Application of circular RNA as colorectal cancer biomarker and therapeutic target

Abstract

The invention relates to application of circular RNA (Ribonucleic Acid) as a colorectal cancer biomarker and a treatment target. In-vivo and in-vitro experiments prove that the migration ability of colorectal cancer cells is remarkably improved in a high-fat environment so as to promote the colorectal cancer process. The circ-PRKACB with remarkably increased expression in colorectal cancer cells in a high-fat environment is obtained through circular RNA sequencing, and exosomes secreted by fat cells are transported and then are taken by the colorectal cancer cells. Clinical samples verify that the expression of the circ-PRKACB in cancer tissues of colorectal cancer patients is higher than that in para-carcinoma tissues, the expression of the circ-PRKACB in serum of colorectal cancer patients is higher than that in serum of non-colorectal cancer patients, the expression quantity is in positive correlation with BMI, and the circ-PRKACB can be used as a biomarker for risk or prognosis evaluation of colorectal cancer. In a mouse colorectal cancer lung metastasis model, circ-PRKACB expression is knocked down, mouse lung metastasis lesions are reduced, the number of survival days is increased, and the circular RNA has the potential of serving as a colorectal cancer treatment target.

Description

Technical Field

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

[0002] Tumor is a complex disease involving a variety of abnormal gene expression regulation. Circular RNA (circRNA) is a special type of non-coding RNA molecule. Unlike traditional linear RNA, circRNA molecules have a closed circular structure, are not affected by RNA exonucleases, are more stably expressed, and are not easily degraded. More and more evidence shows that circular RNA is involved in regulating the occurrence of various tumors, promoting the proliferation of tumor cells, regulating the cell cycle process, etc. If we can deeply understand and discover the role of specific circular RNA in the progression of colorectal cancer, there is hope that we can achieve early detection and diagnosis of colorectal cancer, predict metastasis, and timely and reasonable comprehensive treatment including radical surgery in the clinic, which can greatly improve the survival rate and quality of life of 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 show that exosomes can reshape the tumor microenvironment, affect tumor angiogenesis, tumor growth, and tumor metastasis. Exosomes contain microRNAs, long noncoding RNAs, circular RNAs, and other genes that affect cancer progression, and can deliver various drugs. Therefore, some studies have suggested that they may be used as delivery systems for anti-tumor drugs and genetic tools in cancer treatment. Exosomes are present in all biological fluids and are secreted by all cells, which makes exosomes attractive in liquid biopsies. In the existing technology, the gold standard for colorectal cancer examination is colonoscopy, which is too invasive and difficult for patients to accept. Common tumor markers cannot accurately locate colorectal lesions. The discovery of biomarkers in serum exosomes is of great significance for colorectal cancer screening. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides an application of circular RNA as a biomarker and therapeutic target for colorectal cancer. The present invention confirms through in vivo and in vitro experiments that a high-fat environment significantly improves the migration ability of colorectal cancer cells and thus promotes the progression of colorectal cancer, thereby obtaining a cAMP-dependent protein kinase catalytic subunit β circular RNA (circ-PRKACB) whose expression is significantly increased in colorectal cancer cells under a high-fat environment through circular RNA sequencing screening. The circular RNA can be transported by exosomes secreted by adipocytes and then taken up by colorectal cancer cells. It has been verified by clinical samples of colorectal cancer patients that circ-PRKACB is expressed higher in cancer tissues of colorectal cancer patients than in adjacent tissues, and its expression in the serum of intestinal cancer patients is higher than that of non-intestinal cancer patients, and its expression in serum is positively correlated with the body mass index (BMI) of colorectal cancer patients. In the mouse colorectal cancer lung metastasis model, the expression of circular RNA circ-PRKACB was knocked down, and the lung metastasis lesions of mice were reduced and the number of survival days was increased.

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

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

[0007] The second object of the present invention is to provide a kit for evaluating the risk or prognosis of colorectal cancer, wherein the kit comprises a reagent for detecting the expression amount of circular RNA circ-PRKACB in a sample to be tested.

