Colorectal cancer precise diagnosis and treatment method based on SLK gene

Abstract

The invention relates to the technical field of medical diagnosis, and discloses a kit for detecting colorectal cancer, the kit contains a primer pair used for specific amplification of a No.13 exon of an SLK gene, and the sequence of the primer pair is as follows: an upstream primer: 5 '-GGAGAACAAGAAAGAGTTGTCCA-3', and a downstream primer: 5 '-CTGCCTTCTGCTGCTGGGATG-3'. The kit further comprises a reverse transcription reagent which is used for reversely transcribing the extracted total RNA into cDNA. The early diagnosis of colorectal cancer is realized by detecting the expression condition of the No.13 exon of the SLK gene. The early-stage symptoms of colorectal cancer are not obvious, most patients are confirmed to be in the middle and late stage, and the best treatment opportunity is missed. The specific primer is used for carrying out amplification detection on the No.13 exon of the SLK gene, the molecular marker SLK-LmRNA related to the colorectal cancer can be accurately recognized, abnormal expression can be detected in the early stage of the colorectal cancer, precious treatment time is gained for patients, meanwhile, more accurate and more effective guidance is provided for clinical treatment of the colorectal cancer, and the kit has a good application prospect. The treatment effect is improved.

Description

Technical Field

[0001] The present invention relates to the field of medical diagnosis technology, and specifically to a precise diagnosis and treatment method for colorectal cancer based on the SLK gene. Background Art

[0002] Colorectal cancer (CRC), a common malignant tumor worldwide, has high morbidity and mortality rates, posing a serious threat to human health. Under current medical technology, typical symptoms are often absent in the early stages of colorectal cancer, leading to most patients being diagnosed at advanced stages, missing the optimal time for treatment. At this stage, patients often face a complex treatment process and a poor prognosis, with a five-year relative survival rate of only approximately 64.7%. Furthermore, chemotherapy, the primary treatment for patients with advanced colorectal cancer who have lost the opportunity for surgery, is widely resistant to chemotherapy, posing a significant challenge to clinical treatment.

[0003] Currently, the clinical diagnosis of colorectal cancer relies primarily on traditional imaging, colonoscopy, and histopathology. However, these methods have numerous limitations in early diagnosis, such as insufficient sensitivity for small tumors and an inability to accurately assess tumor malignancy and prognosis. Furthermore, while existing molecular marker detection methods have improved diagnostic accuracy to a certain extent, they still lack highly specific early diagnostic markers, making it difficult to meet the clinical need for accurate early diagnosis of colorectal cancer.

[0004] In terms of treatment, in addition to traditional surgical resection and chemotherapy, a variety of new treatment approaches, such as targeted therapy and immunotherapy, have been continuously explored and applied in recent years. However, due to the complexity and heterogeneity of colorectal cancer, existing treatments still have many problems, such as resistance to targeted therapy drugs and limited indications for immunotherapy, which make it difficult to achieve the expected treatment effect. Therefore, there is an urgent need to develop new diagnostic markers and treatment methods to improve the early diagnosis rate and treatment efficacy of colorectal cancer and improve patient prognosis.

[0005] In summary, the existing technology has obvious deficiencies in the early diagnosis and treatment of colorectal cancer, lacking highly specific and sensitive early diagnostic markers and effective treatment methods. To this end, those skilled in the art have proposed a precise diagnosis and treatment method for colorectal cancer based on the SLK gene to solve the above problems. Summary of the Invention

[0006] In response to the deficiencies of the existing technology, the present invention provides a precise diagnosis and treatment method for colorectal cancer based on the SLK gene, which solves the problems raised in the above-mentioned background technology.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions: a kit for detecting colorectal cancer, the kit containing a primer pair for specifically amplifying exon 13 of the SLK gene, the sequence of the primer pair being:

[0008] Upstream primer: 5'-GGAGAACAAGAGAAAGAGTTGTCCA-3',

[0009] Downstream primer: 5′-CTGCCTTCTGCTGCTGGATG-3′.

