Circular RNA (Ribonucleic Acid) as well as mutant and related application thereof

Abstract

The invention discloses a circular RNA (Ribonucleic Acid) as well as a mutant and related application thereof, and relates to the field of medicines. The invention discloses the new application of hsacirc0069982 for the first time, hsacirc0069982 can be used as an aptamer RNA (Ribonucleic Acid) medicine for resisting hnRNP family proteins, and tumor metastasis can be remarkably inhibited and chemotherapy resistance of tumors can be reversed by increasing the expression quantity of hsacirc0069982; it is found that hsacirc0069982 can directly interact with hnRNP M, depending on a hsacirc0069982 key area, by optimizing and modifying the sequence of the area, an enhanced mutant can be obtained, the sequence is shown as SEQ ID NO: 2-6, and the enhanced mutant shows the hnRNP M binding capacity higher than that of a wild type. Through verification, the hsacirc0069982 and the enhanced mutant thereof can effectively inhibit tumor metastasis and chemotherapy drug resistance, and a new way and means are provided for tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a circular RNA and its mutants and related applications. Background Art

[0002] Tumor metastasis and chemotherapy resistance are the primary factors in cancer mortality. The hnRNP family protein hnRNP M is highly expressed in common tumors such as breast cancer and lung cancer, and its abnormal activation drives tumor metastasis. The hnRNP family proteins widely regulate the gene alternative splicing events related to tumor metastasis. hnRNP M promotes tumor epithelial-mesenchymal transition (EMT) by initiating abnormal splicing of a series of key genes such as CD44 pre-mRNA, thereby promoting tumor metastasis and chemotherapy resistance. Knocking down the expression of hnRNP M using siRNA can significantly inhibit the EMT process of cells and effectively alleviate tumor metastasis and resistance indicators in mice. This shows that hnRNP M is a potential anti-metastatic drug target.

[0003] Currently, there is an urgent need for treatments for metastatic tumors. As key molecules driving tumor metastasis, hnRNP family proteins lack hydrophobic pockets for small molecules to bind to, making them difficult to be targeted by traditional small molecule drugs. Circular RNA, as an endogenous substrate of hnRNP family proteins, can be used as a nucleic acid aptamer to achieve specific targeted inhibition of hnRNPs, making it an ideal drug type.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a circular RNA and its mutants and related applications.

[0006] The present invention is achieved in that:

[0007] In a first aspect, an embodiment of the present invention provides a circular RNA, comprising: any one of circRNA1, circRNA2, circRNA3, circRNA4 and circRNA5; wherein the nucleotide sequences of the circRNA1, the circRNA2, the circRNA3, the circRNA 4 and the circRNA5 are shown in SEQ ID NOs: 2 to 6, respectively.

[0008] In a second aspect, an embodiment of the present invention provides a nucleic acid composition, comprising: at least two of circRNA1, circRNA2, circRNA3, circRNA4 and circRNA5; wherein the nucleotide sequences of the circRNA1, the circRNA2, the circRNA3, the circRNA 4 and the circRNA5 are shown in SEQ ID NOs: 2 to 6, respectively.

[0009] In a third aspect, an embodiment of the present invention provides an isolated nucleic acid encoding the circular RNA described in the preceding embodiment or the nucleic acid composition described in the preceding embodiment.

[0010] In a fourth aspect, an embodiment of the present invention provides a precursor RNA obtained by transcribing the isolated nucleic acid described in the preceding embodiment.

[0011] In a fifth aspect, an embodiment of the present invention provides a vector, which includes: the isolated nucleic acid described in the above embodiment.

[0012] In a sixth aspect, an embodiment of the present invention provides a host cell comprising the vector described in the preceding embodiment.

[0013] In a seventh aspect, an embodiment of the present invention provides a method for preparing the circular RNA or the nucleic acid composition as described in the preceding embodiments, which comprises: culturing the host cell as described in the preceding embodiments.

[0014] In an eighth aspect, an embodiment of the present invention provides a circular RNA derivative, which includes the modified circular RNA described in the preceding embodiment.