[0008] The increased expression level of circular RNA circ-PRKACB in the tested sample reveals that the sample is at risk of colorectal cancer or suggests that the sample has a poor prognosis for 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 circular RNA circ-PRKACB.

[0011] Furthermore, the kit includes primers for amplifying 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 as SEQ ID NO.2, and the nucleotide sequence of the reverse primer is shown as 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 a component for PCR amplification.

[0016] The third object of the present invention is to provide the use of circular RNA circ-PRKACB as a therapeutic target for colorectal cancer, wherein the nucleotide sequence of the circular RNA circ-PRKACB is shown in SEQ ID NO.1.

[0017] The fourth object of the present invention is to provide a drug for treating colorectal cancer, which targets circular RNA circ-PRKACB.

[0018] Furthermore, the colorectal cancer treatment is to reduce the expression of circular RNA circ-PRKACB.

[0019] A fifth object of the present invention is to provide a preparation for reducing the expression of circular RNA circ-PRKACB for use in the preparation of a drug for treating colorectal cancer.

[0020] Furthermore, the preparation includes 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 the 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 the 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 the 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 the 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 the 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 treatment drugs, the nucleotide sequence of the small interfering RNA is shown as 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 as SEQ ID NO.10-11, 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] Beneficial effects of the present invention: The present invention provides the application of circular RNA circ-PRKACB as a biomarker and therapeutic target for colorectal cancer, and the risk of colorectal cancer and the prognosis of the subject to be tested can be evaluated by detecting the expression of circ-PRKACB in the sample to be tested. In a mouse colon cancer lung metastasis model, the expression of circ-PRKACB was knocked down, the lung metastasis lesions of the mice were reduced, and the number of survival days was increased. The circular RNA has the potential to be used as a therapeutic target for colorectal cancer, and the tumor suppression effect can be achieved by targeting circ-PRKACB, thereby producing drugs that inhibit the progression of colorectal tumors, providing a possibility for the clinical treatment of colorectal tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein Figure 1 The high-fat diet-induced obesity mouse colorectal cancer lung metastasis model constructed in Example 1 of the present invention, wherein A is a schematic diagram of the model construction steps, B is a macroscopic photo 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 of co-culturing the supernatant of mouse primary adipocytes induced to differentiate (differentiation group CM) and not induced to differentiate (control group CON) with human colorectal cancer cells in Example 1 of the present invention, wherein A is a schematic diagram of the experimental steps, B is a graphical representation of the migration assay results after the supernatant of the control group and the differentiation group was co-cultured with human colon cancer cells HCT116, colon adenocarcinoma cells RKO and mouse colon cancer cells CMT-93, respectively, with a scale of 50 μm, and C is the statistical results of the number of migrated cells in different experimental groups; Figure 3 This is a graph showing the results of conditioning human intestinal cancer organoids with the culture supernatant of human adipose-derived mesenchymal stem cells of the control group and the differentiation group in Example 1 of the present invention, with a scale of 200 μm; Figure 4 This is a graph showing the migration assay results of the mouse embryonic fibroblasts (3T3-L1) induced differentiation (differentiation group CM) and non-induced differentiation (control group CON) cell supernatants in Example 1 of the present invention after long-term conditional culture of human colorectal cancer cells, with a scale of 50 μm; Figure 5 The nude mouse tail vein lung metastasis model constructed in Example 1 of the present invention, wherein A is a diagram of the model construction steps, B is a diagram of the lung macrophotograph 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 the lung metastasis area; Figure 6It is a heat map of circular RNA sequencing results of human colorectal cancer cell HCT116 after 17 weeks of conditional culture using cell supernatants of mouse embryonic fibroblasts (3T3-L1) induced differentiation (differentiation group CM) and non-induced differentiation (control group CON) in Example 2 of the present invention, wherein control groups 1-3 and differentiation groups 1-3 were repeated in 3 groups in parallel; Figure 7 It is a volcano graph of circular RNA sequencing results of human colorectal cancer cell HCT116 after 17 weeks of conditional culture using cell supernatants of