A peptide circCCDC7 241aa Application in the treatment of prostate cancer

Abstract

The present invention provides a polypeptide circCCDC7 241aa For use in the treatment of prostate cancer, the polypeptide comprises the amino acid sequence shown in SEQ ID NO. 2; or a sequence comprising the amino acid sequence shown in SEQ ID NO. 2 with one or more amino acids substituted, deleted, or added, and having the same function. The present invention discovered that the polypeptide acts on the SLC7A11 protein of tumor cells, inducing ferroptosis in prostate cancer cells by ubiquitination and degradation of the SLC7A11 protein, thereby effectively inhibiting the growth and progression of prostate cancer without significantly damaging normal tissues. While maintaining excellent anti-tumor properties, it also makes up for the shortcomings of traditional chemotherapy drugs with many side effects, and has good prospects for clinical translation.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and specifically relates to a polypeptide circCCDC7 241aa Application in the treatment of prostate cancer. Background Art

[0002] Prostate cancer is the most common malignant tumor of the urinary system in my country and ranks second in mortality among male malignant tumors. Traditional treatments include surgery, endocrine therapy, chemotherapy, and radiotherapy. Surgery primarily targets localized tumors; endocrine therapy can be used for localized tumors and those with distant metastases; and chemotherapy is primarily used for distant metastases and castration-resistant prostate cancer.

[0003] Current treatments have limitations: surgical treatment has limited indications and is only effective for localized prostate cancer; endocrine therapy can easily progress to castration-resistant prostate cancer after 2-3 years of treatment; radiotherapy, often used as a palliative strategy for advanced tumors, fails to eradicate the lesion and can easily cause complications such as damage to surrounding tissues; and chemotherapy drugs lack tumor specificity and can easily kill and damage normal cells. Therefore, there is an urgent need to identify new drugs for the treatment of prostate cancer that can improve their efficacy and target prostate cancer targets. Summary of the Invention

[0004] Based on this, the purpose of the present invention is to provide a polypeptide circCCDC7 241aa Application in the treatment of prostate cancer. The polypeptide is circCCDC7 19-13 It is encoded and can effectively kill prostate cancer cells, inhibit the growth and migration of prostate cancer, and is safe and has no obvious side effects.

[0005] To achieve the above-mentioned purpose, the present invention adopts the following technical solutions.

[0006] A polypeptide for use in preparing a drug for treating prostate cancer, wherein the polypeptide comprises the amino acid sequence shown in SEQ ID NO. 2; or a sequence comprising the amino acid sequence shown in SEQ ID NO. 2 with one or more amino acids substituted, deleted, or added and having the same function.

[0007] The present invention also provides the use of an agent for promoting polypeptide expression in the preparation of a drug for treating prostate cancer, wherein the polypeptide comprises the amino acid sequence shown in SEQ ID NO.2; or a sequence comprising the amino acid sequence shown in SEQ ID NO.2 with one or more amino acids substituted, deleted, or added, and having the same function.

[0008] In some embodiments, the agent that promotes the expression of the polypeptide is circCCDC719-13 Recombinant expression vector; the circCCDC7 19-13 The recombinant expression vector is circCCDC7 19-13 The recombinant expression vector is formed after double enzyme digestion and ligation of the nucleotide and the eukaryotic expression vector; the circCCDC7 19-13 The nucleotide sequence is:

[0009] (1) the nucleotide sequence shown in SEQ ID NO: 1; or

[0010] (2) a sequence that is completely complementary to the nucleotide sequence shown in SEQ ID NO: 1; or

[0011] (3) A sequence having the same function as the nucleotide sequence shown in SEQ ID NO: 1, wherein one or more nucleotides are replaced, deleted or added.

[0012] In some embodiments, the polypeptide or agent can inhibit the proliferation, invasion, or migration of prostate cancer cells.

[0013] In some embodiments, the polypeptide or agent can induce ferroptosis in prostate cancer cells.

[0014] In some embodiments, the polypeptide or agent can induce ferroptosis in prostate cancer cells by ubiquitination and degradation of SLC7A11 protein.

[0015] In some embodiments, the drug is in the form of tablets, injections, capsules, granules, pills, syrups, powders or solutions.

