Application of polypeptides encoded by circular RNA circHDAC5 in the preparation of anticancer drugs

Abstract

This invention relates to the field of oncology technology, specifically to the application of a polypeptide encoded by the circular RNA circHDAC5 in the preparation of anticancer drugs. The amino acid sequence is shown in SEQ ID NO.1. The anticancer drug is used to inhibit colorectal cancer, specifically to inhibit the proliferation and / or metastasis of colorectal cancer cells. This polypeptide is formed by backsplicing exons 13-16 of the HDAC5 gene and can inhibit the proliferation and metastasis of colorectal cancer in both in vivo and in vitro environments. It can effectively inhibit the malignant biological behavior of colorectal cancer cells and can be used to prepare targeted therapeutic drugs for colorectal cancer.

Description

Technical Field

[0001] This invention belongs to the field of oncology, specifically relating to the application of a polypeptide encoded by a circular RNA circHDAC5 in the preparation of anticancer drugs. Background Technology

[0002] Circular RNAs (circRNAs) were once considered byproducts of RNA splicing and lacked any biological function. However, in recent years, with advancements in technologies such as high-throughput sequencing, the important functions of circRNAs have been gradually revealed. Their highly conserved and stable nature allows them to play a crucial role in various human diseases, especially cancer, demonstrating their potential as precision therapeutic targets and molecular markers. Previous studies have suggested that circRNAs regulate gene expression primarily through two mechanisms: first, by acting as a miRNA "sponge" to adsorb miRNAs; and second, by interacting with RNA-binding proteins (RBPs) to form complexes. Research indicates that circRNAs influence the progression of various gastrointestinal tumors, such as pancreatic cancer, liver cancer, and gastric cancer. In colorectal cancer (CRC), circLECRC can act as a "brake signal," inhibiting the excessive activation of oncogenic YAP signaling; while circPPFIA1s can inhibit CRC liver metastasis through the miR-155-5p / CDX1 and HuR / RAB36 pathways.

[0003] Traditionally, circRNAs, as non-coding RNAs, were considered untranslatable. However, recent studies have overturned this understanding, discovering that certain circRNAs containing elements such as the internal ribosome entry site (IRES) can be translated into biologically functional proteins and peptides. For example, circβ-catenin can activate the Wnt pathway by translating peptides, promoting the proliferation of liver cancer cells. circMAPK1 can be translated into a 109-amino acid peptide that inhibits the progression of gastric cancer. Our previous study also confirmed that specific circRNAs can encode short peptides with biological functions that affect the proliferation of colorectal cancer cells. These findings suggest that exploring the mechanisms by which translatable circRNAs regulate CRC liver metastasis is of great value for developing new therapeutic strategies.

[0004] In clinical treatment, current drug therapy for CRC still mainly relies on chemotherapy regimens such as oxaliplatin and 5-fluorouracil. Although these can prolong patient survival, the overall median survival remains limited. With the development of molecular biology, targeted therapy has gradually become a research hotspot. Currently, targeted drugs for colorectal cancer mainly focus on anti-angiogenesis (such as bevacizumab), anti-epidermal growth factor receptor (such as cetuximab), and tyrosine kinase inhibitors (such as regorafenib). However, these existing therapies have problems such as efficacy bottlenecks, drug resistance, and limited applicability. More importantly, the function, mechanism, and coding potential of the circular RNA circHDAC5 in colorectal cancer are still unknown. Therefore, exploring whether circHDAC5 has coding function and the biological effects of its encoded products is of urgent clinical need and significant scientific value for discovering novel therapeutic targets for colorectal cancer. Summary of the Invention

[0005] To address the issue of limited target sites for colorectal cancer, this invention provides the application of a polypeptide encoded by the circular RNA circHDAC5 in the preparation of anticancer drugs.

[0006] The specific technical solution is as follows: First, the present invention provides the application of a polypeptide encoded by circHDAC5 circular RNA in the preparation of anticancer drugs, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO.1.

[0007] SEQ ID NO.1: MPREGSTESESTQEDLEEEDEEDDGEEEEDCIQVKDEEGESGAEEGPDLEEPGAGYKKLFSDAQPLQPLQVYQAPLSLATVPHQALGRTQSSPAAPGGMKSPPDQPVKHLFTTGVVYDTFMLKHQCMCGNTHVHPEHAGRIQSIWSRLQETGLLSKCERIRGRKATLDEI QTVHSEYHTLLYGTSPLNRQKLDSKKLLDPHQDRGAAQAAHHPP.