[0015] In the ninth aspect, an embodiment of the present invention provides a pharmaceutical composition, comprising: a pharmaceutically acceptable carrier and an active ingredient; the active ingredient comprises: a combination of any one or more of the circular RNA described in the preceding embodiments, the nucleic acid composition described in the preceding embodiments, the isolated nucleic acid described in the preceding embodiments, the precursor RNA described in the preceding embodiments, the vector described in the preceding embodiments, the host cell described in the preceding embodiments, and the circular RNA derivatives described in the preceding embodiments.

[0016] In the tenth aspect, an embodiment of the present invention provides an effective ingredient for preparing a product for targeted inhibition of hnRNP protein or for preventing or treating tumors, wherein the effective ingredient comprises any one or more combinations of the following: hsa_circ_0069982, an hsa_circ_0069982 derivative, the hsa_circ_0069982 pre-RNA, a nucleic acid molecule that can be transcribed by a host to form the pre-RNA and processed to form hsa_circ_0069982, the circular RNA described in the preceding embodiments, the nucleic acid composition described in the preceding embodiments, the isolated nucleic acid described in the preceding embodiments, the precursor RNA described in the preceding embodiments, the vector described in the preceding embodiments, the host cell described in the preceding embodiments, the circular RNA derivative described in the preceding embodiments, and the pharmaceutical composition described in the preceding embodiments.

[0017] The present invention has the following beneficial effects:

[0018] The present invention discloses for the first time a new use of circular RNA hsa_circ_0069982, which can be used as an aptamer RNA drug against hnRNP family proteins, and increasing the expression of hsa_circ_0069982 can significantly inhibit tumor metastasis; the present invention finds that hsa_circ_0069982 can directly interact with hnRNP M, depending on the nucleotide sequence of the key region of hsa_circ_0069982, and by optimizing and modifying the sequence of this region, an enhanced mutant can be obtained, the sequence is shown in SEQ ID NO: 2 to 6, and exhibits a stronger binding ability to hnRNP M than the wild-type hsa_circ_0069982. It has been verified that hsa_circ_0069982 and its enhanced mutants effectively inhibit tumor metastasis and chemotherapy resistance, providing new ways and means for tumor targeted therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0020] Figure 1is the interaction between hsa_circ_0069982 and hnRNP M; A is the pulldown experiment of the biotin-labeled probe (AntiSens Probe) targeting hsa_circ_0069982, and Coomassie Brilliant Blue is used to detect the interacting proteins of hsa_circ_0069982; B is the differential bands analyzed by mass spectrometry ( Figure 1 The main protein composition of the black box area in A); C is the RNA Pulldown experiment to verify the interaction between hsa_circ_0069982 and hnRNP family proteins; D is the RIP-PCR detection of the interaction between hnRNPM and hsa_circ_0069982;

[0021] Figure 2 The interaction between hsa_circ_0069982 mutant and hnRNP M; A is a pulldown experiment using a biotin-labeled probe to detect the interaction between the specific region of hsa_circ_0069982 and endogenous hnRNP M; B is a pulldown experiment targeting R 262-126 and R 4378-512 Mutation of key sites in the region was performed to synthesize biotin-labeled S1-S3 (based on R 262 -126 ) and S4-S5 (based on R 4378-512 ); C is the RNA Pulldown experiment to verify the WT probe (composed of R262-126 and R 4378-512 Equal amounts were mixed) and the interaction between S1-S5 and recombinant hnRNP M protein; D is based on the S1-S5 sequence combined into the full-length hsa_circ_0069982 sequence, and introduced into the cells, and RIP-qPCR was used to detect the interaction between hnRNP M and hsa_circ_0069982 mutants;

[0022] Figure 3 The anti-metastatic activity of hsa_circ_0069982 and its mutants; A is the Tranwell assay to detect the anti-metastatic activity of hsa_circ_0069982 in various tumor cells; B is the mutants (A1-A5) of hsa_circ_0069982 prepared by in vitro transcription / cyclization method; C is the in vitro anti-metastatic activity of hsa_circ_0069982 and its mutants; D is the in vivo anti-metastatic activity of hsa_circ_0069982 mutants. One-way ANOVA compares the differences between each group and Vehicle: *, P<0.01; **, P<0.0001. One-way ANOVA compares the differences between each mutant group and WT: #, P<0.001;