[0016] The present invention also provides a drug for treating prostate cancer, comprising an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient is a polypeptide and / or an agent that promotes the expression of the polypeptide;

[0017] The polypeptide comprises the amino acid sequence shown in SEQ ID NO. 2; or a sequence comprising the amino acid sequence shown in SEQ ID NO. 2 with one or more amino acids substituted, deleted or added, and having the same function;

[0018] The agent for promoting polypeptide expression is circCCDC7 19-13 Recombinant expression vector; the circCCDC7 19-13 The recombinant expression vector is circCCDC7 19-13 The recombinant expression vector is formed after double enzyme digestion and ligation of the nucleotide and the eukaryotic expression vector; the circCCDC7 19-13 The nucleotide sequence is:

[0019] (1) the nucleotide sequence shown in SEQ ID NO: 1; or

[0020] (2) a sequence that is completely complementary to the nucleotide sequence shown in SEQ ID NO: 1; or

[0021] (3) A nucleotide sequence as shown in SEQ ID NO: 1 with one or more nucleotides substituted, deleted or added, and having the same function.

[0022] In some embodiments, the auxiliary material includes at least one of an excipient, a filler, a bulking agent, a binder, a humectant, a disintegrant, a solubilizing agent, an adsorbent, a diluent, a solubilizer, an emulsifier, a lubricant, a wetting agent, a suspending agent, a flavoring agent, and a fragrance.

[0023] The present invention also provides a method for detecting circCCDC7 19-13 Use of a reagent for detecting the expression level of circCCDC7 in the preparation of a prostate cancer detection product and / or a prostate cancer prognosis assessment product, characterized in that the circCCDC7 19-13 The nucleotide sequence is:

[0024] (1) the nucleotide sequence shown in SEQ ID NO: 1; or

[0025] (2) a sequence that is completely complementary to the nucleotide sequence shown in SEQ ID NO: 1; or

[0026] (3) A nucleotide sequence as shown in SEQ ID NO: 1 with one or more nucleotides substituted, deleted or added, and having the same function.

[0027] In some embodiments, the above detection of circCCDC7 19-13 The reagent for measuring the expression level comprises an upstream primer as shown in SEQ ID NO.3 and a downstream primer as shown in SEQ ID NO.4: SEQ ID NO.3: 5'-ACAAACTTACCAAGCAGCAG-3'; SEQ ID NO.4: 5'-CACAGCTTTCTTTGTTCGAT-3'.

[0028] This study first discovered circCCDC7 19-13 The expression of circCCDC7 in prostate cancer tissues was lower than that in significant adjacent tissues and was positively correlated with OS and PFS of prostate cancer, suggesting that circCCDC7 19-13 It is an independent prognostic factor for prostate cancer and can be used for auxiliary diagnosis and prognosis evaluation of prostate cancer. 19-13 It can significantly inhibit the proliferation, invasion and migration of prostate cancer. Overexpression of circCCDC719-13 Afterwards, intracellular iron and ROS (reactive oxygen species) accumulated in prostate cancer cells, lipid peroxidation levels increased, mitochondria shrank, and mitochondrial ridges disappeared, which are consistent with the characteristics of ferroptosis.

[0029] Furthermore, the inventors also found that circCCDC7 19-13 Can encode a polypeptide circCCDC7 241aa It acts on the SLC7A11 protein in tumor cells, induces ferroptosis in prostate cancer cells by ubiquitinating and degrading the SLC7A11 protein, thereby effectively inhibiting the growth and progression of prostate cancer. It has no obvious killing effect on normal tissues. While ensuring good anti-tumor properties, it also makes up for the shortcomings of traditional chemotherapy drugs with many side effects, and has good prospects for clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram for finding and predicting circular RNA-encoded proteins.

[0031] Figure 2 Validation of circCCDC7 for clinical samples and Asian clinical cohorts 19-13 The expression of circCCDC7 and its correlation with prognosis; Among them, AC: 31 pairs of cancer and adjacent tissues were used to verify the expression of circCCDC7 19-13 Expression of circCCDC7; DE: Validation of circCCDC7 in 134 pairs of tumor and matched normal tissues 19-13 Expression of; F: survival curve.

[0032] Figure 3 To verify circCCDC7 19-13 Result diagram of protein encoding ability; A: WB test results; B: immunofluorescence test results; C: WB test results; D: mass spectrometry test results.

