CN114836537B - Use of Rbm24 in diagnosis and treatment of colorectal cancer - Google Patents
Use of Rbm24 in diagnosis and treatment of colorectal cancer Download PDFInfo
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- CN114836537B CN114836537B CN202110137668.0A CN202110137668A CN114836537B CN 114836537 B CN114836537 B CN 114836537B CN 202110137668 A CN202110137668 A CN 202110137668A CN 114836537 B CN114836537 B CN 114836537B
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Abstract
The invention discloses the use of Rbm24 in the diagnosis and treatment of colorectal cancer.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to application of Rbm24 in diagnosis and treatment of colorectal cancer.
Background
The incidence and mortality of colorectal cancer are the leading part of Chinese cancer diseases, and diagnosis, molecular typing and treatment thereof are important problems which are urgently needed to be solved clinically. Colorectal cancer can be diagnosed and assisted by methods including pathological biopsy, imaging examination, laboratory testing, and gene sequencing. Early colorectal cancer symptoms are not obvious, however, patients often do not seek medical attention, and therefore the opportunity for early diagnosis and treatment is lost. Most colorectal cancer patients are already at advanced stages of the disease when diagnosed, and distant metastasis occurs. In diagnosed patients, various clinical indicators cannot better predict the therapeutic effect of the patients and the prognosis and survival time, and the specificity and sensitivity are poor. New indicators are therefore needed to assist in molecular typing of colorectal cancer and to predict the therapeutic effect, prognosis and survival time of patients.
At present, the first-line treatment of colorectal cancer is still mainly performed by combining surgery with chemotherapy and radiotherapy, and is simultaneously assisted with targeted treatment, immunotherapy, biological treatment, palliative treatment and the like. Overall, the therapeutic effect of colorectal cancer in china is poor, the 5-year survival rate is slightly increased, but the overall effect is still unsatisfactory, and the occurrence rate of side effects is high, which seriously affects the quality of life of patients, so that the development of new therapeutic means is urgent.
Disclosure of Invention
In view of the problems with the prior art, the present invention provides a marker Rbm24 for colorectal cancer diagnosis and its use in colorectal cancer treatment.
In one aspect, the invention provides the use of Rbm24 as a biomarker in colorectal cancer.
In a specific embodiment, the invention provides the use of a substance for determining the level of Rbm24 in the manufacture of a product for diagnosing colorectal cancer. Specifically, the reagent for detecting the expression level of Rbm24 comprises a reagent for detecting the expression level of mRNA of an Rbm24 gene and/or a reagent for detecting the expression level of Rbm24 protein. In a specific embodiment, the reagent for detecting the mRNA expression level of the Rbm24 gene comprises primers, and/or probes used in real-time quantitative PCR. In a specific embodiment, wherein the reagent for detecting the expression level of an Rbm24 protein comprises an antibody directed against an Rbm24 protein. In a specific embodiment, the product for diagnosing colorectal cancer comprises a kit, a chip or a test paper.
In a second aspect, the invention provides the use of Rbm24 as a drug target in the prevention and treatment of colorectal cancer. In a specific embodiment, the invention provides the use of an agent that upregulates expression of the Rbm24 gene or expression of the Rbm24 protein encoded by the Rbm24 gene in the manufacture of a medicament for the treatment of colorectal cancer.
RBM24 is an RNA binding protein. The inventors found that: RBM24 expression was significantly down-regulated in clinical samples from colorectal cancer patients, and patients with high RBM24 expression survived significantly longer than patients with low RBM24 expression. Experiments in mice have demonstrated that RBM24 knockdown results in abnormal intestinal proliferation in mice and eventually spontaneous colorectal adenomas. These results suggest that RBM24 can be a diagnostic marker for colorectal cancer.
A first object of the present invention is to provide the use of Rbm24 for colorectal cancer diagnosis.
By examining the expression of RBM24 in human CRC tissue and paired adjacent tissue, it was found that: RBM24 expression was significantly reduced in human CRC tissue compared to adjacent tissue, which correlated with TNM staging and patient survival (n=36). However, there is no correlation between RBM24 and other clinical characteristics of CRC patients, including age and gender. Immunohistochemical results showed a significant decrease in RBM24 expression in CRC tumor tissue. qPCR and western blot results showed that RBM24 expression was significantly down-regulated in human CRC tumor tissue.