[0008] The discovery and verification of the specific polypeptide encoded by circHDAC5 have shown that it has a tumor-inhibiting biological function, providing a novel target and active ingredient for the development of new anticancer drugs.

[0009] Furthermore, the circular RNA circHDAC5 is formed by backsplicing of exons 13-16 of the HDAC5 gene. This specific structure is the genetic basis for the production of the functional polypeptide, and this has been verified by sequencing (see Experiment 2), ensuring the certainty and uniqueness of the polypeptide's origin.

[0010] Furthermore, the anticancer drug is used to inhibit colorectal cancer. This use is based on the findings of Experiment 1, namely that circHDAC5 and its encoded polypeptide are significantly underexpressed in colorectal cancer tissues and cell lines, and that this underexpression is associated with poor patient prognosis.

[0011] Furthermore, the anticancer drug is used to inhibit the proliferation and / or metastasis of colorectal cancer cells. The efficacy of this technology has been confirmed in both in vitro and in vivo experiments. In vitro functional experiments and in vivo animal models (Experiment 4) showed that interfering with the expression of this polypeptide significantly promotes the proliferation and metastasis of colorectal cancer cells, while overexpression inhibits these malignant behaviors.

[0012] Furthermore, the metastases are liver metastases and / or lung metastases. This limitation is strongly supported by experimental data. Figure 6 As shown, circHDAC5 was expressed at the lowest level in colorectal cancer liver metastases; Figure 21-22 Animal experiments further demonstrated that interfering with circHDAC5 significantly accelerates the metastasis of cancer cells in the liver and lungs.

[0013] Furthermore, the anticancer drug is a targeted therapy drug. Because the polypeptide originates from a specific circular RNA, with a well-defined sequence and specific function, drugs developed targeting this polypeptide (e.g., by supplementing the polypeptide, mimicking its function, or regulating its expression) possess highly specific and low-side-effect targeted therapy characteristics.

[0014] Furthermore, the drug comprises the polypeptide as well as a pharmaceutically acceptable carrier, diluent, or excipient. Combining the anticancer polypeptide with pharmaceutically acceptable excipients can prepare a stable formulation suitable for clinical administration.

[0015] Furthermore, the dosage form of the drug is an injection, tablet, capsule, or granule. This covers a variety of conventional dosage forms for preparing the polypeptide into a drug. For example, injections are suitable for direct delivery of the active ingredient, while oral dosage forms such as tablets and capsules facilitate patient administration and broaden the applicability of the drug.

[0016] Secondly, the present invention also provides the use of a polypeptide encoded by circular RNA circHDAC5 in the preparation of products for the diagnosis, auxiliary diagnosis or prognostic assessment of colorectal cancer, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO.1.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: it is the first discovery that the 214-amino acid polypeptide encoded by the circular RNA circHDAC5 can effectively inhibit the proliferation and metastasis of colorectal cancer in vivo and in vitro, and has the prospect of being applied to the preparation of targeted drugs for colorectal cancer. Attached Figure Description

[0018] Figure 1 This invention provides experimental 1, which displays heatmaps of circular RNA expression profiles in 5 pairs of CRC tissues and adjacent normal tissues. Figure 2 A schematic diagram of the genome structure; Figure 3 This is a schematic diagram of the box curves for qRT-PCR validation results; Figure 4 A bar chart showing the qRT-PCR validation results of different colorectal cancer cell lines and normal control cell lines; Figure 5 This is a schematic diagram showing the Kaplan-Meier survival curve analysis. Figure 6 A schematic diagram of the results of qRT-PCR verification of circHDAC5 expression changes, where N is adjacent normal tissue, T is primary cancer tissue, and M is liver metastasis tissue; Figure 7 This is a schematic diagram of the agarose gel electrophoresis results; Figure 8 This is a schematic diagram of the results of the Rnase R digestion experiment. In the diagram, Mock represents the blank control group, and Rnase R represents the digestion experiment. Figure 9 A schematic diagram showing the half-life detection results of circHDAC5 and HDAC5 mRNA in DLD-1 cells after treatment with actinomycin D; Figure 10 This is a schematic diagram of the results of PCR verification for nucleocytoplasmic separation. Figure 11 This is a schematic diagram of the results of fluorescence in situ hybridization experiments on the DLD1 cell line; Figure 12 This is a schematic diagram of the open reading frame (ORF) of circHDAC5-214aa; Figure 13 A schematic diagram showing the experimental results of luciferase reporter gene assays for different IRES mutants (WT1, mut1, WT2, mut2); Figure 14 A schematic diagram showing the comparison of results from a dual-luciferase reporter gene experiment; Figure 15 This is a schematic diagram of antibody recognition. Figure 16 This is a graph showing the mass spectrometry detection results; Figure 17 Image of WB test results; Figure 18 The results of the subcutaneous tumor formation experiment in nude mice in Experiment 4; Figure 19 A statistical chart showing the average weight of tumors removed after tumorigenesis experiments; Figure 20A statistical graph showing the changes in tumor volume in each group of nude mice; Figure 21 Schematic diagram for constructing liver and lung metastasis models; Figure 22 A statistical chart showing the number of liver and lung metastases in each group. Detailed Implementation