mouse embryonic fibroblasts (3T3-L1) induced differentiation (differentiation group CM) and non-induced differentiation (control group CON) in Example 2 of the present invention; Figure 8 It is a graph showing the expression level detection results of five circular RNAs after short-term and long-term conditional culture of the supernatants of mouse embryonic fibroblasts (3T3-L1) induced to differentiate (differentiation group CM) and non-induced to differentiate (control group CON) with human colon cancer cells HCT-116 and human colorectal cancer epithelial cells DLD-1 in Example 2 of the present invention; Fig. 9 is a diagram showing the results of dideoxy sequencing (Sanger sequencing) of circ-PRKACB in human colon cancer cell HCT-116 in Example 2 of the present invention; Fig.10 This is an electron microscopic image of exosomes of the exosome detection result in Example 3 of the present invention, with a scale of 100 nm; Fig.11 is a graph showing the nanoparticle tracking analysis results of the exosome detection results in Example 3 of the present invention; Fig.12 This is a graph of the exosome tracer staining results of the exosome detection results in Example 3 of the present invention, with a scale of 50 μm; Fig.13 It is the conditional culture of human intestinal cancer organoids secreted by exosomes from human adipose-derived mesenchymal stem cells in the control group and the differentiation group in Example 3 of the present invention, wherein A is a diagram under an optical microscope, with a scale of 200 μm, and B is a diagram showing the expression level of circular RNA circ-PRKACB in the control group (CON) and the differentiation group (CM) organoids, respectively; Fig.14 3 is a functional experimental result diagram of the exosomes secreted by human adipose-derived mesenchymal stem cells in the control group (CON) and the differentiation group (CM) in Example 3 of the present invention to conditionally culture human colon cancer cells, wherein A is a migration experimental result diagram when the two groups of exosomes are added to human colon cancer cells, with a scale of 100 μm, and B is the specific number of migrated cells in the control group and the differentiation group; C is a result diagram of the expression level of circular RNA circ-PRKACB when the exosomes of the control group and the differentiation group are added to the culture of human colon cancer cells; Fig.15 It is the analysis result of tissue and serum samples of clinical patients with colorectal cancer in Example 4 of the present invention, wherein A is a difference diagram of circular RNA circ-PRKACB expression levels in tumor tissue and adjacent tissue of colorectal cancer patients; B is a difference diagram of circular RNA circ-PRKACB difference multiple levels between cancer and adjacent tissue of colorectal cancer patients with different BMI; C is a difference diagram of circular RNA circ-PRKACB expression levels in serum exosomes of colorectal cancer and non-intestinal cancer patients; D is a difference diagram of circular RNA circ-PRKACB expression levels in serum exosomes of colorectal cancer patients with different BMI; Fig.16 It is the difference in expression of circular RNA circ-PRKACB and mRNA-PRKACB after transient transfection of colorectal cancer cells with small interfering RNA in Example 5 of the present invention; Fig.17 The results of the migration assay after transient transfection of human colorectal cancer cells HCT116 and DLD-1 with small interfering RNA in Example 5 of the present invention, with a scale of 50 μm; Fig.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; Fig.19 This is the colorectal cancer cell migration experiment after stable lentiviral transfection and knockdown of circular RNA circ-PRKACB in Example 5 of the present invention, and the scale is 50 μm; Fig. 20 This is the result of the colorectal cancer cell scratch experiment after stable lentiviral transfection and knockdown of circular RNA circ-PRKACB in Example 5 of the present invention; Fig.21 : is a graph showing the experimental results of the lung metastasis model of mice injected with the tail vein of the HCT116 stable cell line with knockdown of circular RNA circ-PRKACB in Example 5 of the present invention, wherein A is a macroscopic and HE photo of the lung, B is the lung weight to body weight ratio of mice in different stable cell treatment groups, and C is a survival curve of mice in different treatment groups; Fig. 22 This is the step of constructing the high-fat nude mouse fat reduction model in Example 5 of the present invention; Fig.23 is a macroscopic photograph of mouse lungs under high-fat and fat-reduced conditions in Example 5 of the present invention; Fig.24 This is a graph showing the results of HE immunostaining of mouse lungs under high-fat and fat-reduced conditions in Example 5 of the present invention; Fig.25This is a diagram showing the difference in lung metastasis area among different groups of mice in Example 5 of the present invention, wherein PLCDH and circ-PRKACB represent different groups of mice injected with tail veins of different stable cell lines constructed by PLCDH control vector and circular RNA circ-PRKACB overexpressing lentivirus. DETAILED DESCRIPTION