A conditional RBM24 gene knockout mouse model was constructed and tamoxifen (100 mg/kg per day for 5 consecutive days) was intraperitoneally injected into C57BL/6 mice (n=32) for 8 weeks to induce systemic RBM24 knockout. WT mice were injected with the same dose of tamoxifen as a control group. Knock-out efficiency was verified using western blot, qPCR analysis and immunofluorescence. The results showed that RBM24 knockout mice showed a significant increase in BrdU (5-bromodeoxyuridine) positive cells in the gut compared to WT mice. HE staining results indicated that RBM24 knockout mice had thickened colonic epithelium. After 6 months of knockout, the mice were observed to develop colorectal adenomas, and the length of the colorectal was significantly longer than that of wild-type mice.
A second object of the invention is to provide the use of Rbm24 for the treatment of colorectal cancer.
To test the role of RBM24 in CRC cell proliferation, a CCK-8 assay was performed. We have found that overexpression of RBM24 can inhibit cell proliferation. This suggests that RBM24 inhibits CRC cell growth in vitro.
In addition, scratch experiments, transwell migration and invasion assays were performed to determine the role of RBM24 in CRC cell migration and invasion. The results demonstrate that over-expression of RBM24 is inhibited by migration and invasion of colorectal cancer cell line (HCT 116) compared to control cells. Treatment with PI3K-IN-6 (10 nM) or MK-2206 (20 nM) further inhibited the ability of the cells to proliferate, migrate and invade.
The data demonstrate that RBM24 overexpression increases 5-FU or cisplatin-induced apoptosis. Treatment with PI3K-IN-6 or MK-2206 further promoted 5-FU or cisplatin-induced CRC apoptosis after RBM24 overexpression.
In the present application, "colon cancer" refers to cancers and/or tumors that form in the colon tissue (the longest part of the large intestine). Most colon cancers are adenocarcinomas (cancers that originate in cells that produce and release mucus and other fluids). "rectal cancer" refers to cancers and/or tumors that form in the rectal tissue (the last few inches of the large intestine before the anus). "colorectal cancer" in the context of the present application refers to cancers that occur in the colon or rectum.
A "sample" herein is a biological sample isolated from a subject, which may include: for example, but not limited to, whole blood, serum, plasma, blood cells, endothelial cells, tissue biopsies, lymph, ascites, interstitial fluid (also known as "extracellular fluid", including fluids found in the interstitial spaces), bone marrow, cerebrospinal fluid (CSF), saliva, mucus, sputum, sweat, urine or any other secretions, excretions or other body fluids.
The "subject" in the present context is preferably a mammal. The mammal may be a human, non-human primate, mouse, rat, dog, cat, horse or cow, but is not limited to these examples. A non-human mammal may advantageously be used as a subject representing an animal model of colorectal cancer. The subject may be male or female. The subject may be a person previously diagnosed or identified as having colorectal cancer, and optionally, but not necessarily, a person who has undergone colorectal cancer treatment. The subject may also be a non-colorectal cancer subject. The subject may also be one who is diagnosed or identified as having colorectal cancer but exhibits an improvement in disease (e.g., a decrease in tumor size) as a result of receiving one or more colorectal cancer treatments. Alternatively, the subject may be one who has not been previously diagnosed or identified as having colorectal cancer. For example, the subject may be a subject that exhibits one or more colorectal cancer risk factors, or a subject that does not exhibit colorectal cancer risk factors or a subject that is free of symptoms of colorectal cancer. The subject may also be a person suffering from colorectal cancer or at risk of developing colorectal cancer.