[0033] Figure 4 To overexpress circCCDC7 19-13 The results of significantly inhibiting the proliferation, invasion and migration of prostate cancer cells are shown in the figure; A: clone formation ability test result; B: migration ability test result; C: cell proliferation ability test result; D: cell apoptosis test result; F: cell cycle test result.

[0034] Figure 5 To overexpress circCCDC7 19-13 Transcriptome sequencing and bioinformatics analysis results of cell lines; A: Volcano plot of differentially expressed genes; B: GO analysis; C: KEGG analysis; D: GO analysis.

[0035] Figure 6Figure 1 is a result diagram for verifying the ferroptosis phenotype; A: ROS and lipid peroxidation level detection results; B: mitochondrial membrane potential detection results; C: cell death rate detection results; D: intracellular ferrous iron detection results; E: intracellular GSH detection results; F: MDA detection results; G: mitochondrial detection results.

[0036] Figure 7 circCCDC7 241aa Results of the interaction study with SLC7A11 protein; A: IP and mass spectrometry detection results; B: ELISA detection results; C: co-culture immunofluorescence detection results.

[0037] Figure 8 circCCDC7 241aa Figure 2: Research results of downstream molecules regulated at the translation level; A: downstream molecule mRNA detection results; B: downstream molecule protein detection results; C: circCCDC7 241aa Results of IP detection of interaction with SLC7A11.

[0038] Figure 9 circCCDC7 241aa Results of significantly inhibiting prostate cancer progression in vivo; A: tumor growth detection results in mice; B: tumor weight detection results; C: tumor volume detection results. DETAILED DESCRIPTION

[0039] The experimental methods in the following examples of the present invention, where specific conditions are not specified, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0040] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0041] The terms "comprise," "comprising," and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product, or device comprising a series of steps is not limited to the listed steps or modules but may optionally include steps not listed, or other steps inherent to the process, method, product, or device.

[0042] In this application, "plurality" refers to two or more. "And / or" describes the relationship between related objects, indicating that three possible relationships exist. For example, "A and / or B" can mean: A exists alone, A and B exist simultaneously, or B exists alone. The character " / " generally indicates that the related objects are in an "or" relationship.

[0043] The following describes the details in conjunction with specific embodiments.

[0044] Example 1

[0045] To explore the mechanism of prostate cancer occurrence and development, we performed high-throughput sequencing on paired prostate cancer clinical samples of different malignant degrees and found that Circ CCDC7 expression was significantly reduced in prostate cancer tissues. CCDC7 (Coiled-Coil Domain Containing 7) encodes a 7-helical domain protein. We previously found that CCDC7 19-13 There are two forms: Linear CCDC7 19-13 and circCCDC7 19-13 In-depth research revealed that circCCDC7 19-13 It may encode a novel polypeptide that is lowly expressed in the human hormone-insensitive cell lines DU145 and PC3. 19-13 Affects the progression of prostate cancer by encoding polypeptides and secreting them into the extracellular space ( Figure 1 ).

[0046] circCCDC7 19-13 The nucleotide sequence is shown in SEQ ID NO.1:

[0047] SEQ ID NO.1: 5’-TAAGCCTACAAATAATCGAACAAAGAAAGCTGTGAAAACAGTGAAGAAAAAAGACAAAGGAAAATCTGAGGATTCAGAAAAGAAGATGTCTCCAGAAAAAGAGTTTAAAATAAAAGAAGATTTGGATCAAGTACAGAAAGTAGCACGTCTGGAAATTGAGAACAAAGTCCTTCAGGAGCAATTGAAACAGGCTTTACAGGAAGCTGAAAAAGCTAAGCATCAACTTAACTATTTCCTAAATCAAGAGAAGTTACTTAAAAGTGAGGGGAAAACTGAGACAACAATGCAAGTGGGTAATAGTCAAACAAAAGTTAAAGGTGAAGATTCAAAAAATATACCATTGGAGAAAGAAACAAGAAAATCACTGGTTTCAGATTCAGGTGGACAAAGGACAAGTGATAAAATCCAAGAATATCCACAGATCACTGCCCAAAGCGGAAGACTGATTGAAAAGAGCTCAGAGAAGAAACGATCTAGCCCTGCTATTTCAGATCTTTCACAGATCCTTAAGTCTCAAGATGAATCAGCATTTTTAGAGAGTTCAAATGAAGTTTCAGTTGCTGAAAACCAATCCTATAAATCTCCATCAGAGACCCATGATAAATCACTTACAACAGTATCATCCAGCAAAGAAGTTCAAGATTCACTGTCTGTTGGAACATTGGCTCAAAAAAACGAAACAGTGATATCACCATTCATTTTACCTCCTGTTCTTACAGAAAGTAAAAAGGCTGATGTTTCAGAAGAACAATTACAAAAGATGACTGAAGAACAAACTTACCAAGCAGCAGAAAAATCTCAAG-3’。