[0019] The technical solution of the present invention will be described in detail below through experiments and embodiments, but the scope of protection of the present invention is not limited to the experiments and embodiments described.

[0020] The tissue samples used in the following experiments and examples were from patients who underwent colorectal cancer surgery. The patients and their families were fully informed of the purpose and procedure of the study before the operation and signed informed consent forms.

[0021] The colorectal cancer cell lines SW480, DLD-1, LoVo, HT29, HCT116, CaCo2 and the normal control cell line NCM460 used were all purchased from the Shanghai Cell Bank.

[0022] The cell culture medium used was prepared with DMEM / F12, 10% fetal bovine serum, 1% penicillin and 1% streptomycin, all purchased from Vicente Biotechnology Co., Ltd.; TRIzol used for RNA extraction was purchased from Invitrogen; and reagents used for qRT-PCR were all purchased from Nanjing Novozymes Co., Ltd.

[0023] The amino acid / nucleotide sequences involved are: SEQ ID NO.1: MPREGSTESESTQEDLEEEDEEDDGEEEEDCIQVKDEEGESGAEEGPDLEEPGAGYKKLFSDAQPLQPLQVYQAPLSLATVPHQALGRTQSSPAAPGGMKSPPDQPVKHLFTTGVVYDTFMLKHQCMCGNTHVHPEHAGRIQSIWSRLQETGLLSKCERIRGRKATLDEI QTVHSEYHTLLYGTSPLNRQKLDSKKLLDPHQDRGAAQAAHHPP.

[0024] Experiment 1: Screening for differentially expressed circular RNA circHDAC5 with translational potential in colorectal cancer, and validating its expression in tissues and cells. This experiment first detected the expression of circular RNA in 72 colorectal cancer and paired adjacent normal tissue samples using high-throughput sequencing. Bioinformatics analysis using the circRNADb database and IRES finder software revealed that the expression of circular RNA circHDAC5 was downregulated in colorectal cancer tissues and that it possessed translational potential. Subsequently, 72 colorectal cancer and paired adjacent normal tissue samples were collected, and RNA was extracted from the colorectal tissues and cells. The expression level of circular RNA circHDAC5 was verified using qRT-PCR.

[0025] like Figure 1-6 The image shows the expression analysis results of the circular RNA circHDAC5 in colorectal cancer tissues and cells. Among them, Figure 1 A heatmap is used to display the circular RNA expression profiles of five pairs of CRC tissues and adjacent normal tissues, with red representing high expression and green representing low expression. It shows that circHDAC5 exhibits a low expression trend in CRC tissues (adjacent normal tissues are represented by green clusters, while CRC tissues show a reverse distribution of red clusters) and possesses translational potential.

[0026] Figure 2 This is a schematic diagram of the genome structure. hsa_circ_0005055 (i.e., circHDAC5) is formed by backsplicing of exons 13-16 of the HDAC5 gene (red lines mark the circRNA linker region), and its chromosome location is chromosome 17 (Chr17). The pie chart on the right shows the sequence composition of circHDAC5 (different colors represent exon / intron regions).

[0027] Figure 3 Box plots were used to compare the relative expression levels of circHDAC5 in cancer (CRC) tissue and adjacent normal tissue. The results showed that circHDAC5 expression in CRC tissue was significantly lower than that in adjacent normal tissue (**, P<0.01).