The amount of Rbm24 protein, peptide, nucleic acid, polymorphism, metabolite, or other analyte in the test sample can be measured and compared to normal control levels. The term "normal control level" refers to the level of one or more Rbm24 proteins, nucleic acids, polymorphisms, metabolites or other analytes or Rbm24 index that typically occur in a subject as follows: subjects who are not and are not likely to have colorectal cancer, e.g., the young subjects are monitored until they are older and are not found to develop colorectal cancer or associated disease sequelae, such as ulcerative colitis, inflammatory bowel disease, and/or crohn's disease, relative to samples collected from longitudinal studies of the young subjects. The normal control level may be a range or an index. Alternatively, the normal control level may be a pattern database of previously tested subjects. A change in the level of one or more Rbm24 proteins, nucleic acids, polymorphisms, metabolites or other analyte samples derived from the subject as compared to a normal control level may be indicative of the subject suffering from or being at risk of developing colorectal cancer. In contrast, when the method is administered prophylactically, a similar level of one or more Rbm24 protein, nucleic acid, polymorphism, metabolite, or other analyte sample derived from the subject as compared to a normal control level may indicate that the subject does not have colorectal cancer or has little or no risk of developing colorectal cancer.
The reference value may refer to a value obtained from a control subject or population whose cancer status is known (i.e., diagnosed or identified as having colorectal cancer, or not diagnosed or identified as having colorectal cancer). The reference value may be an index value or a reference value, such as "normal control level" as defined herein. The reference sample or index or baseline value may be taken from or derived from one or more subjects who have received anti-cancer therapy, radiation therapy or chemotherapy; or from or derived from one or more subjects at low risk of developing colorectal cancer; or from or derived from a subject exhibiting an improvement in colorectal cancer risk factors as a result of receiving treatment. Alternatively, the reference sample or index value or baseline value may be taken from or derived from one or more subjects who have not received anti-cancer therapy, radiation therapy or chemotherapy. For example, samples may be taken from subjects receiving initial treatment for colorectal cancer and subsequent treatment for colorectal cancer to monitor treatment progress. The reference values may also include values derived from risk prediction algorithms or calculation indices of colorectal cancer population studies, such as those disclosed herein. The reference value may also include a value from a subject or population that has suffered from polyps, ulcerative colitis, inflammatory bowel disease, and/or crohn's disease but has not suffered from colorectal cancer.
The difference in the level or amount of Rbm24 (which may be an "effective amount") measured by the methods of the invention may include an increase or decrease in the level or amount of Rbm24 as compared to a normal control level, reference value, index value, or baseline value. An increase or decrease in the amount of Rbm24 relative to a reference value may indicate progression of colorectal cancer; delay, progression, development or alleviation of colorectal cancer; an increased or decreased risk of developing colorectal cancer or its associated complications. An increase or decrease may be indicative of success of one or more colorectal cancer treatment regimens; or may indicate an improvement or regression of colorectal cancer risk factors. The increase or decrease may be, for example, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the reference value or normal control level.
The gene expression disclosed herein can be measured at the RNA level using any method known in the art. For example, northern hybridization assays using probes that specifically recognize one or more of these sequences can be used to determine gene expression. Alternatively, expression may be measured using reverse transcription PCR assays (RT-PCR), for example, using primers specific for differentially expressed sequences. RNA may also be quantified using, for example, other target amplification methods (e.g., TMA, SDA, NASBA) or signal amplification methods (e.g., bDNA), and the like. Preferably, the expression level of the biomarkers of the invention is detected by real-time PCR, as also described in PCT/US02/38806 (published as WO 03/048377; therianos et al, (2004) am. J. Pathol.164 (3): 795-806, the contents of which are incorporated herein by reference).
Alternatively, metabolites or fragments of the Rbm24 proteins and nucleic acids can be measured. Metabolites may be detected by a variety of methods known to those skilled in the art, including: refractive index spectroscopy (RI), ultraviolet spectroscopy (UV), fluorescence analysis, radiochemical analysis, near infrared spectroscopy (near-IR), nuclear magnetic resonance spectroscopy (NMR), light scattering analysis (LS), mass spectrometry, pyrolysis mass spectrometry, nephelometry, dispersive raman spectroscopy (DISPERSIVE RAMAN spec), gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF), surface Enhanced Laser Desorption Ionization (SELDI) mass spectrometry, ion jet spectroscopy in combination with mass spectrometry, capillary electrophoresis, NMR and IR detection. (see, WO 04/056456 and WO04/088309, the entirety of which is incorporated by reference). In this regard, other Rbm24 analytes may be measured using the detection methods described above or other methods known to the skilled artisan.