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

[0034] Example 1: High-fat environment promotes the progression of colorectal cancer (1) 4-6 week old C57BL / 6 mice were purchased and divided into two groups, and fed a high-fat diet and a normal diet, respectively. After 4 weeks, mouse colon cancer cells MCA38 were injected into the tail vein of the mice. After 3 weeks of high-fat feeding, the mice were killed. The experimental process is as follows: Figure 1 As shown in A, the lungs of the two groups of mice were observed. Figure 1 As shown in B, the number of lung metastases in the high-fat diet group mice increased significantly. HE staining experiments were performed on the lung tissues of the two groups of mice: the mice were killed and the lung tissues were fixed in formaldehyde, dehydrated with gradient alcohol, embedded in paraffin, trimmed and sliced ​​into 3 mm slices, dewaxed with xylene, and hydrated with 100%, 95%, 90% and 80% ethanol in sequence, then stained with hematoxylin and eosin, and sealed. The results are shown in Figure 1. Figure 1 As shown in center C, mice fed a high-fat diet had more and larger lung metastases than mice fed a normal diet, suggesting that high-fat feeding promotes the metastasis of colorectal cancer.

[0035] (2) 3-4 week old C57BL / 6 mice were purchased, and primary mouse adipocytes were isolated from subcutaneous adipose tissue. The isolated primary adipocytes were divided into two groups and seeded into the lower chamber of a 24-well plate. DMEM complete medium was used for culture. One group was induced to differentiate into mature adipocytes by adding phosphodiesterase inhibitor IBMX, insulin INS and dexamethasone DEX, namely the differentiation group (CM group). The other group was not induced to differentiate as the control group (CON group). The process was as follows: Figure 2 As shown in A. Human colon cancer cells HCT116, colon adenocarcinoma cells RKO, and mouse colon cancer cells CMT-93 were seeded into the upper chamber of a 24-well plate and co-cultured with primary adipocytes from mice in the differentiation group and control group for 28 hours. Migration assays were performed. The results are shown in Figure 2 As shown in B and C, the migration ability of colon cancer cells co-cultured with differentiation group cells was significantly higher than that of colon cancer cells co-cultured with control group cells, that is, 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 above, namely, one group was an induced differentiation group (CM group) and the other group was a control group (CON group) without induced differentiation. The supernatants produced by the two groups of cells were used to treat human colorectal cancer organoids, i.e., conditional culture was performed. The results are shown in Figure 2. Figure 3 As shown, colorectal cancer organoids cultured using the supernatant of differentiation group 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 above, namely, one group was an induced differentiation group (CM group) and the other group was a control group (CON group) without induced differentiation. The cell supernatants of the differentiation group and the control group were used to treat human colorectal cancer cells HCT116 and mouse colon cancer cells CMT-93, respectively, for conditional culture for 12 weeks. The results of the migration assay were as follows: Figure 4 As shown, the migration ability of colorectal cancer cells cultured with the supernatant of mature adipocytes in the differentiation group was significantly higher than that in the control group.