[0023] Figure 4 Figure 3 is the sensitizing effect of hsa_circ_0069982 and its mutants on chemotherapy-resistant cells; A is the sensitizing effect of hsa_circ_0069982 on chemotherapy-resistant cells of breast cancer, lung cancer, colorectal cancer and gastric cancer detected by MTT; B is the comparison of the IC50 enhancement effect of hsa_circ_0069982 and its mutants on chemotherapy-resistant cells of lung cancer, breast cancer and colorectal cancer. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0025] Circular RNA (circRNA) is a type of single-stranded RNA molecule that forms a covalently closed structure through "reverse shearing" and has high conservation and tissue / temporal expression specificity. Compared with linear RNA, circRNA is not easily degraded by nucleases because it has no 5' and 3' ends, and can be stably present in the tumor microenvironment, blood, urine, saliva and other body fluids through exosomes; and it has the characteristics of low immunogenicity, making it an ideal type of RNA drug. In terms of molecular mechanism, circRNA exerts biological functions by acting as a "miRNA sponge", "protein decoy", encoding small peptides, etc.

[0026] In view of the problems existing in the prior art, the inventors of the present application have discovered for the first time that hsa_circ_0069982 can effectively target hnRNP family proteins as an aptamer, which can occupy the RNA binding pocket of hnRNP family proteins and weaken the latter's ability to recognize other functional RNAs. As an aptamer, hsa_circ_0069982 itself has the advantages of high stability and low immunogenicity, and is an ideal type of RNA drug.

[0027] On the one hand, an embodiment of the present invention provides a circular RNA, comprising: any one of circRNA1, circRNA2, circRNA3, circRNA4 and circRNA5; wherein the nucleotide sequences of the circRNA1, the circRNA2, the circRNA3, the circRNA 4 and the circRNA5 are shown in SEQ ID NOs: 2 to 6, respectively.

[0028] The present invention example first discovered that hsa_circ_0069982 can directly interact with hnRNP M, depending on the nucleotide sequence of the key region of hsa_circ_0069982. hnRNP M is one of the most important molecules for regulating gene alternative splicing. hnRNP M promotes epithelial-mesenchymal transition and cancer metastasis of tumor cells by initiating alternative splicing reprogramming of downstream genes. The present invention found that hsa_circ_0069982 can significantly reverse the alternative splicing reprogramming downstream of hnRNP M, and mutating the key sequence of hsa_circ_0069982 can significantly change its regulation of alternative splicing events downstream of hnRNP M.

[0029] The embodiments of the present invention optimize and transform the sequence of the key region of hsa_circ_0069982 to obtain enhanced mutants 1 to 5 (circRNA1 to 5), the sequences of which are shown in SEQ ID NOs: 2 to 6. Mutants 1 to 5 exhibit stronger hnRNP M binding ability than wild-type hsa_circ_0069982. It has been verified that hsa_circ_0069982 has good anti-metastatic activity and can effectively improve or reverse the chemotherapy resistance of tumors. Mutants 1 to 5 have higher anti-metastatic activity than the wild type, can more effectively inhibit tumor metastasis, and more effectively improve or reverse the chemotherapy resistance of tumors, thereby improving the therapeutic effect of chemotherapy.

[0030] On the other hand, an embodiment of the present invention provides a nucleic acid composition, comprising: at least two of circRNA1, circRNA2, circRNA3, circRNA4 and circRNA5; wherein the nucleotide sequences of the circRNA1, the circRNA2, the circRNA3, the circRNA 4 and the circRNA5 are shown in SEQ ID NOs: 2 to 6, respectively.

[0031] In some embodiments, the at least two can be a combination of any two, any three, any four, and any five of circRNA1, circRNA2, circRNA 3, circRNA4, and circRNA5.

[0032] On the other hand, an embodiment of the present invention provides an isolated nucleic acid encoding the circular RNA described in the above embodiment or the nucleic acid composition described in any of the above embodiments.

[0033] On the other hand, an embodiment of the present invention provides a precursor RNA, which is obtained by transcription of the isolated nucleic acid described in the above embodiment. The precursor RNA can form the circular RNA (any one of circRNA1 to 5) described in the above embodiment by an in vitro or in vivo cyclization method. The in vitro cyclization method can specifically be chemical ligation, enzymatic ligation, or PIE cyclization based on self-splicing of type I introns.

