Application of a circular RNA as a biomarker and therapeutic target for colorectal cancer

Abstract

The application relates to application of circular RNA as a colorectal cancer biomarker and treatment target, in-vivo and in-vitro experiments prove that a high-fat environment significantly improves the migration ability of colorectal cancer cells and further promotes the colorectal cancer process. Circular RNA sequencing obtains circ-PRKACB which is significantly increased in the colorectal cancer cells in a high-fat environment, and is taken by the colorectal cancer cells after being transported by the exosomes secreted by fat cells. Through clinical sample verification, the expression of circ-PRKACB in the cancer tissues of colorectal cancer patients is higher than that in the paracancer tissues, the expression in the serum of the intestinal cancer patients is higher than that in the non-intestinal cancer patients, and the expression amount is positively correlated with BMI, and the circular RNA can be used as a biomarker for evaluating the risk or prognosis of colorectal cancer. In a mouse colorectal cancer lung metastasis model, the expression of circ-PRKACB is knocked down, lung metastasis lesions of the mice are reduced, and the survival days are increased, so the circular RNA has the potential to be used as a colorectal cancer treatment target.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of a circular RNA as a biomarker and therapeutic target for colorectal cancer. Background Technology

[0002] Cancer is a complex disease involving the regulation of various abnormal gene expression. Circular RNAs (circRNAs) are a special class of non-coding RNA molecules. Unlike traditional linear RNAs, circRNAs have a closed circular structure, are not affected by RNA exonucleases, are more stable in expression, and are less prone to degradation. Increasing evidence suggests that circRNAs participate in the regulation of various tumors, promoting tumor cell proliferation and regulating cell cycle progression. A deeper understanding and discovery of the specific role of circRNAs in the progression of colorectal cancer holds promise for early detection, diagnosis, and prediction of metastasis in clinical practice, enabling timely and appropriate comprehensive treatment, including radical surgery. This could significantly improve the survival rate and quality of life for colorectal cancer patients, and prolong their survival time.

[0003] Exosomes are vesicle structures with an average size of 100 nanometers that can transport proteins, lipids, and nucleic acids. Research data indicates that exosomes can remodel the tumor microenvironment, influencing tumor angiogenesis, tumor growth, and tumor metastasis. Exosomes contain microRNAs, long non-coding RNAs, circular RNAs, and other genes that affect cancer progression, and can deliver various drugs; therefore, some studies suggest they may serve as delivery systems for anti-tumor drugs and genetic tools in cancer treatment. Exosomes are present in all bodily fluids and secreted by all cells, making them attractive for liquid biopsies. Currently, the gold standard for colorectal cancer screening is colonoscopy, which is highly invasive and difficult for patients to tolerate. Common tumor markers cannot accurately locate colorectal lesions. Therefore, the discovery of biomarkers in serum exosomes is of great significance for colorectal cancer screening. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides an application of circular RNA as a biomarker and therapeutic target for colorectal cancer. This invention demonstrates through in vivo and in vitro experiments that a high-fat environment significantly enhances the migration ability of colorectal cancer cells, thereby promoting colorectal cancer progression. Through circular RNA sequencing, a cAMP-dependent protein kinase catalytic subunit β-circular RNA (circ-PRKACB) was identified, showing significantly elevated expression in colorectal cancer cells under a high-fat environment. This circular RNA can be transported by exosomes secreted by adipocytes and taken up by colorectal cancer cells. Validation using clinical colorectal cancer patient samples showed that circ-PRKACB expression was higher in cancerous tissues than in adjacent normal tissues, and its expression in the serum of colorectal cancer patients was higher than in non-colorectal cancer patients. Furthermore, serum expression was positively correlated with the body mass index (BMI) of colorectal cancer patients. In a mouse model of colorectal cancer lung metastasis, knockdown of circ-PRKACB expression reduced lung metastatic lesions and increased survival days in mice.

[0005] The first object of the present invention is to provide the application of circular RNA circ-PRKACB as a biomarker for colorectal cancer, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0006] Furthermore, the expression level of the circular RNA circ-PRKACB was specifically upregulated in the serum and cancer tissue of colorectal cancer patients.

[0007] A second objective of this invention is to provide a kit for assessing the risk or prognosis of colorectal cancer, the kit comprising reagents for detecting the expression level of circular RNA circ-PRKACB in a sample to be tested.