[0048] I. circCCDC7 19-13 Its expression in tumor tissues is lower than that in adjacent tissues

[0049] 1. Detect the expression of circCCDC7 19-13 in prostate cancer tissues and adjacent tissues

[0050] qPCR was used to validate circCCDC7 in 31 pairs of cancer and adjacent normal tissues from our hospital and 134 pairs of tumor and matched normal tissues from Changhai Hospital. 19-13 The expression of the gene was detected using a SYBR kit, and the operation steps were carried out according to the kit instructions. The detection primers included the upstream primer shown in SEQ ID NO.4 and the downstream primer shown in SEQ ID NO.5: SEQ ID NO.4: 5'-ACAAACTTACCAAGCAGCAG-3'; SEQ ID NO.5: 5'-CACAGCTTTCTTTGTTCGAT-3'.

[0051] like Figure 2 As shown, 31 pairs of cancer and adjacent tissue clinical samples from our hospital ( Figure 2 AC) and 134 pairs of tumor and matched normal tissues from Changhai Hospital ( Figure 2 DE) confirmed circCCDC7 19-13 The expression of TNF-α in prostate cancer tissue is significantly lower than that in adjacent tissues, and is positively correlated with OS and PFS of prostate cancer ( Figure 2 F), suggesting that circCCDC7 19-13 It is an independent prognostic factor for prostate cancer.

[0052] 2. circCCDC7 19-13 The expressive potential

[0053] To study circCCDC7 19-13 The present invention has the potential to express circCCDC7 19-13 The Flag plasmid sequence was inserted before the predicted stop codon of the nucleotide sequence for the detection of circCCDC7 19-13 The corresponding detection antibody is CST's FLAG@M2 antibody #14793. The recombinant expression vector was constructed and transfected into prostate cancer cell lines PC3 and DU145. Western blotting, immunofluorescence, and mass spectrometry were used for analysis.

[0054] 1. Construction and transfection of fusion sequence carrying Flag plasmid

[0055] The artificially synthesized sequence is shown as nucleotide sequence SEQ ID NO.3 (fusion sequence carrying Flag plasmid):

[0056] SEQ ID NO.3: 5’-AAAACGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGTAAGCCTACAAATAATCGAACAAAGAAAGCTGTGAAAACAGTGAAGAAAAAAGACAAAGGAAAATCTGAGGATTCAGAAAAGAAGATGTCTCCAGAAAAAGAGTTTAAAATAAAAGAAGATTTGGATCAAGTACAGAAAGTAGCACGTCTGGAAATTGAGAACAAAGTCCTTCAGGAGCAATTGAAACAGGCTTTACAGGAAGCTGAAAAAGCTAAGCATCAACTTAACTATTTCCTAAATCAAGAGAAGTTACTTAAAAGTGAGGGGAAAACTGAGACAACAATGCAAGTGGGTAATAGTCAAACAAAAGTTAAAGGTGAAGATTCAAAAAATATACCATTGGAGAAAGAAACAAGAAAATCACTGGTTTCAGATTCAGGTGGACAAAGGACAAGTGATAAAATCCAAGAATATCCACAGATCACTGCCCAAAGCGGAAGACTGATTGAAAAGAGCTCAGAGAAGAAACGATCTAGCCCTGCTATTTCAGATCTTTCACAGATCCTTAAGTCTCAAGATGAATCAGCATTTTTAGAGAGTTCAAATGAAGTTTCAGTTGCTGAAAACCAATCCTATAAATCTCCATCAGAGACCCATGATAAATCACTTACAACAGTATCATCCAGCAAAGAAGTTCAAGATTCACTGTCTGTTGGAACATTGGCTCAAAAAAACGAAACAGTGATATCACCATTCATTTTACCTCCTGTTCTTACAGAAAGTAAAAAGGCTGATGTTTCAGAAGAACAATTACAAAAGATGACTGAAGAACAAACTTACCAAGCAGCAGAAAAATCTCAAG-3’。

[0057] The synthetically obtained nucleotides and the Plenti-Cir vector were double digested (5’ end: EcoRI; 3’ end: BamHI), and then ligated to obtain circCCDC7. 19-13Overexpression vector containing Flag sequence. 19-13 The overexpression vector and the control vector Plenti-Cir were co-transfected with the lentiviral packaging plasmid into 293T cells. After 48 h of transfection, the supernatant was collected, concentrated and purified to obtain the circCCDC7-containing 19-13 Virus solutions containing overexpression vector and control vector sequences.

