG-mut (only overexpression of RNA level) three kinds of overexpression plasmids were transfected into tumor cells for 8 hours, then the culture medium was replaced with fresh culture medium and cultured for 7 days. Finally, the cell colonies were stained with crystal violet.

[0055] As shown in the results, Figure 2As shown, it indicates that circPIAS1 plays a pro-cancer role mainly depends on its encoded novel protein, rather than the RNA level function, and also proves that circPIAS1-108aa has a high target potential.

[0056] Example 3 The proliferation ability of tumor cells after using specific inhibitor of circPIAS1-108aa is significantly inhibited

[0057] In the colony formation experiment, ten tumor cells were inoculated into 6-well plates at a density of 2x10 3 cells per well. After 8 hours of transfection with ASOs of circPIAS1-108aa inhibitor, the culture medium was replaced with fresh culture medium and cultured for 7 days. Finally, the cell colonies were stained with crystal violet.

[0058] The results are shown in Figure 3 As shown, after down-regulating circPIAS1 by ASOs, the number of tumor cell clones is significantly reduced. It indicates that the inhibitor of circPIAS1 can significantly inhibit the growth of tumors.

[0059] Example 4 The target protein circPIAS1-108aa inhibitor can significantly enhance the anti-tumor effect of immune drugs

[0060] In this experiment, 6-8-week-old C57BL / 6J male and female mice (SPF) were used as research objects, and 2x10 5 B16-F10 cells were subcutaneously injected to construct a melanoma cancer model. When the tumor of C57 mice grew to 50mm 3 , it was divided into four groups for drug treatment, and this date was recorded as day 0. PD-1 inhibitor and IgG negative control, from day 0, every 3 days, 2mg / kg intraperitoneal injection, a total of five times. ASO-circPIAS1 and ASO-NC were used to specifically inhibit circPIAS1-108aa, from day 3, every 3 days, 10nmol intratumoral injection, a total of four times. During the period, the body weight and tumor volume of mice were observed and recorded, and finally the tumor tissues of each group were collected on day 14 for analysis.

[0061] The results are shown in Figure 4 As shown, using the inhibitor of target circPIAS1-108aa can significantly enhance the inhibitory effect of immune drugs on tumors.

Claims

1. Use of an inhibitor of circPIAS1-108aa in the preparation of an antitumor drug, characterized in that, the inhibitor of circPIAS1-108aa is an antisense oligonucleotide ASO; the antisense oligonucleotide ASO is any one of the following nucleotide sequences: #1: 5'-GCAGAAATTGGAAGACATC-3', SEQ ID NO. 2 #2: 5'-ATTGGAAGACATCAGACAA-3', SEQ ID NO. 3 #3: 5'-GAAGACATCAGACAACAGT-3', SEQ ID NO. 4 #4: 5'-GACTGCAGAAATTGGAAGAC-3', SEQ ID NO. 5 #5: 5'-AGACATCAGACAACAGTCAG-3', SEQ ID NO. 6; the tumor is lung cancer, liver cancer, gastric cancer, melanoma, pancreatic cancer, breast cancer, nasopharyngeal carcinoma, colorectal cancer, esophageal cancer or prostate cancer.

2. Use of a pharmaceutical composition in the preparation of an antitumor medicament, characterized in that, the pharmaceutical composition comprises the circPIAS1-108aa inhibitor of claim 1 and a pharmaceutically acceptable carrier; and the tumor is lung cancer, liver cancer, gastric cancer, melanoma, pancreatic cancer, breast cancer, nasopharyngeal carcinoma, colorectal cancer, esophageal cancer or prostate cancer.

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