[0008] Elevated expression of circular RNA circ-PRKACB in the test sample indicates a risk of colorectal cancer or suggests a poor prognostic effect of the sample on colorectal cancer.

[0009] Furthermore, the sample to be tested is a serum sample or a tissue sample.

[0010] Furthermore, exosomes were extracted from the serum samples to identify the expression level of the circular RNA circ-PRKACB.

[0011] Furthermore, the kit includes primers for amplifying the circular RNA circ-PRKACB.

[0012] Furthermore, the primers include a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown in SEQ ID NO.2, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.3.

[0013] Specifically, the forward primer is 5'-ACCAGAGGAAGTGAAAGAGTTT-3'.

[0014] Specifically, the reverse primer is 5'-TTCAAAATCTTCAAGTCCGGCA-3'.

[0015] Furthermore, the colorectal cancer detection kit also includes components for PCR amplification.

[0016] A third objective of this invention is to provide the application of circular RNA circ-PRKACB as a therapeutic target for colorectal cancer, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0017] A fourth objective of this invention is to provide a colorectal cancer treatment drug that targets the circular RNA circ-PRKACB.

[0018] Furthermore, the colorectal cancer treatment involves reducing the expression of the circular RNA circ-PRKACB.

[0019] A fifth objective of this invention is to provide the use of formulations that reduce the expression of circular RNA circ-PRKACB in the preparation of medicaments for the treatment of colorectal cancer.

[0020] Furthermore, the formulation comprises small interfering RNA or short hairpin RNA.

[0021] Furthermore, the nucleotide sequence of the small interfering RNA is shown in SEQ ID NO.4-5, SEQ ID NO.6-7 or SEQ ID NO.8-9, and the nucleotide sequence of the short hairpin RNA is shown in SEQ ID NO.10-11 or SEQ ID NO.12-13.

[0022] Furthermore, the small interfering RNA includes siRNA#1, siRNA#2, or siRNA#3, and the short hairpin RNA includes shRNA#2 or shRNA#3.

[0023] Preferably, the formulation comprises short hairpin RNA shRNA#2 or shRNA#3.

[0024] Specifically, the forward sequence of siRNA#1 is 5'-AGAGGAAGTGAAAGAGUUUtt-3' (shown in SEQ ID NO.4), and the reverse sequence is 5'-AAACUCUUUCACUUCCUCUtt-3' (shown in SEQ ID NO.5).

[0025] Specifically, the forward sequence of siRNA#2 is 5'-GAGGAAGUGAAAGAGUUUCtt-3' (shown in SEQ ID NO.6), and the reverse sequence is 5'-GAAACUCUUUCACUUCCUCtt-3' (shown in SEQ ID NO.7).

[0026] Specifically, the forward sequence of siRNA#3 is 5'-AGGAAGUGAAAGAGUUUCUtt-3' (shown in SEQ ID NO.8), and the reverse sequence is 5'-AGAAACUCUUUCACUUCCUtt-3' (shown in SEQ ID NO.9).

[0027] Specifically, the forward sequence of shRNA#2 is 5'-CCGGGAGGAAGUGAAAGAGUUUCCTCGAGGAAACUCUUUCACUUCCUCTTTTT-3' (shown in SEQ ID NO.10), and the reverse sequence is 5'-AATTAAAAAGAGGAAGUGAAAGAGUUUCCTCGAGGAAACUCUUUCACUUCCUC-3' (shown in SEQ ID NO.11).

[0028] Specifically, the forward sequence of shRNA#3 is 5'-CCGGAGGAAGUGAAAGAGUUUCUCTCGAGAGAAACUCUUUCACUUCCUTTTTT-3' (shown in SEQ ID NO.12), and the reverse sequence is 5'-AATTAAAAAAGGAAGUGAAAGAGUUUCUCTCGAGAGAAACUCUUUCACUUCCU-3' (shown in SEQ ID NO.13).

[0029] The sixth object of the present invention is to provide the use of small interfering RNA or short hairpin RNA in the preparation of colorectal cancer therapeutic drugs, wherein the nucleotide sequence of the small interfering RNA is shown in SEQ ID NO.4-5, SEQ ID NO.6-7 or SEQ ID NO.8-9, and the nucleotide sequence of the short hairpin RNA is shown in SEQ ID NO.10-11 or SEQ ID NO.12-13.