[0038] (5) 3T3-L1 cells (mouse embryonic fibroblasts) were divided into two groups as above, one for induced differentiation (CM group) and the other for non-induced differentiation control (CON group). The culture supernatants of the differentiation group and control group cells were used to treat human colorectal cancer cells HCT116 for 17 weeks, resulting in long-term treated HCT116 cells (CM / CON). 2×10 6 The cells obtained after culture were injected into mice through the tail vein to establish a nude mouse tail vein lung metastasis model. The mice were killed after 9 weeks. The experimental process was as follows: Figure 5 As shown in A. The mouse lungs were observed and HE staining was performed. The experimental results are shown in Figure 5 As shown in Figure B, compared with the control group, the number of lung metastases in mice cultured with colorectal cancer cells for a long time with the supernatant of the differentiation group increased. The proportion of lung tissue weight in the body weight of mice and the area of ​​lung metastases in mice were calculated respectively. The results are shown in Figure 2. Figure 5 As shown in C, after tail vein injection of HCT116 cells cultured in the differentiation group, the weight of the mouse lung tissue and the proportion of body weight increased significantly, and the area of ​​lung metastasis increased significantly compared with the control group, indicating that the supernatant secreted by mature adipocytes stably promoted the progression of human colon cancer cells HCT116.

[0039] Example 2: Circular RNA circ-PRKACB is highly expressed in colorectal cancer cells (1) Mouse embryonic fibroblasts 3T3-L1 were divided into two groups, one group was induced to differentiate into mature adipocytes, namely the differentiation group (CM group), and the other group was not induced to differentiate as the control group (CON group). The culture supernatant of the cells in the differentiation group and the control group was used to treat human colorectal cancer cells HCT116 for 17 weeks. After the culture was completed, three groups of human colon cancer cells HCT116 in the CM group and the CON group were set up for repeated circular RNA sequencing. The heat map and volcano map results are shown in the following figure. Figure 6 and Figure 7 As shown, the contents of circular RNA 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 environment were significantly higher than those in the control group.

[0040] (2) The culture supernatants of the differentiation group and the control group were used to treat human colon cancer cells HCT-116 and human colorectal cancer epithelial cells DLD-1 for short-term (3 weeks) and long-term (13 weeks) to detect the expression levels of circular RNA circ-PRKACB, circ-RAPGEF, circ-CCSER2, circ-GLIS3 and circ-NECTIN3. The results are shown in Figure 8 As shown in the figure, only the expression of circ-PRKACB and circ-RAPGEF5 in the differentiated group colorectal cancer cells was stably higher than that in the control group. Considering that the abundance of circ-RAPGEF5 in colorectal cancer cells is too low, its direct biological effect may be relatively small, so subsequent studies focus on the role of circ-PRKACB in the progression of colorectal cancer.

[0041] (3) The circular RNA circ-PRKACB (hsa_circ_0004083) in human colon cancer cell HCT116 was sequenced by dideoxy sequencing (Sanger sequencing). The results are as follows: Fig. 9 As shown, this circular RNA is stably present in human colorectal cancer cells.

[0042] Example 3: Exosomes transport circular RNA circ-PRKACB to colorectal cancer cells 1. The culture supernatant of mouse primary adipocytes was subjected to electron microscopy and NTA (nanoparticle tracking analysis) experiments. The results were as follows: Fig.10 and Fig.11 As shown, it is confirmed that exosomes exist in the supernatant of primary adipocyte culture. The PKH67 (exosome tracer dye) experiment was performed on the supernatant of primary human adipose-derived mesenchymal stem cells. The results are shown in Fig.12 As shown, exosomes were confirmed to enter human colon cancer cells HCT-116.

[0043] The steps of the PKH67 exosome tracer dye experiment are as follows: (1) Exosomes were quantified using bicinchoninic acid (BCA) to obtain the exosome concentration; (2) Preparation of dye working solution: Mix PKH67 and universal membrane marker diluent (Diluent C) at a ratio of 1:10 and use immediately after preparation; (3) Add 200 μg of exosomes to 15 μL of dye working solution in a brown tube (protected from light), vortex to mix for 1 minute, and then 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) Extract exosomes again using an exosome extraction kit (protected from light) to remove excess dye; (6) 2×10 in 24-well plate 5 The HCT116 cells were seeded on the cell slides, and after 24 hours, the exosome dye complex was incubated with the HCT116 cells in a 37°C 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 min, and wash the cells three times with PBS buffer (wash on a shaker for 3 min each time, protected from light); (8) Add 4',6-diamidino-2-phenylindole (DAPI) dye to stain the cell nucleus (protect from light and incubate on a shaker for 30 minutes), discard the staining solution and wash three times with PBS, protect from light for 3 minutes each time; (9) Place the cell slide upside down on a glass slide, seal the slide and store it away from light, and take fluorescent photos using a confocal microscope.