[0058] PC3 and DU145 cells were routinely cultured, and the virus solution was used to infect PC3 and DU145 cells, and stable expression strains were obtained by screening.

[0059] Western blotting detection: collecting circCCDC7 19-13 The overexpression cell lines and control stable expression cell lines were lysed to extract proteins and then analyzed by Western blotting.

[0060] Immunofluorescence detection: collecting circCCDC7 19-13 For overexpression cell lines and control stable expression cell lines, cell slides were prepared and then immunofluorescence detection was performed.

[0061] Mass spectrometry detection: To verify the existence of predicted encoded proteins in nature, supernatants from wild-type 293T cells and PC3 cells were collected, concentrated and lyophilized, and verified by mass spectrometry.

[0062] like Figure 3 As shown, Western blotting confirmed circCCDC7 19-13 Can encode protein ( Figure 3 A). Immunofluorescence results suggest that the encoded novel protein is mainly located in the cytoplasm ( Figure 3 B). The supernatant of the concentrated and freeze-dried wild-type cells was verified by mass spectrometry to detect the corresponding amino acid sequence at the predicted molecular weight position, confirming the existence of the predicted exocrine protein encoded in nature ( Figure 3 CD).

[0063] The above results confirmed that circCCDC7 19-13 It has the potential to encode a polypeptide and the encoded polypeptide is localized in the Golgi apparatus. In the present invention, the polypeptide is named circCCDC7 241aa , which comprises the amino acid sequence shown in SEQ ID NO.2:

[0064] SEQ ID NO.2: MSPEKEFKIKEDLDQVQKVARLEIENKVLQEQLKQALQEAEKAKHQLNYFLNQEKLLKSEGKTETTMQVGNSQTKVKGEDSKNIPLEKETRKSLVSDSGGQRTSDKIQEYPQITAQSG RLIEKSSEKKRSSPAISDLSQILKSQDESAFLESSNEVSVAENQSYKSPSETHDKSLTTVSSSKEVQDSLSVGTLAQKNETVISPFILPPVLTESKKADVSEEQLQKMTEEQTYQAAEKSQEN.

[0065] Example 2

[0066] This example studies the overexpression of circCCDC7 19-13 Effects and mechanisms on prostate cancer cells.

[0067] The following experimental groups were set up: PC3 cells circCCDC7 19-13 Overexpression group, DU145 cells circCCDC7 19-13 Overexpression group, PC3 cell control vector group, DU145 cell control vector group. The overexpression group is indicated by 19-13 OE, and the control vector group is indicated by VECTOR.

[0068] PC3 cells and DU145 cells were cultured and infected according to the method described in Example 1. After 48 h of infection, cell proliferation and viability were detected using a CCK8 kit in strict accordance with the instructions. Clone formation ability was detected using crystal violet staining. Migration ability was detected using a scratch wound test. Cell cycle and apoptosis were detected using flow cytometry.

[0069] like Figure 4 As shown, circCCDC7 in PC3 cells 19-13 Overexpression group, DU145 cells circCCDC7 19-13 The proliferation, invasion and migration abilities of the overexpression group were significantly lower than those of the PC3 cell control vector group and the DU145 cell control vector group ( Figure 4 A~C), but there was no significant change in cell cycle and cell apoptosis ( Figure 4 D) Overexpression of circCCDC7 in prostate cancer cells 19-13 It can significantly inhibit the proliferation, invasion and migration of prostate cancer, but there is no statistical difference in the effect on cell cycle and cell apoptosis.