[0030] Preferably, the nucleotide sequence of the short hairpin RNA is as shown in SEQ ID NO.10-11 or SEQ ID NO.12-13.

[0031] The beneficial effects of this invention are: This invention provides the application of the circular RNA circ-PRKACB as a biomarker and therapeutic target for colorectal cancer. The risk and prognosis of colorectal cancer in test subjects can be assessed by detecting the expression level of circ-PRKACB in the sample. Knockdown of circ-PRKACB expression in a mouse model of colon cancer lung metastasis reduced lung metastases and increased survival days. This circular RNA has the potential to serve as a therapeutic target for colorectal cancer. Targeting circ-PRKACB can achieve anti-tumor effects, thereby enabling the production of drugs that inhibit the progression of colorectal tumors, potentially providing a basis for clinical treatment of colorectal cancer. Attached Figure Description

[0032] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein... Figure 1 This is a mouse model of colorectal cancer lung metastasis induced by high-fat diet and obesity constructed in Example 1 of the present invention. A is a schematic diagram of the model construction steps, B is a gross photograph of the lungs of mice in the high-fat diet and normal diet groups, and C is the HE immunostaining results of the lungs of mice in the high-fat diet and normal diet groups. Figure 2 This is an experiment in Example 1 of the present invention, in which mouse primary adipocytes were induced to differentiate (differentiation group CM) and not induced to differentiate (control group CON) cell supernatants were co-cultured with human colorectal cancer cells. A is a schematic diagram of the experimental steps, B is a graph showing the migration measurement results of the control group and differentiation group supernatants after co-culturing with human colon cancer cells HCT116, colon adenocarcinoma cells RKO and mouse colon cancer cells CMT-93, respectively, with a scale bar of 50 μm, and C is the statistical result of the number of migrating cells in different experimental groups; Figure 3 This is a diagram showing the results of conditionally culturing human colorectal cancer organoids in the culture supernatant of human adipose-derived mesenchymal stem cells from the control group and the differentiation group in Example 1 of this invention. The scale bar is 200 μm. Figure 4 This is a graph showing the results of long-term conditional culture of human colorectal cancer cells in mouse embryonic fibroblasts (3T3-L1) supernatant after induced differentiation (differentiation group CM) and non-induced differentiation (control group CON) in Example 1 of the present invention. The scale bar is 50 μm. Figure 5 This is a nude mouse tail vein lung metastasis model constructed in Embodiment 1 of the present invention, wherein A is a diagram of the model construction steps, B is a gross lung photograph and HE staining results, and C is a statistical diagram of the proportion of lung weight to mouse body weight and a statistical diagram of lung metastasis area. Figure 6This is a heatmap of circular RNA sequencing results of human colorectal cancer cells HCT116 after 17 weeks of conditional culture using supernatant of mouse embryonic fibroblasts (3T3-L1) induced differentiation (differentiation group CM) and uninduced differentiation (control group CON). The control group 1-3 and the differentiation group 1-3 are 3 parallel replicates. Figure 7 This is a volcano plot of circular RNA sequencing results of human colorectal cancer cells HCT116 after 17 weeks of conditional culture using supernatants of mouse embryonic fibroblasts (3T3-L1) induced differentiation (differentiation group CM) and uninduced differentiation (control group CON). Figure 8 This is a graph showing the results of detecting the expression levels of five circular RNAs in the supernatants of mouse embryonic fibroblasts (3T3-L1) after induction of differentiation (differentiation group CM) and non-induction of differentiation (control group CON) and human colon cancer cells HCT-116 and human colorectal cancer epithelial cells DLD-1 under short-term and long-term conditional culture, respectively, in Example 2 of the present invention. Figure 9 This is a diagram showing the dideoxy sequencing (Sanger sequencing) results of circ-PRKACB in human colon cancer cells HCT-116 in Example 2 of this invention; Figure 10 This is an electron micrograph of exosomes from the exosome detection results in Example 3 of the present invention, with a scale bar of 100 nm; Figure 11 This is a graph showing the nanoparticle tracking analysis results of the exosome detection results in Example 3 of this invention; Figure 12 This is an image of the exosome tracer staining results from the exosome detection in Example 3 of this invention, with a scale bar of 50 μm; Figure 13 This is a conditional culture of human colorectal cancer organoids from exosomes secreted by human adipose-derived mesenchymal stem cells in the control group and the differentiation group in Example 3 of the present invention. A is an illustration