[0044] Second, the 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, namely the differentiation group (CM group), and the other group was not induced to differentiate as the control group (CON group). Exosomes were extracted from the cell supernatant of the differentiation group and the control group, respectively, and cultured in human intestinal cancer organoids for 4 days. Under the microscope, it can be seen that the organoids in the differentiation group grew faster, and the exosomes in the differentiation group promoted the growth of human intestinal cancer organoids, such as Fig.13 As shown in A. The expression level of circular RNA circ-PRKACB in human colorectal cancer organoids under different treatments was determined. The results are shown in Fig.13 As shown in B, after the differentiation group was cultured with exosomes, the expression level of circular RNA circ-PRKACB in human colorectal cancer organoids was significantly higher than that in the control group.

[0045] 3. The isolated primary human adipose-derived mesenchymal stem cells were divided into two groups as above, namely, one group was induced differentiation group (CM group) and the other group was not 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, and the migration assay was performed on the cultured cells. The results are as follows Fig.14 As shown in A and B in Figure 3, the migration ability of human colorectal cancer cells in the differentiation group after exosome culture was increased. The expression level of circular RNA circ-PRKACB in HCT116 cells after exosome culture in the two groups was measured. The results are shown in Fig.14 As shown in Figure C, the expression level of circular RNA circ-PRKACB in colorectal cancer cells treated with exosomes in the differentiation 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 levels of circular RNA circ-PRKACB in cancer tissues and adjacent tissues of 74 clinical colorectal cancer patients were statistically analyzed. Fig.15 As shown in A, the expression level of circ-PRKACB in cancer tissues was significantly higher than that in adjacent adjacent tissues.

[0047] (2) The difference in the expression levels of circular RNA circ-PRKACB between cancer and adjacent tissues of 74 patients with colorectal cancer and different body mass indexes (BMI) was statistically analyzed. Among them, 19 patients had a BMI higher than 24, and 55 patients had a BMI lower than 24. Fig.15 As shown in Figure 2B, 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-colon cancer patients and 60 colon cancer patients, exosomes were extracted from them, and the expression level of circular RNA circ-PRKACB in exosomes was detected. The results are as follows: Fig.15 As shown in Figure C, the expression level of circ-PRKACB in serum exosomes of colorectal cancer patients was significantly higher than that of non-colorectal cancer patients, suggesting the promoting role of circ-PRKACB in colorectal cancer.

[0049] (4) Serum samples from 60 patients with colorectal cancer were collected, exosomes were extracted from them, 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 level lower than 25. The results are as follows Fig.15As shown in D, the expression level of circular RNA circ-PRKACB in serum exosomes of patients with BMI higher than 25 was also significantly higher than that of patients with low BMI. This suggests that in patients with colorectal cancer, the high-fat environment induces more circ-PRKACB to be taken up by colorectal cancer cells, thereby further promoting the progression of cancer.

[0050] Example 5: Circular RNA circ-PRKACB as a therapeutic target for colorectal cancer (1) siRNA si-circPRKACB #1-3 were designed 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] The expression level of circular RNA circ-PRKACB was knocked down by transient transfection with siRNA in human colorectal cancer cells HCT116. Fig.16 As shown, it can be seen that the three siRNAs will not affect the expression of mRNA-PRKACB while knocking down the expression level of circular RNA circ-PRKACB.

[0052] (2) Migration assays were performed in human colorectal cancer cells HCT116 and DLD-1. The results are as follows: Fig.17 and Fig.18 As shown in the figure, after transient transfection and knockdown of circular RNA circ-PRKACB expression in human colorectal cancer cells, the cell migration ability was significantly reduced. The migration experiment results show that the migration ability of human colorectal cancer cells with circular RNA expression knocked down by si-circPRKACB#2 and si-circPRKACB#3 sequences was more significantly reduced.