Drawings
Fig. 1: IHC detects representative pictures of RBM24 expression in paracancerous and neoplastic tissues of colorectal cancer patients.
Fig. 2: IHC score statistics of RBM24 expression in paracancerous and neoplastic tissues of colorectal cancer patients.
Fig. 3: the RBM24 detected by qPCR provided by the invention is down-regulated in tumor tissues of colorectal cancer patients.
Fig. 4: the expression of the RBM24 detected by qPCR in tumor tissues of colorectal cancer patients is related to TNM staging.
Fig. 5: the expression of RBM24 in tumor tissue of colorectal cancer patients by qPCR detection correlates with patient survival time.
Fig. 6: RBM24 was down-regulated in tumor tissue of colorectal cancer patients as measured by western blotting.
Fig. 7: WB detects RBM24 knockout mice knockout efficiency.
Fig. 8: qPCR detects RBM24 knockout mice knockout efficiency.
Fig. 9: after RBM24 knockout, HE staining indicated significant intestinal proliferation.
Fig. 10: after RBM24 knockdown, the intestinal length increases significantly.
Fig. 11: after RBM24 knockout, HE staining indicates spontaneous tumor nodules in the gut.
Fig. 12: RBM24 inhibits proliferation of human colorectal cancer cells HCT 116.
Fig. 13: RBM24, in conjunction with small molecule inhibitors, inhibits proliferation of human colorectal cancer cells HCT 116.
Fig. 14: overexpression of RBM24 in human colorectal cancer cells increases the proportion of 5-Fu induced apoptosis.
Fig. 15: overexpression of RBM24 in human colorectal cancer cells increases the proportion of cisplatin-induced apoptosis.
Fig. 16: after RBM24 is over-expressed, small molecule inhibitor treatment synergistically elevates the proportion of drug-induced apoptosis.
Fig. 17: RBM24 inhibits migration and invasion of human colorectal cancer cells HCT 116.
Fig. 18: RBM24, in conjunction with small molecule inhibitors, inhibits migration of human colorectal cancer cells HCT 116.
Fig. 19: RBM24, in conjunction with small molecule inhibitors, inhibits migration and invasion of human colorectal cancer cells HCT 116.
Detailed Description
The terms used herein have meanings commonly understood by one of ordinary skill in the art to which the present invention pertains. Terms such as "a," "an," and "the" are not intended to refer to only a singular entity, but rather include the general category in which a particular embodiment may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not limit the invention unless outlined in the claims.
Examples
Example 1 expression of RBM24 in clinical colorectal cancer patient samples.
1.1 Clinical sample collection.
The method comprises the following steps: tumor samples and corresponding paracancerous samples from 36 subjects were collected at a first hospital affiliated with the Xiamen university (clinical information is shown in Table 1) with prior acquisition of hospital ethical committee approval and patient informed consent. To qualify for inclusion in the study, each patient was evaluated individually, and a party who had to meet the following criteria could be included in the study: 1) The treatment such as clinical radiotherapy or chemotherapy is not carried out before the operation; 2) Colorectal cancer is diagnosed through clinical pathology; 3) No other history of significant disease has previously been presented; 4) The basic data of the patients are complete, including clinical case parameters such as gender, age, smoking history, family history, operation time, operation mode, pathological diagnosis, TNM stage, tumor differentiation degree, lymph node metastasis, current status, disease-free survival time, total survival time and the like. Surgical excision is required, and after clinical specimens are obtained, liquid nitrogen flash-freezing is immediately performed, and the specimens are then stored in a-80 ℃ freezer until use.
Results: a total of 36 clinical colorectal cancer patients meeting the inclusion criteria were obtained and detailed clinical information is shown in table 1.
Table 1: clinical pathology in 36 colorectal cancer patients.
1.2 Immunohistochemical results showed a significant decrease in RBM24 expression in human CRC tissue compared to adjacent tissue.