under an optical microscope with a scale bar of 200 μm, and B is a graph showing the expression level of circular RNA circ-PRKACB in organoids of the control group (CON) and the differentiation group (CM), respectively. Figure 14 This is a diagram showing the functional experimental results of exosomes secreted by human adipose-derived mesenchymal stem cells in the control group (CON) and the differentiated group (CM) of the present invention in Example 3. In Figure A, the migration results of exosomes added to human colon cancer cells are shown, with a scale bar of 100 μm. Figure B shows the specific number of migrating cells in the control group and the differentiated group. Figure C shows the expression level of circular RNA circ-PRKACB when exosomes from the control group and the differentiated group are added to human colon cancer cell culture. Figure 15 This is the analysis result of tissue and serum samples from clinical patients with colorectal cancer in Example 4 of the present invention. A shows the difference in expression levels of circular RNA circ-PRKACB between tumor tissue and adjacent tissue in colorectal cancer patients; B shows the fold difference in circ-PRKACB expression levels between cancerous and adjacent tissues in colorectal cancer patients with different BMIs; C shows the difference in expression levels of circular RNA circ-PRKACB in serum exosomes between colorectal cancer and non-colorectal cancer patients; D shows the difference in expression levels of circular RNA circ-PRKACB in serum exosomes in colorectal cancer patients with different BMIs. Figure 16 This refers to the difference in expression between circular RNA circ-PRKACB and mRNA-PRKACB after transient transfection of colorectal cancer cells with small interfering RNA in Example 5 of this invention. Figure 17 The results of the migration assay performed after transient transfection of human colorectal cancer cells HCT116 and DLD-1 with small interfering RNA in Example 5 of this invention are shown in the scale bar, which is 50 μm. Figure 18 This is the statistical result of the number of migrating cells after transient transfection of human colorectal cancer cells HCT116 and DLD-1 with small interfering RNA in Example 5 of the present invention; Figure 19 This is the colorectal cancer cell migration experiment after stable transfection with lentivirus knockdown of circular RNA circ-PRKACB in Example 5 of the present invention, with a scale bar of 50 μm; Figure 20 This is the result of a scratch assay on colorectal cancer cells after stable transfection with lentivirus knockdown of circular RNA circ-PRKACB in Example 5 of this invention. Figure 21 This is a figure showing the experimental results of the mouse tail vein lung transfer model of HCT116 cells with knocked-down circular RNA circ-PRKACB injected in Example 5 of the present invention. A is a gross lung and HE photo, B is the lung weight-to-body weight ratio of mice in different stable cell treatment groups, and C is the survival curve of mice in different treatment groups. Figure 22 This refers to the steps in constructing the high-fat nude mouse fat reduction model in Embodiment 5 of the present invention; Figure 23 These are gross lung photographs of mice under high-fat and low-fat conditions in Example 5 of this invention; Figure 24 This is an image showing the HE immunostaining results of mouse lungs under high-fat and low-fat conditions in Example 5 of this invention; Figure 25This is a graph showing the difference in lung metastasis area among different groups of mice in Example 5 of the present invention. PLCDH and circ-PRKACB represent different groups of mice injected via tail vein into different stable cell lines constructed by PLCDH control vector and circ-PRKACB overexpression lentivirus. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0034] Example 1: High-fat environment promotes the progression of colorectal cancer (1) Purchased 4-6 week old C57BL / 6 mice and divided them into two groups, feeding them a high-fat diet and a normal diet, respectively. After 4 weeks, the mice were injected with mouse colon cancer cells MCA38 via the tail vein. After 3 weeks of high-fat feeding, the mice were sacrificed. The experimental procedure is as follows: Figure 1 As shown in Figure A, the lungs of the two groups of mice were observed, and the results are as follows. Figure 1 As shown in Figure B, the number of lung metastases was significantly increased in the high-fat diet group of mice. HE staining experiments were performed on lung tissues from both groups of mice: after euthanasia, lung tissue was fixed in formaldehyde, dehydrated with a gradient of alcohols, embedded in paraffin, trimmed to 3 mm, dewaxed in xylene, and then hydrated sequentially with 100%, 95%, 90%, and 80% ethanol. Hematoxylin and eosin staining were then performed, followed by mounting. The results are shown in Figure B. Figure 1 As shown in Figure C, mice fed a high-fat diet had more and larger lung metastases than mice fed a normal diet, suggesting that a high-fat diet promotes the metastasis of colorectal cancer.