[0070] Next, we overexpressed circCCDC7 19-13Transcriptome sequencing and bioinformatics analysis of cells revealed that overexpression of circCCDC7 19-13 Pathways related to post-oxidative stress, lipid peroxidation, and iron transport were significantly activated ( Figure 5 ): Figure 5 A is the volcano plot of differentially expressed genes; Figure 5 B-D are the results of KEGG and GO analysis, indicating overexpression of circCCDC7 19-13 After that, the differentially expressed genes were enriched in pathways such as lipid metabolism, iron transport, and mitochondrial activity. Therefore, we speculated that circCCDC7 19-13 Does it function by inducing tumor ferroptosis?

[0071] Furthermore, we verified the ferroptosis phenotype.

[0072] 1. Detection of ROS content and lipid peroxidation levels

[0073] The following experimental groups were set up: PC3 cells circCCDC7 19-13 Overexpression group (19-13 OE), PC3 cell control vector group (VECTOR), PC3 cell circCCDC7 19-13 Overexpression combined with ferroptosis inhibitor FER1 treatment group (19-13 OE+FER1), PC3 cell control vector combined with ferroptosis inhibitor FER1 treatment group (VEC+FER1).

[0074] PC3 cells were cultured and infected according to the method described in Example 1. 48 hours after infection, cells were treated with FER1 or solvent. The final concentration of FER1 in the culture system was 0.4 μM. Following treatment, cells in each group were cultured for 24 hours. ROS levels in each group were measured using a DCFH probe, and lipid peroxidation levels were measured using a Lipid C11 probe.

[0075] 2. Mitochondrial Membrane Potential Detection

[0076] The following experimental groups were set up: PC3 cells circCCDC7 19-13 Overexpression group (19-13), PC3 cell control vector group (Vector), and positive control group (Positive Control). For the positive control group, CCCP (10 mM) was added to the cell culture medium at a recommended dilution ratio of 1:1000, diluted to 10 μM, and treated for 20 minutes. PC3 cells were cultured and infected according to the method described in Example 1. After infection, cells in each group were cultured for 48 hours, and the mitochondrial membrane potential of each group was measured using a JC-1 probe.

[0077] 3. Treatment with ferroptosis inhibitors, necrosis inhibitors, and apoptosis inhibitors

[0078] The following experimental groups were set up: PC3 / DU145 cell control vector group (19-13 OE-), PC3 / DU145 cell CirCCDC7 19-13 Overexpression group (19-13 OE+), PC3 / DU145 cells circCCDC7 19-13 Overexpression combined with ferroptosis inhibitor FER1 treatment group (19-13 OE+Fer1+), PC3 / DU145 cells circCCDC7 19-13 Overexpression combined with necrosis inhibitor Nec1 treatment group (19-13 OE+Nec1+), PC3 / DU145 cells circCCDC7 19-13 Overexpression combined with the apoptosis inhibitor Z-VAD treatment group (19-13 OE+Z-VAD+). The final concentrations of Fer1, Nec1, and Z-VAD in the culture system in each group were 0.4uM, 30uM, and 40uM, respectively. PC3 cells were cultured and infected according to the method described in Example 1. After 48 hours of infection, the cells in each group were treated with drugs or solvents. Cell viability was assessed by CCK8 assay 24 hours after treatment.

[0079] 4. Intracellular ferrous iron accumulation

[0080] The following experimental groups were set up: PC3 cells circCCDC7 19-13 Overexpression group (19-13 OE) and PC3 cell control vector group (NC). 72 hours after cell transfection, each group of cells were detected using 1. Phen green SK probe flow cytometry; 2. Iron detection kit (ferrozine microplate method).

[0081] 5. Detection of intracellular GSH content

[0082] The following experimental groups were set up: PC3 cells circCCDC7 19-13 Overexpression group (19-13 OE), PC3 cell control vector group (Vector). PC3 cells were cultured and infected according to the method described in Example 1. After infection, the cells of each group were cultured for 72 hours, and the GSH content in each group of cells was detected using a GSH detection kit (S0052, Beyotime).

[0083] 6. Detection of intracellular lipid peroxidation product MDA

[0084] The following experimental groups were set up: PC3 cells circCCDC7 19-13Overexpression group (19-13 OE) and PC3 cell control vector group (VECTOR). PC3 cells were cultured and infected according to the method described in Example 1. After infection, cells in each group were cultured for 72 hours, and the MDA content in each group was measured using a lipid oxidation (MDA) detection kit (S0131S, Beyotime).