The method comprises the following steps: intestinal tissue was fixed in 4% paraformaldehyde for more than 48 hours. The PBS was washed 3 times for 5 minutes each. Then dehydrated with 10% sucrose solution, followed by gradient dehydration with 20% and 30% sucrose solution in sequence. After OCT embedding, quick freezing was performed using liquid nitrogen. The slide was sliced using a frozen microtome (Leica) to a thickness of 5um and stored in a freezer at-80 ℃. After tissue sections were obtained as described previously, H 2O2 was added and incubated at room temperature for 10 minutes. After PBS wash, antigen retrieval was performed and blocked with goat serum for 1h. Specific primary antibody (RBM 24 antibody, abcam: ab94567,1:300, overnight incubation at 4 ℃ C.) was added followed by HRP conjugated secondary antibody, incubation at room temperature for 1 hour, washing with PBS, adding DAB chromogenic solution, reaction at room temperature for 5 minutes, counterstaining with hematoxylin staining solution for 30 seconds followed by gradient alcohol dehydration and taking photographs in a sealed package.
Results: the immunohistochemical results showed a significant decrease in RBM24 expression in human CRC tissue compared to adjacent tissue (fig. 1; fig. 2).
1.3 MRNA for RBM24 was significantly down-regulated in colorectal cancer tissues
The method comprises the following steps: and taking a proper amount of tissue or cell samples, adding a TRIzol reagent according to the instruction of the kit, and performing subsequent extraction operation to obtain total RNA. The concentration and purity of total RNA were measured using an ultraviolet spectrophotometer, and the ratio of A260/A280 was 1.6-2.0, which was considered acceptable. Then cDNA is synthesized by using a reverse transcription kit, and the operation is performed according to the specification of a fluorescent dye kit of qPCR, and corresponding primers are added to detect the relative expression level of the target gene. The final result, GAPDH gene, was normalized as an internal control.
Results: we found that mRNA of RBM24 was significantly down-regulated in colorectal cancer tissues (fig. 3).
1.4 The lower the RBM24 expression, the higher the TNM staging.
The method comprises the following steps: after clinical samples and corresponding clinical information are collected as described above, the expression levels of RBM24 in each patient are detected, and the expression levels of RBM24 in patients with different TNM stages are collated for statistical analysis.
Results: the expression level of RBM24 in the tissues of patients with TNM stage I/II was significantly higher than in patients with TNM stage III/IV (n=36), i.e. the higher the TNM stage, the lower the RBM24 expression (fig. 4).
1.5 Patient survival for RBM24 low expression is shorter.
The method comprises the following steps: after collecting clinical samples and corresponding clinical information as described above, the expression level of RBM24 in each patient was examined, and all patients were classified into two groups of RBM24 high-expression or low-expression. The relationship between RBM24 expression levels and time to live was analyzed using K-M statistical methods.
Results: the expression level of RBM24 was correlated with patient survival (n=36), i.e., patient survival with higher RBM24 expression was longer and patient survival with lower RBM24 expression was shorter (fig. 5).
1.6 Protein detection RBM24 was significantly down-regulated in colorectal cancer tissues.
The method comprises the following steps: a suitable amount of tissue was placed in 300ul of lysate (1% PMSF was added in advance), the tissue was minced, ground, sonicated, and centrifuged at 12000 Xg for 15 minutes to obtain the supernatant. After protein concentration was measured by BCA method, 5X Loading Buffer was added, boiled at 100℃for 10 minutes, and stored at-20 ℃. SDS-PAGE laminated gel and separation gel are prepared. The loaded 30ug protein sample per well was subjected to electrophoresis under appropriate conditions. The proteins were then transferred to PVDF membranes by wet transfer, 5% skim milk was blocked for 1 hour, followed by a first antibody incubation, and a 4 degree shaker overnight. The antibody concentration was RBM24 antibody (Abcam: ab94567, 1:1000); beta-actin antibodies: santa cruz, SC-47778,1: 3000). After TBST washing, the membrane was incubated with HRP-conjugated secondary antibody for 1 hour at room temperature. The secondary antibody is goat anti-mouse (Boschner organism, BA1075, 1:2000) or goat anti-rabbit (Boschner organism, BA1054, 1:2000). And (3) adding a chemiluminescent kit working solution for exposure after washing the membrane to obtain protein strips. Beta-actin or GAPDH is considered as an internal reference.