[0035] (2) Purchase 3-4 week old C57BL / 6 mice, isolate primary adipocytes from subcutaneous adipose tissue, and divide the isolated primary adipocytes into two groups. Seed them into the lower chamber of a 24-well plate and culture them in DMEM complete medium. One group was supplemented with phosphodiesterase inhibitor IBMX, insulin INS, and dexamethasone DEX to induce differentiation into mature adipocytes, i.e., the differentiation group (CM group). The other group was not induced to differentiate and served as the control group (CON group). The process is as follows. Figure 2 As shown in Figure A. Human colon cancer cells HCT116, colon adenocarcinoma cells RKO, and mouse colon cancer cells CMT-93 were seeded into the upper chambers of a 24-well plate, respectively, and co-cultured with primary adipocytes from the differentiation group and control group mice for 28 hours. Migration assays were then performed, and the results are shown in Figure A. Figure 2 As shown in Figures B and C, colon cancer cells co-cultured with differentiated cells showed significantly higher migration ability than colon cancer cells co-cultured with the control group, indicating that mature adipocytes promoted the migration ability of colon cancer cells.

[0036] (3) Primary human adipose-derived mesenchymal stem cells isolated from human adipose tissue were divided into two groups as shown above: one group was the induced differentiation group (CM group), and the other was the non-induced differentiation control group (CON group). The supernatant produced by the two groups of cells was used to treat human colon cancer organoids, i.e., conditional culture was performed. The results are as follows: Figure 3 As shown, colorectal cancer organoids cultured using the supernatant of differentiated cells grew significantly faster than those in the control group, suggesting that a high-fat environment promotes the progression of colorectal cancer.

[0037] (4) 3T3-L1 cells (mouse embryonic fibroblasts) were divided into two groups as shown above: one group was the induced differentiation group (CM group), and the other was the non-induced differentiation control group (CON group). Human colorectal cancer cells HCT116 and mouse colon cancer cells CMT-93 were treated with the cell supernatant from the differentiation group and the control group, respectively, for conditioned culture for 12 weeks. The migration assay results are shown below. Figure 4 As shown, colorectal cancer cells cultured in the supernatant of mature adipocytes from the differentiation group had significantly higher migration ability than those in the control group.

[0038] (5) 3T3-L1 cells (mouse embryonic fibroblasts) were divided into two groups as described above: one group was the induced differentiation group (CM group), and the other was the non-induced differentiation control group (CON group). Human colorectal cancer cells HCT116 were treated with the culture supernatant of the differentiation group and the control group for 17 weeks, becoming long-term treated HCT116 cells (CM / CON). 2×10⁻⁶ cells were then used to treat HCT116 cells. 6 The cultured cells were injected into mice via the tail vein to establish a nude mouse tail vein lung metastasis model. The mice were sacrificed after 9 weeks. The experimental procedure is as follows: Figure 5 As shown in Figure A. Mouse lungs were observed and HE staining was performed; the results are shown below. Figure 5 As shown in Figure B, compared with the control group, mice with colorectal cancer cells cultured in the supernatant of the differentiation group for a long period of time had increased lung metastases. The percentage of lung tissue weight in the total body weight of mice and the area of ​​lung metastases were calculated, and the results are shown below. Figure 5 As shown in Figure C, after tail vein injection of HCT116 cells cultured in the differentiation group, the lung tissue weight and body weight ratio of mice increased significantly, and the area of ​​lung metastases increased significantly compared with the control group. This indicates that the supernatant secreted by mature adipocytes stably promotes the progression of human colon cancer cells HCT116.

[0039] Example 2: High expression of circular RNA circ-PRKACB in colorectal cancer cells (1) Mouse embryonic fibroblasts 3T3-L1 were divided into two groups: one group was induced to differentiate into mature adipocytes (CM group), and the other group was not induced to differentiate as the control group (CON group). Human colorectal cancer cells HCT116 were treated with the culture supernatant of the differentiated group and the control group for 17 weeks. After culture, three replicates of circular RNA sequencing were performed on human colon cancer cells HCT116 in both the CM and CON groups. The heatmap and volcano plot results are shown below. Figure 6 and Figure 7 As shown, the levels of circular RNAs circ-PRKACB (hsa_circPRKACB_006), circ-RAPGEF (hsa_circRAPGEF5_005), circ-CCSER2, circ-GLIS3 (hsa_circGLIS3_003), and circ-NECTIN3 in colorectal cancer cells under high-fat conditions were significantly higher than those in the control group.