[0085] 7. Mitochondrial Detection

[0086] The following experimental groups were set up: PC3 cells circCCDC7 19-13 Overexpression group (19-13 OE), PC3 cell control vector group (VECTOR), and Erastin treatment group (ferroptosis inducer 10 μM, positive control). PC3 cells were cultured and infected according to the method described in Example 1. After 72 hours of culture, mitochondrial characteristics of each group were observed using transmission electron microscopy.

[0087] The results showed that overexpression of circCCDC7 19-13 Prostate cancer cells meet the characteristics of ferroptosis ( Figure 6 ). Figure 6 A: DCFH probe detection indicated overexpression of circCCDC7 19-13 Later, intracellular ROS increased in prostate cancer cells, and Lipid C11 probe detection indicated an increase in lipid peroxidation levels; Figure 6 B: Ko525 probe detection suggests overexpression of circCCDC7 19-13 Alterations in mitochondrial membrane potential in prostate cancer cells; Figure 6 C: Overexpression of circCCDC7 19-13 Inhibition of tumor cell activity: ferroptosis inhibitor (Fer1) can rescue, but necrosis inhibitor (Nec1) and apoptosis inhibitor (Z-VAD) cannot rescue; Figure 6 D: Colorimetric assay to detect intracellular ferrous iron accumulation. The results showed that overexpression of circCCDC7 19-13 After that, the ferrous content in prostate cancer cells increased significantly; Figure 6 E: Overexpression of circCCDC7 19-13 Later, GSH in prostate cancer cells decreases, making them susceptible to ferroptosis; Figure 6 F: Overexpression of circCCDC7 19-13 Later, the intracellular lipid peroxidation product MDA in prostate cancer cells increased; Figure 6 G: Transmission electron microscopy suggests overexpression of circCCDC7 19-13 Later, the mitochondria of prostate cancer cells shrank and the mitochondrial cristae disappeared.

[0088] Example 3

[0089] Our results confirmed that overexpression of circCCDC719-13 Prostate cancer cells undergo ferroptosis, and circCCDC7 19-13 Can encode the polypeptide circCCDC7 241aa Next, we will study the mechanism of ferroptosis in prostate cancer cells.

[0090] Detection of circCCDC7 by IP and mass spectrometry 241aa Whether there is interaction between β-catenin and SLC7A11.

[0091] The following experimental groups were set up: PC3 cells circCCDC7 19-13 Overexpression group (Flag-19-13), PC3 cell control vector group (VECTOR). PC3 cells were cultured and infection experiments were performed according to the method described in Example 1. After infection, the cells of each group were cultured for 48 hours, and the supernatant was collected and circCCDC7 was detected by ELISA. 19-13 The amount of the encoded protein.

[0092] PC3 cell circCCDC7 19-13 The overexpression stable strain (19-13 OE) and the control vector stable expression strain (VECTOR) were co-cultured with wild-type PC3 cells. After 48 h of culture, the expression of circCCDC7 was detected by immunofluorescence. 241aa Whether it binds to SLC7A11.

[0093] The results are as follows Figure 7 As shown, IP and mass spectrometry suggest that circCCDC7 241aa Interacts with SLC7A11 ( Figure 7 A), SLC7A11 is located in the cell membrane and is an important molecule for the transport of cystine and glutamate inside and outside the cell. Cystine is the raw material for the synthesis of GSH (glutathione) by cells, and GSH is an important antioxidant in cells. System Xc-(SLC7A11 / SLC3A2) on the cell membrane can transport extracellular cystine into the cell and transport intracellular glutamate out of the cell. Glutathione peroxidase (GPX4) converts reduced GSH into oxidized GSH, while reducing lipid hydroperoxides to corresponding lipid alcohols or reducing free hydrogen peroxide to water. GSH depletion leads to GPX4 inactivation, causing lipid peroxide accumulation to trigger ferroptosis. Elisa suggests that circCCDC7 241aa Can be secreted outside the cell and may act through autocrine secretion ( Figure 7 B) Exuded circCCDC7 was confirmed by co-culture and immunofluorescence assays 241aa Can bind to SLC7A11 on the WT tumor cell membrane ( Figure 7C). The above results suggest that circCCDC7 241aa SLC7A11 is regulated at the post-translational level.

[0094] Next, we investigated the post-translational modifications of SLC7A11.