[0028] Figure 4 The bar chart shows the results of qRT-PCR verification of the relative expression level of circHDAC5 in different colorectal cancer cell lines and normal control cell lines. It can be seen that the expression of circHDAC5 in CRC cell lines (especially DLD-1) is lower than that in normal colonic epithelial cells (such as NCM460), that is, the expression of circHDAC5 is downregulated in colorectal cancer cell lines.

[0029] Figure 5The Kaplan-Meier survival curve analysis shows that CRC patients with high circHDAC5 expression had significantly shorter survival than those with low expression (P=0.005), suggesting that circHDAC5 is associated with poor prognosis in CRC. This indicates a negative correlation between circHDAC5 and the prognosis of colorectal cancer.

[0030] Figure 6 This is a schematic diagram showing the results of qRT-PCR detection of the relative expression levels of circHDAC5 in adjacent normal tissue (N), primary colorectal cancer tissue (T), and liver metastasis tissue (M) (10 cases each). The results show that the expression level of circHDAC5 was lowest in liver metastasis (M).

[0031] Experiment 2: Verifying that the circular RNA circHDAC5 is formed by backsplicing of exons 13-16 and is mainly located in the cytoplasm. After Experiment 1 verified the low expression of circular RNA circHDAC5 in colorectal cancer, this experiment verified the circular structure of circHDAC5.

[0032] Sanger sequencing was used to verify that circHDAC5 is formed by backsplicing of exons 13-16. Agarose gel electrophoresis and RNase R digestion experiments confirmed that circHDAC5 is indeed a circular structure. All reagents and equipment used in the Sanger sequencing experiments were purchased from Nanjing Qingke Biotechnology Co., Ltd.

[0033] A probe for the circular RNA circHDAC5 was constructed, and its subcellular localization was verified using fluorescence in situ hybridization (FISH). Nuclear and cytoplasmic RNA were extracted using a nucleocytoplasmic RNA isolation kit for PCR to further verify the subcellular localization of circHDAC5. The probes and kits used in the FISH experiment were purchased from Guangzhou Ribobio Biotechnology Co., Ltd. (catalog number C10910). The nucleocytoplasmic RNA isolation kit was purchased from Nanjing Beyotime Biotechnology Co., Ltd.

[0034] FISH test procedure (follow the steps outlined in the kit instructions): 1. Cell Culture With 3*10 5 After the cells are evenly seeded in a 6-well plate at a density per well for 24 hours, the cell confluence reaches 60%-70%.

[0035] 2. Cell fixation and permeability Wash cells with PBS for 5 min, then fix with 4% paraformaldehyde at room temperature for 10 min, wash cells with PBS for 5 min, repeat 3 times. Add 1 ml of pre-cooled permeabilization buffer to each well, let stand at 4 degrees for 5 min, discard the permeabilization buffer, and then wash cells with PBS for 5 min, repeat 3 times.

[0036] 3. Probe detection Add 200 μL of prehybridization buffer to each well and block at 37°C for 30 min. In the dark, add 2.5 μL of 20 μM FISH ProbeMix stock solution to 100 μL of hybridization buffer. Discard the prehybridization buffer and add 100 μL of probe hybridization buffer containing the probe. In the dark, hybridize overnight at 37°C. In the dark, wash cells three times with hybridization wash I at 42°C for 5 min each time. In the dark, wash cells once with hybridization wash II at 42°C. In the dark, wash cells once with hybridization wash III at 42°C. In the dark, wash cells with PBS and incubate at room temperature for 5 min.

[0037] 4.DNA staining Protect from light, add 1 ml of DAPI staining solution and stain for 10 min, wash cells three times with PBS for 5 min each time, and then examine and photograph under a confocal microscope.

[0038] 5. Verification of the circHDAC5 circular structure and subcellular localization.

[0039] Specifically, Figure 7 The results are from Agarose gel electrophoresis. Using cDNA (reverse transcription product) and gDNA (genomic DNA) as templates, circHDAC5 (primers across exon linker regions) and GAPDH (linear RNA control) were amplified by PCR. The results showed that a circHDAC5 band was amplified in cDNA (cDNA lane), while no band was found in gDNA (excluding genomic DNA contamination), verifying the circularization characteristic of circHDAC5; GAPDH bands were found in both cDNA and gDNA (normal linear RNA amplification).