[0010] Preferably, the kit further comprises a reverse transcription reagent for reverse transcribing the extracted total RNA into cDNA.

[0011] The SLK gene-based precise diagnosis and treatment method for colorectal cancer includes the following steps:

[0012] Extracting total RNA from the sample to be tested;

[0013] Use the reverse transcription reagent in the kit to reverse transcribe the total RNA into cDNA;

[0014] PCR amplification was performed using the primer pairs in the kit to obtain the amplified product;

[0015] The amplified product was subjected to agarose gel electrophoresis to observe whether there was a band at 229 bp in the gel. If there was a band, it indicated that SLK-L mRNA was present in the sample to be tested, and there was a risk of colorectal cancer.

[0016] Preferably, the method further comprises the step of detecting using a nucleic acid probe specific for exon 13 of the SLK gene, wherein the probe is coupled with a detectable group selected from a chromophore, a chemiluminescent group, a fluorophore or an isotope.

[0017] Preferably, the sample to be tested is a human tissue or blood sample.

[0018] Preferably, the method further comprises the step of quantitatively analyzing the detection results, and evaluating the severity of colorectal cancer by detecting the expression level of SLK-L mRNA.

[0019] A precise diagnosis and treatment method for colorectal cancer based on the SLK gene, further comprising the following steps:

[0020] Design and synthesize sgRNA targeting exon 13 of the SLK gene;

[0021] The sgRNA and CRISPR / Cas9 gene knockout plasmid were co-transfected into colorectal cancer cells;

[0022] Use puromycin to screen stable strains;

[0023] Total cellular RNA was extracted and reverse transcribed;

[0024] RT-qPCR was performed on the reverse transcribed cDNA to detect the knockout efficiency of sgRNA;

[0025] The Transwell experiment was used to detect changes in the invasive ability of colorectal cancer cells after knocking out exon 13 of the SLK gene. If the invasive ability is reduced, it indicates that the knockout is effective and has the potential to treat colorectal cancer.

[0026] Preferably, the sequence of the sgRNA is: 5'-CCTACAGTGTATAAGCATGC-3', and the screening concentration of puromycin is 0.2 μg / mL.

[0027] The present invention provides a precise diagnosis and treatment method for colorectal cancer based on the SLK gene. It has the following beneficial effects:

[0028] 1. The present invention achieves early diagnosis of colorectal cancer by detecting the expression of exon 13 of the SLK gene. The early symptoms of colorectal cancer are not obvious, and most patients are diagnosed in the middle or late stages, missing the best time for treatment. The present invention uses specific primers to amplify and detect exon 13 of the SLK gene, which can accurately identify the colorectal cancer-related molecular marker SLK-LmRNA, and can detect abnormal expression in the early stages of colorectal cancer, thereby buying valuable treatment time for patients, significantly improving the sensitivity and specificity of diagnosis, and effectively avoiding missed diagnosis and misdiagnosis.

[0029] 2. The precise diagnosis and treatment method of the present invention can quantitatively analyze the expression of SLK-L mRNA and then assess the severity of colorectal cancer. This feature allows doctors to develop personalized treatment plans based on the specific condition of each patient. By deeply understanding the molecular characteristics related to colorectal cancer in patients, doctors can accurately judge the patient's response to different treatment methods, such as surgery and chemotherapy, providing more accurate and effective guidance for the clinical treatment of colorectal cancer, improving treatment efficacy, reducing unnecessary treatment side effects, and improving patients' treatment tolerance and quality of life.

[0030] 3. This invention not only offers advantages in diagnosis but also innovatively provides new approaches for the treatment of colorectal cancer. On the one hand, by designing a sgRNA targeting exon 13 of the SLK gene and combining it with CRISPR / Cas9 gene knockout technology, this exon can be effectively knocked out, reducing the invasiveness of colorectal cancer cells and providing a potential target and method for gene therapy. On the other hand, by screening for small molecule compounds, particularly antisense oligonucleotides, that can affect Exon 13 skipping of SLK mRNA, a new avenue for drug development has been opened up, potentially overcoming challenges such as chemotherapy resistance in existing colorectal cancer treatments. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The ExonSkipDB database of the present invention provides a schematic diagram of the isoform abundance and PSI value of single exon skipping events of the SLK gene in various cancers.