[0053] According to the sequences of si-circPRKACB#2 and si-circPRKACB#3, sh-circPRKACB#2 and sh-circPRKACB#3 were designed, and their nucleotide sequences were shown in SEQ ID NO.10-11 and SEQ ID NO.12-13, respectively, and used to construct cell lines with stable knockdown of circular RNA circ-PRKACB expression in human colorectal cancer cell HCT116, and migration assay and scratch assay were performed, and the results were shown respectively. Fig.19 and Fig. 20 As shown in the figure, it can be seen that after stable knockdown of circular RNA circ-PRKACB expression, the cell migration ability was significantly reduced.

[0054] A mouse tail vein lung metastasis model with knockdown of circ-PRKACB expression was constructed. The three HCT116 cell stable lines, lentiviral vector LV3, sh-circPRKACB#2, and sh-circPRKACB#3, were injected into the tail vein of BALB / c-nude mice. Fig.21 As shown, it was found that the lung weight to body weight ratio of mice in the sh-circPRKACB#2 and sh-circPRKACB#3 groups was decreased, the HE cancer lesions were smaller, and the survival time was longer.

[0055] The high-fat nude mouse fat loss model was constructed. The construction process was as follows: Fig. 22 As shown, the PLDCH control vector was used to construct the overexpression cell stable strain HCT116 PLDCH, and the circular RNA circ-PRKACB overexpression lentivirus was used to construct the overexpression cell stable strain. 3-5 week old BALB / c male nude mice were purchased and fed with high-fat food for 2 weeks and then divided into two groups. HCT116PLDCH and HCT116 circ-PRKACB overexpression stable strain cells were injected through the tail vein, respectively. After feeding high-fat food for 4 weeks, one group continued to be fed with high-fat food, and the other group was replaced with normal mouse food for fat reduction. After 5 weeks, the mice were killed and the lung metastasis was observed. The gross condition of the lungs is shown in the figure. Fig.23 HE staining experiment was performed on mouse lung tissue, and the results are shown in Fig.24 and Fig.25 As shown, mice in the group overexpressing circular RNA circ-PRKACB had more lung metastases, while mice in the group overexpressing circ-PRKACB who underwent fat loss had significantly fewer lung metastases. This confirmed that the content of circ-PRKACB in serum increased under a high-fat environment, and was further taken up by tumor cells through exosomes. When the high-fat diet was changed to a normal diet, the content of circ-PRKACB in serum decreased, and colorectal cancer metastasis was significantly reduced. Fat loss alleviated the promotion of circular RNA circ-PRKACB on the progression of colorectal cancer.

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

[0057] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. Application of circular RNA as a biomarker for colorectal cancer, characterized by: The nucleotide sequence of the circular RNA is shown in SEQ ID NO.

1.

2. A kit for assessing the risk or prognosis of colorectal cancer, characterized in that: The kit comprises a reagent for detecting the expression amount of circular RNA in a sample to be tested, and the nucleotide sequence of the circular RNA is shown in SEQ ID NO.

1.

3. The kit according to claim 2, characterized in that: The kit includes primers for amplifying circular RNA, wherein the primers include a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown as SEQ ID NO.2, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.

3.

4. Application of circular RNA as a therapeutic target for colorectal cancer, characterized in that: The nucleotide sequence of the circular RNA is shown in SEQ ID NO.

1.

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

1.

6. The drug for treating colorectal cancer according to claim 5, characterized in that: The colorectal cancer therapeutic drug reduces the expression of circular RNA.

7. Use of a preparation for reducing circular RNA expression in the preparation of a drug for treating colorectal cancer, wherein the nucleotide sequence of the circular RNA is shown in SEQ ID NO.

1.

8. The use according to claim 7, characterized in that: The agent includes small interfering RNA or short hairpin RNA.

9. The use according to claim 8, characterized in that: 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.

10. Use of small interfering RNA or short hairpin RNA in the preparation of a drug for treating colorectal cancer, characterized in that: 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.

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