[0040] Figure 8 This is a schematic diagram of the RNase R digestion experiment. Mock represents the untreated control group, and RNase R represents the digestion-treated group. The results showed that the linear RNA internal reference (GAPDH) was significantly degraded after RNase R digestion, while the circHDAC5 band remained stable, verifying the resistance of its circular structure to RNase R.

[0041] Figure 9The half-lives of circHDAC5 and HDAC5 mRNA in DLD-1 cells after treatment with actinomycin D were determined. The results showed that the half-life of circHDAC5 (red curve) was longer than that of HDAC5 mRNA (blue curve), consistent with the anti-degradation properties of circular RNA. This result validates the stability of circHDAC5.

[0042] Figure 10 This is a schematic diagram of the results of nuclear-cytoplasmic separation PCR verification. GAPDH and U6 are both internal controls. The relative expression level of circHDAC5 in the cytoplasm and the nucleus was detected by qRT-PCR. The results show that circHDAC5 is mainly expressed in the cytoplasm (red column is higher than blue column) rather than in the nucleus.

[0043] Figure 11 This is a schematic diagram of the fluorescence in situ hybridization (FISH) results for the DLD1 cell line, showing the immunofluorescence staining results. DAPI (blue) labels the cell nucleus, while circHDAC5 (green) shows low co-localization with the nucleus, further confirming that circHDAC5 is mainly located in the cytoplasm.

[0044] Experiment 3: Verifying that the circular RNA circHDAC5 can encode a polypeptide of 214 amino acids. The circular RNA circHDAC5 possesses two essential prerequisites for translation: the IRES element and the ORF sequence. In this experiment, wild-type and mutant vectors with the IRES element were constructed, and the activity of the IRES element was detected using a dual-luciferase reporter gene assay. All vectors and kits used in the experiment were purchased from Shanghai Jikai Gene Co., Ltd.

[0045] The applicant discovered that the amino acid sequence of the polypeptide encoded by the circular RNA circHDAC5 contained the amino acid recognition region of the HDAC5 antibody. Therefore, the applicant used a commercially available antibody against HDAC5 to perform a Western blotting experiment to verify the existence of the polypeptide, and then further verified the amino acid sequence of the polypeptide using mass spectrometry.

[0046] Western blot assay (routine detection method): First, clean the glass plate with deionized water, dry it, fix the glass plate, pour the prepared lower layer adhesive along the side until it is about 2-3 cm from the top, then add 1 ml of isopropyl alcohol, let it stand for 30 minutes, after the lower layer adhesive has completely solidified, discard the top isopropyl alcohol, add the prepared upper layer adhesive, slowly insert the comb, being careful to avoid air bubbles, let it stand for 20 minutes, after the upper layer adhesive has solidified, gently pull out the comb.

[0047] Insert the glass plate into the electrophoresis tank, fill it with electrophoresis buffer, and add the marker and protein sample to the wells in sequence. First, adjust the voltage to 80 V for electrophoresis. After the protein has reached the separating gel, adjust the voltage to 120 V and continue electrophoresis. Stop the electrophoresis when the target protein reaches the bottom. Cut the gel according to the molecular weight of the target protein.

[0048] Cut a PVDF membrane to the appropriate size, soak it in methanol, place it on the cut protein gel, and immerse it in the transfer solution. Then, install it in the order from anode to cathode, add it to the transfer tank, place an ice pack, and transfer the membrane at a constant current of 250 mA on ice for 90 minutes.

[0049] Blocking: Place the transferred membrane into the prepared blocking solution and incubate at room temperature on a shaker for 2 hours. Then wash three times with TBST solution for 5 minutes each time. Incubate with primary antibody overnight at 4°C on a shaker. The next day, wash the sample three times with TBST solution on a shaker for 10 minutes each time. Incubate the corresponding secondary antibody at room temperature for 2 hours.

[0050] The image was then washed three times with TBST solution for 10 minutes each time. Next, a developer was prepared, and the image was exposed in a developing apparatus under light-protected conditions. The image was then saved and grayscale values ​​were analyzed.