[0034] On the other hand, an embodiment of the present invention provides a vector, which includes: the isolated nucleic acid described in the above embodiment.

[0035] The recombinant vector is an expression vector or a cloning vector, preferably an expression vector, and may refer to any recombinant polynucleotide construct, which may directly or indirectly (such as packaged into a virus) introduce the target DNA fragment into a host cell by means of transformation, transfection or transduction, and express the target gene. One type of vector is a plasmid, i.e., a circular double-stranded DNA molecule, which can connect the target DNA fragment to a plasmid ring. Another type of vector is a viral vector, which can connect and package the target DNA fragment into a viral genome (such as adenovirus, adeno-associated virus, retrovirus, slow virus, oncolytic virus). After these vectors enter the host cell, the target gene can be expressed.

[0036] On the other hand, an embodiment of the present invention provides a host cell comprising the vector described in the above embodiment.

[0037] On the other hand, an embodiment of the present invention provides a method for preparing the circular RNA as described in the above embodiment or the nucleic acid composition as described in the above embodiment, which comprises: culturing the host cell as described in the above embodiment.

[0038] On the other hand, an embodiment of the present invention provides a circular RNA derivative, which includes the modified circular RNA described in any of the aforementioned embodiments.

[0039] In some embodiments, the modification includes a combination of any one or more of the following modifications: sugar modification of nucleotides, modification of the connection mode between nucleotides, cholesterol modification, locked nucleotide modification, peptide modification, lipid modification, halogen modification, hydrocarbon modification and nucleic acid modification.

[0040] In some embodiments, the sugar modification of the nucleotide includes any one or more of 2-O-methyl sugar modification, 2-O-methoxyethyl sugar modification, 2-O-alkyl sugar modification, 2-fluoro sugar modification, sugar ring modification and locked nucleotide modification.

[0041] In some embodiments, the modification of the connection mode between the nucleotides includes any one or more of phosphorothioate modification and phosphoalkylation modification.

[0042] On the other hand, an embodiment of the present invention provides a pharmaceutical composition, comprising: a pharmaceutically acceptable carrier and an active ingredient; the active ingredient comprises: the circular RNA described in the preceding embodiment, the nucleic acid composition described in any preceding embodiment, the isolated nucleic acid described in any preceding embodiment, the precursor RNA described in any preceding embodiment, the vector described in any preceding embodiment, the host cell described in any preceding embodiment, and any one or more combinations of the circular RNA derivatives described in the preceding embodiments.

[0043] In some embodiments, the pharmaceutical composition further comprises a delivery vehicle for delivering the active ingredient.

[0044] In some embodiments, the delivery vehicle comprises any one or more of liposomes, liposome nanoparticles (LNPs), metal nanoparticles (AuNPs), hydrogels, and exosomes.

[0045] In some embodiments, the delivery vector includes: an unmodified targeting vector or a chemically modified targeting vector. In some embodiments, the chemical modification includes glycosylation / cholesterol modification to enhance target organ uptake, PEG-lipid modification to enhance stability, and modification with folic acid, glucose, PD-L1, HER2, EGFR, FGFR, or other specific ligands or receptors to enhance tumor cell targeting ability.

[0046] In some embodiments, the pharmaceutical composition further comprises an optional anti-tumor drug.

[0047] In some embodiments, the anti-tumor drug includes: any one or more of a chemotherapeutic agent and a multi-target kinase inhibitor. Optionally, the chemotherapeutic agent includes (but is not limited to): doxorubicin, epirubicin, etoposide, cisplatin, oxaliplatin. Optionally, the multi-target kinase inhibitor includes (but is not limited to) sorafenib, gefitinib, and defactinib.

[0048] In some embodiments, the pharmaceutically acceptable carrier includes, but is not limited to, water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof.

[0049] The pharmaceutical composition of the present invention can be formulated into any possible dosage form. In some embodiments, the dosage form is an injection. In other embodiments, the pharmaceutical composition is a nanoformulation.