[0095] We first explored the changes in the transcription and translation levels of ferroptosis pathway-related molecules by PCR and WB and found that overexpression of circCCDC7 19-13 After 14 h, the RNA and protein levels of downstream molecules such as GPX4 and NOX2 were significantly decreased, while the transcription level of its upstream molecule SLC7A11 did not change significantly ( Figure 8 A), but SLC7A11 was significantly reduced at the translation level ( Figure 8 B) Further studies showed that overexpression of circCCDC7 19-13 After that, SLC7A11 is ubiquitinated at the K48 site.

[0096] The above results suggest that circCCDC7 241aa SLC7A11 is ubiquitinated and degraded through the K48 ubiquitination site, thereby inducing ferroptosis in prostate cancer.

[0097] Example 4

[0098] This example studies circCCDC7 241aa tumor suppressor effect.

[0099] 4-5 week old male mice (BALB / c-nu) were selected and divided into the following groups: PC3 cell control vector group (VECTOR), PC3 cell circCCDC7 19-13 Overexpression group (19-13 OE), PC3 cell circCCDC719-13 overexpression combined with ferroptosis inhibitor FER1 treatment group ( 19-13 The VECTOR and VEC+Fer1 groups were treated with the PC3 cell control vector stably expressed strain prepared in Example 1 by subcutaneous injection; the 19-13 OE and 19-13 OE+Fer1 groups were treated with the PC3 cell circCCDC7 prepared in Example 1 by subcutaneous injection. 19-13 Overexpression stable strains. The VEC+Fer1 and 19-13 OE+Fer1 groups were simultaneously injected with 2 mg / kg of Fer1 via the tail vein; the VECTOR and 19-13 OE groups were injected with an equal volume of PBS. PC3 cells in each group also express fluorescent protein, which can be used to monitor tumor formation.

[0100] like Figure 9As shown, compared with the VECTOR group, the tumor growth in the 19-13 OE group was significantly inhibited ( Figure 9 A), tumor weight was significantly reduced, but Fer1 treatment was able to restore tumor growth ( Figure 9 B); The tumor volume was significantly reduced, but Fer1 treatment was able to restore tumor growth ( Figure 9 C).

[0101] In summary, circCCDC7 19-13 The expression of circCCDC7 in prostate cancer tissues was lower than that in significant adjacent tissues and was positively correlated with OS and PFS of prostate cancer, suggesting that circCCDC7 19-13 Overexpression of circCCDC7 is an independent prognostic factor for prostate cancer 19-13 After circCCDC7, prostate cancer cells undergo ferroptosis. 19-13 Can encode a polypeptide circCCDC7 241aa It acts on the SLC7A11 protein in tumor cells, induces ferroptosis in prostate cancer cells by ubiquitinating and degrading the SLC7A11 protein, thereby effectively inhibiting the growth and progression of prostate cancer. It has no obvious killing effect on normal tissues. While ensuring good anti-tumor properties, it also makes up for the shortcomings of traditional chemotherapy drugs with many side effects, and has good prospects for clinical transformation.

[0102] The technical features of the above-described embodiments can be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0103] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Use of an agent for promoting polypeptide expression in the preparation of a drug for treating prostate cancer, characterized in that: The polypeptide comprises the amino acid sequence shown in SEQ ID NO.2; The agent that promotes the expression of the polypeptide is circCCDC7 19-13 recombinant expression vector; the circCCDC7 19-13 The recombinant expression vector is circCCDC7 19-13 The recombinant expression vector is formed after double enzyme digestion and ligation of the nucleotide and the eukaryotic expression vector; the circCCDC7 19-13 The nucleotide sequence is the nucleotide sequence shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that The polypeptide can inhibit the proliferation, invasion or migration of prostate cancer cells.

3. The use according to claim 1, characterized in that The polypeptide can induce ferroptosis in prostate cancer cells.

4. The use according to claim 1, wherein The polypeptide can induce ferroptosis in prostate cancer cells by ubiquitin-degrading SLC7A11 protein.

5. The use according to any one of claims 1 to 4, characterized in that The dosage form of the medicine is injection.

6. Detection of circCCDC7 19-13 The use of a reagent for measuring expression levels in the preparation of a prostate cancer detection product or a prostate cancer prognosis assessment product is characterized in that: circCCDC7 19-13 The nucleotide sequence is: the nucleotide sequence shown in SEQ ID NO: 1.

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