[0040] (2) Human colon cancer cells HCT-116 and human colorectal cancer epithelial cells DLD-1 were treated with culture supernatant from the differentiation group and control group for short-term (3 weeks) and long-term (13 weeks) treatment, and the expression levels of circular RNAs circ-PRKACB, circ-RAPGEF, circ-CCSER2, circ-GLIS3 and circ-NECTIN3 were detected. The results are as follows: Figure 8 As shown, only circ-PRKACB and circ-RAPGEF5 showed consistently higher expression levels in differentiated colorectal cancer cells compared to the control group. Considering the low expression abundance of circ-RAPGEF5 in colorectal cancer cells, its direct biological impact may be relatively small. Therefore, subsequent studies focused on the role of circ-PRKACB in colorectal cancer progression.

[0041] (3) Dideoxy sequencing (Sanger sequencing) was performed on the circular RNA circ-PRKACB (hsa_circ_0004083) in human colon cancer cells HCT116. The results are as follows: Figure 9 As shown, this circular RNA is stably present in human colorectal cancer cells.

[0042] Example 3: Exosome transport of circular RNA circ-PRKACB to colorectal cancer cells I. Electron microscopy and NTA (nanoparticle tracking analysis) experiments were performed on the culture supernatant of primary mouse adipocytes. The results are as follows: Figure 10 and Figure 11 As shown, exosomes were confirmed to be present in the supernatant of primary adipocyte culture. PKH67 (exosome tracer dye) assays were performed on the supernatant of human primary adipocyte mesenchymal stem cell culture, and the results are as follows: Figure 12 As shown, this confirms that exosomes enter human colon cancer cells HCT-116.

[0043] The steps for the PKH67 exosome tracer dye experiment are as follows: (1) The concentration of exosomes was quantitatively obtained using protein diquinoline carboxylic acid (BCA); (2) Preparation of dye working solution: Mix PKH67 with general membrane labeling diluent (DiluentC) at a ratio of 1:10 and use immediately after preparation; (3) Add 200 μg of exosomes to 15 μL of dye working solution into a brown tube (protected from light), vortex to mix for 1 minute, and then let stand and incubate for 10 minutes. (4) Add 6 mL of 1×PBS to the incubated exosome dye complex and mix well to terminate the incubation; (5) Use the exosome extraction kit to extract exosomes again (protected from light) to remove excess dye; (6) 2×10 in a 24-well plate 5 HCT116 cells were seeded onto cell slides, and after 24 hours, exosome dye complexes were incubated with HCT116 cells at 37°C in a cell incubator in the dark for 12 hours. (7) Wash the cells twice with PBS, fix the cells with 4% formaldehyde on a shaker at room temperature for 20 minutes, and wash the cells three times with PBS buffer (wash on a shaker for 3 minutes each time, protected from light). (8) Add 4',6-diamidinyl-2-phenylindole (DAPI) dye to stain cell nuclei (protect from light, incubate on a shaker for 30 minutes), discard the staining solution and wash 3 times with PBS, protecting from light for 3 minutes each time; (9) Place the cell slide upside down on a glass slide, seal it, and store it in the dark. Take a fluorescence photograph using a confocal microscope.

[0044] II. Primary human adipose-derived mesenchymal stem cells isolated from human adipose tissue were divided into two groups: one group was induced to differentiate into mature adipocytes (CM group), and the other group was not induced to differentiate as a control group (CON group). Exosomes were extracted from the cell supernatant of the differentiation group and the control group, respectively, and cultured as human colorectal cancer organoids for 4 days. Under a microscope, it was observed that the growth rate of organoids in the differentiation group was faster, and the exosomes in the differentiation group promoted the growth of human colorectal cancer organoids. Figure 13 As shown in Figure A. The expression level of the circular RNA circ-PRKACB in human colorectal cancer organoids under different treatments was measured, and the results are shown in Figure A. Figure 13 As shown in Figure B, after exosome culture in the differentiated group, the expression level of circular RNA circ-PRKACB in human colorectal cancer organoids was significantly higher than that in the control group.