[0050] On the other hand, an embodiment of the present invention also provides the use of an effective ingredient in preparing a product for targeted inhibition of hnRNP protein or for preventing or treating tumors, wherein the effective ingredient comprises any one or more combinations of the following: hsa_circ_0069982, an hsa_circ_0069982 derivative, the hsa_circ_0069982 pre-RNA, a nucleic acid molecule that can be transcribed by a host to form the pre-RNA and processed to form hsa_circ_0069982, the circular RNA as described in the preceding embodiments, the nucleic acid composition as described in any preceding embodiment, the isolated nucleic acid as described in any preceding embodiment, the precursor RNA as described in any preceding embodiment, the vector as described in any preceding embodiment, the host cell as described in any preceding embodiment, the circular RNA derivative as described in any preceding embodiment, and the pharmaceutical composition as described in any preceding embodiment.

[0051] In some embodiments, the nucleotide sequence of hsa_circ_0069982 is as shown in SEQ ID NO:1.

[0052] In some embodiments, the hsa_circ_0069982 derivative includes a modified hsa_circ_0069982. The modification is the same as the modification described in any of the preceding embodiments and will not be repeated here.

[0053] In some embodiments, the pre-RNA of hsa_circ_0069982 is selected from any one or more of ENST00000325278.7; ENST00000395759.6; ENST00000359107.10; ENST00000423607.6 and ENST00000429335.5. The pre-RNA can be processed in the host to form hsa_circ_0069982.

[0054] In some embodiments, the hnRNP protein comprises any one of hnRNP M, hnRNP L, hnRNP U, hnRNP K and hnRNP A2B1.

[0055] In some embodiments, the tumor includes any one or more of a primary tumor, a metastatic tumor, and a drug-resistant recurrent tumor.

[0056] In some embodiments, the tumor includes any one or more combinations of breast cancer, endometrial cancer, ovarian cancer, cervical cancer, lung cancer, liver cancer, kidney cancer, gastric cancer, intestinal cancer, and head and neck cancer.

[0057] In some embodiments, the resistance comprises resistance to a chemotherapeutic drug.

[0058] In some embodiments, the chemotherapy drugs include any one or more of doxorubicin, cisplatin and oxaliplatin.

[0059] "Treatment" as used herein includes preventing or alleviating a condition, reducing the rate at which a condition develops or progresses, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0060] For cancer, "treatment" can refer to inhibiting or slowing the growth, reproduction, or metastasis of tumors or malignant cells, or some combination of the above. For tumors, "treatment" includes eliminating all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of the tumor, or some combination of the above.

[0061] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0062] Example 1 Preparation of relevant materials

[0063] To obtain the full-length sequence of hsa_circ_0069982, the corresponding DNA sequence was amplified by PCR using cell cDNA as a template, and then inserted into the expression vector pLaccase2-puro by homologous recombination to obtain the overexpression plasmid pL-hsa_circ_0069982 for subsequent transfection and virus packaging; the parent gene of hsa_circ_0069982 and its flanking intron sequence were amplified by PCR using cell genomic DNA as a template, and then inserted into the T7 expression vector to obtain pT7-hsa_circ_0069982. TM The hsa_circ_0069982 precursor RNA sequence was obtained using the T7 transcription kit (Invitrogen, #AM1312) for subsequent transfection; the hsa_circ_0069982 sequence was cloned into the T7 expression vector and expressed using MEGAscript TMA large number of linear hsa_circ_0069982 molecules were obtained by in vitro transcription using the T7 transcription kit (Invitrogen, #AM1333). After purification by ethanol / ammonium acetate, they were annealed with complementary Splint sequences in vitro and ligated with T4 DNA ligase (NEB, #M0202T). The ligated products were treated with TUBO DNase and RNase R (YEASEN, #14606ES86) to remove the residual DNA template and linear RNA molecules, respectively. The products were purified by HPLC to obtain in vitro circularized hsa_circ_0069982 molecules for subsequent efficacy evaluation.

[0064] In order to obtain hsa_circ_0069982-related overexpression plasmid mutants, pL-hsa_circ_0069982 was used as a template, and primers containing different mutation sites were used for PCR amplification to obtain pL-hsa_circ_0069982 plasmids containing specific site mutations; in order to prepare hsa_circ_0069982 mutant molecules in vitro, pT7-hsa_circ_0069982 was used as a template, and primers containing different mutation sites were used for PCR amplification to obtain pT7-hsa_circ_0069982 plasmids containing specific site mutations, and then the in vitro transcription / circularization steps were performed according to the above process to obtain hsa_circ_0069982 mutant molecules (A1~A5, the sequences are shown in SEQ ID NO:2~6 respectively).