[0051] like Figure 12-17 As shown, circHDAC5 can encode a 214aa short peptide. Figure 12 This is a schematic diagram of the open reading frame (ORF) of circHDAC5-214aa. The ORF of circHDAC5 is 706 nt in length and is expected to translate into a 214-amino acid protein (circHDAC5-214aa), containing the IRES (internal ribosome entry site) sequence. Database analysis results show that circHDAC5 meets the requirements for encoding a polypeptide.

[0052] Figure 13 The results of luciferase reporter gene assays for different IRES mutants (WT1, mut1, WT2, mut2) are presented. Luciferase activity (relative light units) was measured using pRL-TK as an internal control. The results showed that the luciferase activity of IRES WT1 and WT2 was significantly higher than that of mut1 and mut2 (**, P<0.01), suggesting that IRES is a key element in circHDAC5 translation. Dual-luciferase reporter gene assays revealed that luciferase activity was significantly reduced after mutation of the 7-152 nt IRES sequence. Figure 14 and Figure 13 The experimental content was consistent, further verifying the necessity of IRES for circHDAC5 translation.

[0053] Figure 15This diagram illustrates antibody recognition, showing the amino acid sequence (214 aa) of circHDAC5-214aa and the antibody recognition site (e.g., the epitope of the ab1439 antibody, whose amino acid sequence is shown in SEQ ID NO.2: QPHQDRGAGAQAAHHPP). Since the 214 aa polypeptide sequence encoded by circHDAC5 contains the recognition site of the HDAC5 antibody (ab1439), this antibody is used to detect the 214 aa polypeptide.

[0054] Figure 16 The image shows the results of mass spectrometry (MS) analysis. After transfection with the circHDAC5 IRES mutant and overexpression vector, mass spectrometry analysis revealed an amino acid sequence, as shown in SEQ ID NO.3: KKLLDPHQDRGAAQAAHHPP, which is consistent with the 214 aa short peptide sequence. This indicates that circHDAC5 encodes a 214 aa polypeptide. The peptide sequence of the circHDAC5 translation product was verified (matching the theoretical sequence).

[0055] Figure 17 The image shows the results of Western blotting (WB). Western blotting validated the expression of circHDAC5-214aa. To further verify the expression level of the 214 aa peptide produced by circHDAC5 translation, overexpression of circHDAC5 (214 aa) yielded a protein band of approximately 21 kDa (consistent with the expected molecular weight), while the empty vector control group did not show this band. Tubulin was used as an internal control. The 214 aa short peptide was detected in DLD-1 cells using Western blotting.

[0056] This experiment demonstrates that the circular RNA circHDAC5 can encode a 214-amino acid polypeptide, which is named circHDAC5-214aa.

[0057] Experiment 4: In vivo experiments verified that the circular RNA circHDAC5 exerts its function of inhibiting the malignant biological behavior of colorectal cancer cells by encoding a 214-amino acid polypeptide.

[0058] In this experiment, plasmids for stable knockdown of circHDAC5 and control plasmids were constructed. After transfection into DLD-1 cells, subcutaneous tumorigenesis experiments and liver and lung metastasis models were performed in nude mice for validation. All plasmids used in the experiment were purchased from Shanghai Jikai Gene Co., Ltd.

[0059] Subcutaneous tumor formation experiment in nude mice: Five-week-old female BALB / c nude mice were purchased from the Animal Center of Nanjing Medical University and housed there. Cells from different transfection treatments were removed, the culture medium was discarded, the cells were washed twice with PBS, digested with trypsin, and transferred to 10 ml EP tubes. The cells were then centrifuged, and the supernatant was discarded. The cells were resuspended in PBS, counted, and the final cell concentration was adjusted to 100,000 / 100 μL. 100 μL of the cell suspension was subcutaneously injected into the axilla of each nude mouse. One week after injection, the long and transverse diameters of the subcutaneous tumor were measured and recorded every other day. Four weeks later, the mice were euthanized by cervical dislocation, and tumor samples were obtained. The tumor samples were photographed, recorded, and preserved.

[0060] Lung metastasis model: Five-week-old BALB / c nude female nude mice with NOD / SCID immunodeficiency underwent the necessary cell digestion, mixing, and counting. Cells were resuspended in PBS solution to adjust cell concentration. The cell suspension (5 × 10⁻⁶ cells / mL) was then used. 5 Cells (50 μl / 50 μl) were injected into the tail vein of mice, and the health status of the mice was closely observed. Small animal in vivo imaging technology was used to observe the growth of lung metastases. Mice were sacrificed after 5 weeks, and lung tissue was obtained for observation and H&E staining to assess the degree of lung metastasis.