[0045] III. The isolated primary human adipose-derived mesenchymal stem cells were divided into two groups as shown above: one group was the induced differentiation group (CM group), and the other was the non-induced differentiation control group (CON group). Exosomes were extracted from the cell supernatant of the differentiation group and the control group, respectively, and cultured with human colorectal cancer cells HCT116. Cell migration assays were performed on the cultured cells, and the results are as follows: Figure 14 As shown in Figures A and B, the migration ability of human colorectal cancer cells after exosome culture in the differentiated group was increased. The expression level of circular RNA circ-PRKACB in HCT116 cells after exosome culture in both groups was measured, and the results are shown in Figures B and C. Figure 14 As shown in C, the expression level of circular RNA circ-PRKACB in colorectal cancer cells treated with exosomes in the differentiated group was significantly higher than that in the control group.

[0046] Example 4: Clinical samples show differential expression of circular RNA circ-PRKACB (1) The expression level of circular RNA circ-PRKACB in cancerous tissue and adjacent tissue of 74 clinical colorectal cancer patients was statistically analyzed. The results are as follows: Figure 15 As shown in A, the expression level of circ-PRKACB in cancerous tissue was significantly higher than that in adjacent normal tissue.

[0047] (2) The fold difference in expression levels of circular RNA circ-PRKACB between cancerous and adjacent tissues was statistically analyzed in 74 clinical colorectal cancer patients with different body mass indexes (BMIs). Among them, 19 patients had a BMI higher than 24, and 55 patients had a BMI lower than 24. The results are as follows: Figure 15 As shown in B, the expression level of circ-PRKACB in cancer tissues of patients with high BMI was significantly higher than that of patients with low BMI.

[0048] (3) Serum samples were collected from 21 non-colorectal cancer patients and 60 colorectal cancer patients. Exosomes were extracted from the samples, and the expression level of circular RNA circ-PRKACB in the exosomes was detected. The results are as follows: Figure 15 As shown in C, the expression level of circ-PRKACB in the serum exosomes of colorectal cancer patients was significantly higher than that of non-colorectal cancer patients, suggesting the promoting effect of circ-PRKACB in colorectal cancer.

[0049] (4) Serum samples were collected from 60 patients with colorectal cancer. Exosomes were extracted from the samples, and the expression level of circular RNA circ-PRKACB in the exosomes was detected. Among them, 19 patients had a BMI higher than 25, and 41 patients had a BMI lower than 25. The results are as follows: Figure 15As shown in Figure D, the expression level of the circular RNA circ-PRKACB in serum exosomes of patients with a BMI higher than 25 was significantly higher than that of patients with a low BMI. This suggests that in colorectal cancer patients, a high-fat environment induces greater uptake of circ-PRKACB by colorectal cancer cells, thereby further promoting cancer progression.

[0050] Example 5: Circular RNA circ-PRKACB as a therapeutic target for colorectal cancer (1) Design siRNA si-circPRKACB #1-3 to knock down the expression of circular RNA circ-PRKACB in human colorectal cancer cells. The nucleotide sequences are shown in SEQ ID NO.4-5, SEQ ID NO.6-7 and SEQ ID NO.8-9, respectively.

[0051] In human colorectal cancer cells HCT116, transient transfection with siRNA knocked down the expression level of circular RNA circ-PRKACB, and the results were as follows: Figure 16 As shown, the three siRNAs can knock down the expression level of circular RNA circ-PRKACB without affecting the expression of mRNA-PRKACB.

[0052] (2) Migration assays were performed on human colorectal cancer cells HCT116 and DLD-1, respectively. The results are as follows: Figure 17 and Figure 18 As shown, transient transfection with knockdown of circular RNA circ-PRKACB in human colorectal cancer cells significantly reduced cell migration ability. The migration assay results indicate that the reduction in migration ability was more pronounced in human colorectal cancer cells whose circular RNA expression was knocked down using si-circPRKACB#2 and si-circPRKACB#3 sequences.

[0053] Based on the sequences of si-circPRKACB#2 and si-circPRKACB#3, sh-circPRKACB#2 and sh-circPRKACB#3 were designed, with nucleotide sequences shown in SEQ ID NO.10-11 and SEQ ID NO.12-13, respectively. These were used to construct a cell line in human colorectal cancer cells HCT116 with stable knockdown of the circular RNA circ-PRKACB. Migration assays and scratch assays were performed, and the results are shown below. Figure 19 and Figure 20 As shown, stable knockdown of circular RNA circ-PRKACB expression significantly reduced cell migration ability.