[0065] Example 2 hsa_circ_0069982 and its mutants as aptamers binding hnRNP M

[0066] To verify the function of hsa_circ_0069982 as an aptamer of hnRNP M, this example used a biotin-labeled probe specifically targeting the cyclization site of hsa_circ_0069982 to perform RNA pulldown coupled protein spectrum experiments, confirming that hnRNP M, hnRNP R, and hnRNP U are the main interacting proteins of hsa_circ_0069982 ( Figure 1 AB in Figure ), and hnRNP M has the strongest binding to hsa_circ_0069982 ( Figure 1 Subsequently, RNA-binding protein immunoprecipitation coupled with quantitative PCR (RIP-PCR) was performed using anti-hnRNP M antibody to confirm the endogenous interaction between hnRNP M and hsa_circ_0069982 ( Figure 1 ).

[0067] As an RNA binding protein, hnRNP M can specifically bind to RNA molecules rich in GU motifs. To this end, this example analyzed the nucleotide sequence of hsa_circ_0069982 by bioinformatics to find the potential region (R1 11-61 , R2 62-126 , R3 217-317 , R4 378-512 , R5 580-662 ; The superscript numbers represent their base positions in hsa_circ_0069982) synthesized probes to confirm region R2 62-126 and R4 378-512 Has high affinity for hnRNP M ( Figure 2 A); at the same time, area R2 62-126 and R4 378-512 Site-directed mutagenesis of key motifs in Figure 2 B) to verify its affinity with endogenous hnRNP M ( Figure 2 C); In view of the fact that the base mutations in S1-S5 can enhance the binding with hnRNP M, the present invention constructed hsa_circ_0069982 mutant isomers (circRNA1-5, the sequences are shown in SEQ ID NO: 2-6 respectively) containing the above mutation sites based on S1-S5, synthesized the hsa_circ_0069982 mutants and introduced them into H1299 cells, and verified the binding ability of the mutants with hnRNP M by RIP-qPCR.

[0068] Example 3 Pharmacodynamics and safety evaluation of hsa_circ_0069982 and its mutants

[0069] The RNA delivery system (RNA-LNP) was constructed using lipid nanoparticles (LNP), the most mature nucleic acid drug carrier currently studied; Tranwell experiments in various tumor cells confirmed that hsa_circ_0069982 can effectively inhibit tumor migration ( Figure 3 A in the figure). Subsequently, the hsa_circ_0069982 mutants (A1 to A5) with modified hnRNP M binding key sites were subjected to in vitro transcription / circularization ( Figure 3 B), the circular RNA prepared in vitro by LNP was transfected into tumor cells, and the anti-migration ability of the mutants was evaluated by Tranwell assay. The results showed that compared with the wild-type hsa_circ_0069982, the anti-migration activity of A1 to A5 mutants was improved to varying degrees ( Figure 3C in Figure ). Subsequently, the anti-metastatic activity of hsa_circ_0069982 and its mutants was further confirmed in a breast cancer tail vein lung metastasis model ( Figure 3 D).

[0070] Example 4 Sensitization effect of hsa_circ_0069982 and its mutants on chemotherapy-resistant tumors

[0071] The RNA-LNP in Example 3 was used to deliver hsa_circ_0069982 and its mutants. First, the wild-type hsa_circ_0069982 was transfected, and the drug half-inhibitory concentration (IC50) was calculated using MTT in a variety of chemotherapy-resistant tumor cells (MDA-MB-231 / Dox, A549 / DDP, HCT116 / Oxaliplatin, SGC7901 / DDP) to evaluate its sensitization effect on the drug-resistant phenotype.

[0072] Among them, Dox is doxorubicin, DDP is cisplatin, and Oxaliplatin is oxaliplatin.