Results: we found that RBM24 was significantly down-regulated in colorectal cancer tissue (fig. 6).
Example 2.Rbm24 knockout mice develop spontaneous colorectal cancer.
2.1 RBM24 knockout mouse construction.
The method comprises the following steps: tamoxifen-induced whole-body RBM24 knockout mice were further constructed on a C57BL/6 background using the Cre-Loxp system by inserting Loxp sites at both ends of the second and third exons of the RBM24 gene. The genetic disruption of RBM24 after tamoxifen induction was confirmed by PCR reaction of genomic DNA using specific primers for RBM24, showing significant reduction in mRNA and protein levels of RBM24 in colon tissue by qPCR and western blot analysis. All mice were kept free of specific pathogens, fed at a 12:12 hour light/dark cycle, and received regular food and ad libitum. All experimental procedures involving animal levels were performed according to animal protocols approved by the experimental animal center at the university of Xiamen.
Results: the genome PCR result shows that the genome of RBM24 is destroyed, and the construction success of RBM24 knockout mice is preliminarily confirmed. The mRNA and protein of RBM24 were subsequently demonstrated to be significantly down-regulated in knockout mice (FIG. 7; FIG. 8).
2.2 HE staining confirmed abnormal proliferation of the mouse intestinal tract after RBM24 knockout.
The method comprises the following steps: the mice are killed by cervical dislocation, the colon and the rectum of the mice are taken out, PBS is extracted by a 20ml syringe for 3 times, the intestinal contents are guaranteed to be washed cleanly, the intestinal tracts are placed on an operation table, scissors are used for cutting along the long diameter of the intestinal tracts, the intestinal tracts are flattened, intestinal tract swelling is observed, and the intestinal tracts are photographed and archived. Intestinal tissue was fixed in 4% paraformaldehyde for more than 48 hours. The PBS was washed 3 times for 5 minutes each. Then dehydrated with 10% sucrose solution, followed by gradient dehydration with 20% and 30% sucrose solution in sequence. After OCT embedding, quick freezing was performed using liquid nitrogen. The slide was sliced using a frozen microtome (Leica) to a thickness of 5um and stored in a freezer at-80 ℃. After tissue sections were obtained as described above, hematoxylin stain (60 ℃) was stained for 60s and 1% ethanol hydrochloride was used for 3s. Washing with tap water for 15 minutes, after the slice returns to blue, dyeing with eosin solution for 60s, washing with distilled water for 2s, further dehydrating sequentially with ethanol xylene, and taking photos with a neutral resin sealing piece.
Results: HE staining results indicated that RBM24 knockdown mice had increased colonic epithelial thickening. After 6 months of knockout we observed that mice developed colorectal adenomas and that the length of the colorectal was significantly longer than wild-type mice (fig. 10; fig. 11).
Example 3. Overexpression of rbm24 suppresses colorectal cancer cell malignancy phenotype.
3.1 RBM24 overexpression inhibits HCT116 cell proliferation.
The method comprises the following steps: human colorectal cancer HCT116 cells were purchased from the Shanghai cell bank of the national academy of sciences and frozen by the laboratory. Cells were cultured in DMEM medium containing 10% FBS and 1% penicillin mixed solution at 37 ℃,5% CO 2 and saturated humidity.
To construct RBM24 overexpressing cell lines, we amplified the CDS region of RBM24 and cloned into the multicloning site of pcdna3.1 plasmid to construct a plasmid for RBM24 transient expression. At the same time we cloned the CDS region of RBM24 into the lentiviral packaged core plasmid and packaged it into lentiviral particles. After 48h of infection of cells with lentiviral particles, 1ug/ml puromycin was used for 72h of screening to obtain pure RBM24 overexpressing cell lines. Expression levels of RBM24 were detected using qPCR and western blotting to determine overexpression efficiency.