[0054] A mouse model of lung metastasis via tail vein was constructed by knocking down circ-PRKACB expression. Three stable HCT116 cell lines—lentiviral vectors LV3, sh-circPRKACB#2, and sh-circPRKACB#3—were injected into the tail vein of BALB / c-nude mice. The results were as follows: Figure 21 As shown, mice in the sh-circPRKACB#2 and sh-circPRKACB#3 groups had a lower lung-to-body weight ratio, smaller HE lesion area, and longer survival time.

[0055] Constructing a high-fat nude mouse fat loss model, the construction process is as follows: Figure 22 As shown, the stable overexpression cell line HCT116 PLDCH was constructed using the PLDCH control vector, and a stable overexpression cell line was constructed using a lentivirus overexpressing circular RNA circ-PRKACB. Male BALB / c nude mice aged 3-5 weeks were purchased and fed a high-fat diet for 2 weeks, then divided into two groups. HCT116PLDCH and HCT116 circ-PRKACB overexpression stable cell lines were injected via tail vein, respectively. After 4 weeks of feeding a high-fat diet, one group continued to be fed a high-fat diet, while the other group was switched to a normal mouse diet for fat reduction. After 5 weeks, the mice were sacrificed, and lung metastasis was observed. The gross lung condition is shown in the figure. Figure 23 As shown in the figure. HE staining experiment was performed on mouse lung tissue, and the results are as follows. Figure 24 and Figure 25 As shown, mice overexpressing the circular RNA circ-PRKACB had more lung metastases, while mice in the fat-reduced circ-PRKACB overexpression group had significantly fewer lung metastases. This confirms that serum circ-PRKACB levels increase under a high-fat environment, and are further taken up by tumor cells via exosomes. When the diet was changed from high-fat to normal, serum circ-PRKACB levels decreased, and colorectal cancer metastasis was significantly reduced. Fat reduction alleviated the promoting effect of circ-PRKACB on colorectal cancer progression.

[0056] The above experimental results reveal that the circular RNA circ-PRKACB has the potential to serve as a therapeutic target for colorectal cancer.

[0057] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A kit for assessing the risk or prognosis of colorectal cancer, characterized in that: The kit contains reagents for detecting the expression level of circular RNA in a sample to be tested, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The reagent kit according to claim 1, characterized in that: The kit includes primers for amplifying circular RNA, the primers including a forward primer and a reverse primer, the nucleotide sequence of the forward primer being shown in SEQ ID NO.2, and the nucleotide sequence of the reverse primer being shown in SEQ ID NO.

3.

3. A drug for treating colorectal cancer, characterized in that: The colorectal cancer treatment drug targets circular RNA, the nucleotide sequence of which is shown in SEQ ID NO.

1. The colorectal cancer treatment drug reduces the expression of circular RNA. The treatment drug includes small interfering RNA or short hairpin RNA, the nucleotide sequence of which is shown in SEQ ID NO.4-5, SEQ ID NO.6-7 or SEQ ID NO.8-9, and the nucleotide sequence of which is shown in SEQ ID NO.10-11 or SEQ ID NO.12-13.

4. The application of agents that reduce circular RNA expression in the preparation of drugs for the treatment of colorectal cancer, characterized in that, The nucleotide sequence of the circular RNA is shown in SEQ ID NO.

1. The formulation includes small interfering RNA or short hairpin RNA. The nucleotide sequence of the small interfering RNA is shown in SEQ ID NO.4-5, SEQ ID NO.6-7 or SEQ ID NO.8-9. The nucleotide sequence of the short hairpin RNA is shown in SEQ ID NO.10-11 or SEQ ID NO.12-13.

5. The application of small interfering RNA or short hairpin RNA in the preparation of drugs for colorectal cancer treatment, characterized in that: The nucleotide sequences of the small interfering RNA are shown in SEQ ID NO.4-5, SEQ ID NO.6-7 or SEQ ID NO.8-9, and the nucleotide sequences of the short hairpin RNA are shown in SEQ ID NO.10-11 or SEQ ID NO.12-13.

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