[0073] The results showed that wild-type hsa_circ_0069982 had a certain degree of improvement in the drug resistance of various tumor cells ( Figure 4 A in the figure). Subsequently, the above experiment was repeated with hsa_circ_0069982 mutants to compare the improvement effect of mutants on the IC50 of drug-resistant strains. It was found that compared with wild-type hsa_circ_0069982, hsa_circ_0069982 mutants (A1-A5) with modified hnRNP M binding key sites could further reduce the IC50 of drug-resistant strains ( Figure 4 The above results indicate that hsa_circ_0069982 can improve the chemotherapy resistance of drug-resistant cells, and its mutant can further enhance the above effect.

[0074] The sequence information designed in this application is as follows.

[0075]

[0076]

[0077]

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A circular RNA, characterized in that It includes: any one of circRNA1, circRNA2, circRNA 3, circRNA4 and circRNA5; wherein the nucleotide sequences of the circRNA1, the circRNA 2, the circRNA3, the circRNA4 and the circRNA5 are shown in SEQ ID NOs: 2 to 6, respectively.

2. A nucleic acid composition, characterized in that It comprises: at least two of circRNA1, circRNA 2, circRNA 3, circRNA4 and circRNA5; wherein the nucleotide sequences of the circRNA1, the circRNA 2, the circRNA3, the circRNA4 and the circRNA5 are shown in SEQ ID NOs: 2 to 6, respectively.

3. An isolated nucleic acid, characterized in that It encodes the circular RNA according to claim 1 or the nucleic acid composition according to claim 2.

4. A precursor RNA, characterized in that It is obtained by transcription of the isolated nucleic acid according to claim 3.

5. A carrier, characterized in that It includes: The isolated nucleic acid of claim 3.

6. A host cell, characterized in that It contains the vector according to claim 4.

7. The method for preparing the circular RNA according to claim 1 or the nucleic acid composition according to claim 2, characterized in that: It includes: Cultivate the host cell according to claim 6.

8. A circular RNA derivative, characterized in that It includes: The modified circular RNA according to claim 1; Optionally, the modification includes any one or more of the following modifications: sugar modification of nucleotides, modification of the connection between nucleotides, cholesterol modification, locked nucleotide modification, peptide modification, lipid modification, halogen modification, hydrocarbon modification and nucleic acid modification; Optionally, the sugar modification of the nucleotide includes any one or more of 2-O-methyl sugar modification, 2-O-methoxyethyl sugar modification, 2-O-alkyl sugar modification, 2-fluoro sugar modification, sugar ring modification and locked nucleotide modification; Optionally, the modification of the connection mode between the nucleotides includes any one or more of phosphorothioate modification and phosphoalkylation modification.

9. A pharmaceutical composition, characterized in that It includes: Pharmaceutically acceptable carriers and active ingredients; The active ingredient comprises: any one or more combinations of the circular RNA of claim 1, the nucleic acid composition of claim 2, the isolated nucleic acid of claim 3, the precursor RNA of claim 4, the vector of claim 5, the host cell of claim 6, and the circular RNA derivative of claim 8; Optionally, the pharmaceutical composition further comprises a delivery vehicle for delivering the active ingredient; Optionally, the delivery vehicle comprises any one or more of liposomes, liposome nanoparticles, metal nanoparticles, hydrogels and exosomes.

10. Use of an active ingredient in the preparation of a product for targeted inhibition of hnRNP protein or for the prevention or treatment of tumors, characterized in that: The active ingredient comprises any one or more of the following combinations: hsa_circ_0069982, hsa_circ_0069982 derivatives, hsa_circ_0069982 pre-RNA, a nucleic acid molecule that can be transcribed by a host to form the pre-RNA and processed to form hsa_circ_0069982, the circular RNA according to claim 1, the nucleic acid composition according to claim 2, the isolated nucleic acid according to claim 3, the precursor RNA according to claim 4, the vector according to claim 5, the host cell according to claim 6, the circular RNA derivative according to claim 8, and the pharmaceutical composition according to claim 9; Optionally, the tumor includes: any one or more of a primary tumor, a metastatic tumor, and a drug-resistant recurrent tumor; Optionally, the tumor includes any one or more combinations of breast cancer, endometrial cancer, ovarian cancer, cervical cancer, lung cancer, liver cancer, kidney cancer, gastric cancer, intestinal cancer, and head and neck cancer.

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