After the cells of each group are inoculated into a 96-well plate according to the density of 1X 10 5 cells per well and cultured overnight, a CCK-8 reagent with the volume fraction of 10% is added in the corresponding time, after the cells are further incubated for 2 hours, the absorbance value of the solution is detected at 450nm by using an enzyme-labeled instrument, a cell-free culture medium and the CCK-8 reagent are used as negative controls, and a cell proliferation curve is drawn.
Results: we have found that overexpression of RBM24 can inhibit cell proliferation. This suggests that RBM24 inhibited CRC cell growth in vitro (fig. 12). Treatment with PI3K-IN-6 or MK-2206 further inhibited the ability of the cells to proliferate (FIG. 13).
3.2 RBM24 overexpression promotes chemotherapy-induced apoptosis of HCT 116.
The method comprises the following steps: after each group of cells was cultured to logarithmic growth phase and seeded at a density of 5×10 5 cells/well per well in 6-well plates for adaptation overnight, the cells were continued to be cultured to a confluency of 50% -60%. After treatment with cisplatin or without cisplatin for the corresponding time, cells were collected by pancreatin digestion, washed 2 times with PBS and the supernatant discarded. Cells were fixed with 75% ethanol for 30min, and after washing and resuspension, cells were treated with PI dye and rnase a in the dark for 30min, washed again, and cell density was adjusted to 1 x 10 6 cells/ml when resuspension was performed. Cell cycle distribution and apoptosis rate were measured using a flow cytometer and the results were analyzed using FlowJo software. Results: the data demonstrate that RBM24 overexpression increases 5-FU or cisplatin-induced apoptosis (fig. 14; fig. 15). Treatment with PI3K-IN-6 or MK-2206 further promoted 5-FU or cisplatin-induced CRC apoptosis after RBM24 overexpression (FIG. 16).
3.3 RBM24 overexpression inhibits HCT116 migration and invasion.
The method comprises the following steps: for the migration experiments, the cell density was adjusted to 5X 10 5 cells/ml after each cell group was cultured as described above and resuspended in serum-free medium. 200ul of the cell suspension was added to the upper chamber of the Transwell chamber and 500ul of complete medium was added to the lower chamber. After the assembly of the upper cavity and the lower cavity is completed, the culture plate is placed in a cell culture box for continuous incubation for 12 hours. After the upper chamber was removed, the non-migrated cells on the upper layer of the polycarbonate membrane were erased by using a cotton swab, and the cells were fixed with 75% ethanol for 10min. After staining with 0.1% crystal violet dye at 37 ℃ for 30min, the migrated cells were photographed. At least 5 fields per group were selected for statistical analysis of the number of migrating cells.
For the invasive experiments, 100ul of matrigel solution with a concentration of 5mg/ml was previously added to the upper chamber of the Transwell chamber, after incubation at 37℃for at least 30min, the supernatant was aspirated, and the cell suspension was added to matrigel, followed by the above procedure to obtain the results of cell invasion.
Results: over-expression of RBM24 inhibited migration and invasion of colorectal cancer cell line (HCT 116) compared to control cells (fig. 17). Treatment with PI3K-IN-6 or MK-2206 further inhibited the ability of the cells to proliferate, migrate and invade (FIG. 18; FIG. 19).
Claims (6)
1. Use of an agent for detecting the expression level of Rbm24 in the manufacture of a product for diagnosing colorectal cancer.
2. The use according to claim 1, wherein the reagent for detecting the expression level of Rbm24 comprises a reagent for detecting the expression level of Rbm24 gene mRNA and/or a reagent for detecting the expression level of Rbm24 protein.
3. The use according to claim 2, wherein the reagent for detecting the expression level of the Rbm24 gene mRNA comprises primers, and/or probes used in real-time quantitative PCR.
4. The use of claim 2, wherein the reagent for detecting the expression level of an Rbm24 protein comprises an antibody directed against an Rbm24 protein.
5. The use of claim 1, wherein the product comprises a kit, chip or test paper.
6. Use of an agent that upregulates expression of the Rbm24 gene or expression of an Rbm24 protein encoded by the Rbm24 gene in the manufacture of a medicament for the treatment of colorectal cancer.
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