[0061] Liver metastasis model: Five-week-old BALB / c nude female nude mice with NOD / SCID immunodeficiency underwent the necessary cell digestion, mixing, and counting. Cells were resuspended in PBS solution to adjust cell concentration. Following aseptic principles, the mice's abdomen was opened after anesthesia, and the cell suspension (5 × 10⁻⁶ cells / mL) was then placed inside. 5 Cells (50 μl / 50 μl) were injected into the spleen of mice. The abdomen was sutured post-surgery. The health status of the mice was closely monitored. Liver metastasis growth was observed using small animal in vivo imaging techniques. Mice were sacrificed after 5 weeks, and liver tissue was obtained for observation and H&E staining to assess the degree of liver metastasis.

[0062] like Figure 18-22 As shown, the effect of circHDAC5 on the progression and metastasis of colorectal cancer can be verified in vivo. Figure 18 The results of the subcutaneous tumor formation experiment in nude mice are clearly visible. The tumor growth volume in the sh-circ group (circHDAC5 knockdown group) is larger than that in the sh-ctrl group (control group), suggesting that circHDAC5 knockdown can promote tumor growth in vivo. Figure 19 The average weight of the dissected tumors after the tumorigenesis experiment is shown. The average tumor weight in the sh-circ group was significantly higher than that in the sh-ctrl group, quantitatively confirming the promoting effect of circHDAC5 knockdown on tumor growth. Figure 20The growth curves of tumor volume over time were displayed. The tumor volume growth rate in the sh-circ group was significantly faster than that in the sh-ctrl group, further validating the aforementioned growth-promoting effect during the dynamic process. DLD-1 cells transfected with stably knocked-down circHDAC5 and control cells were injected into the splenic vein and tail vein of mice, respectively, to establish liver metastasis and lung metastasis models. Figure 21 These are the experimental results of liver and lung metastasis models. Gross observation and HE staining revealed that the sh-circ group mice had more liver and lung metastatic nodules than the sh-ctrl group (marked by the yellow dashed box in the figure). The results indicate that knockdown of circHDAC5 accelerates tumor metastasis. Figure 22 Statistical charts were generated for the number of liver and lung metastatic lesions in each group, and quantitative analysis was performed on the number of liver and lung metastatic nodules. The results showed that the number of liver and lung metastatic nodules in the sh-ctrl group was significantly lower than that in the sh-circ group, indicating that circHDAC5 knockdown (i.e., loss of function of this peptide) can promote liver / lung metastasis of colorectal cancer cells, thus verifying that the peptide encoded by circHDAC5 has the function of inhibiting metastasis.

[0063] Different groups in this in vivo experiment collectively verified that the circular RNA circHDAC5 functions by encoding a 214-amino acid polypeptide to inhibit the malignant biological behavior of colorectal cancer cells.

[0064] Unless otherwise specified, all technologies mentioned above refer to existing technologies.

[0065] As described above, although the invention has been shown and described with reference to specific preferred experiments and embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. Application of a polypeptide encoded by a circular RNA (circHDAC5) in the preparation of an anticancer drug, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The circular RNA circHDAC5 is formed by backsplicing of exons 13-16 of the HDAC5 gene.

3. The application according to claim 1, characterized in that, The anticancer drug mentioned is used to inhibit colorectal cancer.

4. The application according to claim 3, characterized in that, The anticancer drug is used to inhibit the proliferation and / or metastasis of colorectal cancer cells.

5. The application according to claim 4, characterized in that, The metastases are liver metastases and / or lung metastases.

6. The application according to claim 1, characterized in that, The anticancer drug mentioned is a targeted therapy drug.

7. The application according to claim 1, characterized in that, The drug comprises the polypeptide as well as a pharmaceutically acceptable carrier, diluent, or excipient.

8. The application according to claim 7, characterized in that, The dosage form of the drug is injection, tablet, capsule or granule.

9. The use of a polypeptide encoded by a circular RNA circHDAC5 in the preparation of products for the diagnosis, auxiliary diagnosis, or prognostic assessment of colorectal cancer, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.1.

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