[0032] Figure 2 This is a schematic diagram showing the PSI values ​​of SLKExon13 jumping in tumor samples and their matched normal samples provided by TCGA.

[0033] Figure 3 This is a schematic diagram showing the expression of two transcripts of the SLK gene in colorectal cancer tissues and normal colorectal tissues detected by qPCR according to the present invention.

[0034] Figure 4 This is a schematic diagram showing the use of qPCR to detect the interference efficiency of sgRNA on SLK13 exon in the present invention.

[0035] Figure 5 This is a schematic diagram showing the effect of knocking out SLK exon 13 on the invasion and migration of colorectal cancer cells according to the present invention. DETAILED DESCRIPTION

[0036] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the present specification. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0037] Please see the attached Figure 1 -Attached Figure 5 , an embodiment of the present invention provides a precise diagnosis and treatment method for colorectal cancer based on the SLK gene.

[0038] Example 1

[0039] Differential expression of gene spliceosomes of the present invention

[0040] 1. Sample collection:

[0041] Colorectal cancer tissue samples were collected from patients with colorectal cancer who had undergone surgery. Thirty-four pairs of colorectal cancer tissue and adjacent normal tissue were collected. All patients were confirmed to have colorectal cancer by pathological examination. Other eligibility criteria included: no prior treatment, no concurrent malignancies, no concurrent hormone-related diseases, and complete clinical data.

[0042] Specifically, the samples collected were from patients who had undergone surgical treatment for colorectal cancer, and all patients had undergone pathological examination to ensure sample accuracy. Furthermore, the enrolled patients had not received any other treatment prior to admission and had no concurrent malignancies or hormone-related diseases. These requirements helped ensure the reliability of the study results and avoid confounding factors that could interfere with the conclusions. Furthermore, complete clinical data were required to ensure sufficient information for analysis and research.

[0043] 2. Verification at the mRNA level

[0044] 2.1 Extraction of tissue RNA:

[0045] Total RNA was extracted from tissues using the Trizol one-step method, and the concentration of the RNA solution was determined by reading the absorbance (OD) at 260 nm and 280 nm using a Nanodrop ND-1000. The integrity of the RNA was checked by electrophoresis on a 1% formaldehyde denaturing agarose gel and observation under ultraviolet light.

[0046] 2.2 Reverse transcription

[0047] Use a reverse transcription kit and reverse transcription buffer to synthesize cDNA from 1 μg of total RNA. For each sample, use 1 μg of total RNA as template RNA in a 25 μl reaction. Add the following components to a PCR tube: DEPC water, 5× reverse transcription buffer, 10 mmol / l dNTPs, 0.1 mmol / l DTT, 30 μmol / l igodT, 200 U / μl MMLVRT, and template RNA. Incubate at 42°C for 1 hour, 72°C for 10 minutes, and briefly centrifuge.

[0048] Specifically, first, total RNA from the tissue was extracted using the Trizol one-step method. This method can effectively isolate RNA while removing impurities such as protein and DNA. The concentration of the extracted RNA solution was measured using a Nanodrop ND-1000 instrument, which determines the concentration by measuring the absorbance value at a specific wavelength. In addition, the integrity of the RNA was checked by formaldehyde denaturing agarose gel electrophoresis. This step ensures that the extracted RNA is of qualified quality and has not been degraded and can be used for subsequent experimental operations.

[0049] Next comes the reverse transcription step, where the extracted total RNA is reverse transcribed using a reverse transcription kit to synthesize cDNA. In this process, total RNA serves as a template, and reverse transcription buffer and other reagents work together to convert RNA into cDNA. The reaction system includes multiple components, such as reverse transcription buffer, dNTPs, DTT, OligodT, and MMLV-RT, which work together to ensure a smooth reverse transcription reaction. Reaction conditions are optimized to ensure efficient reverse transcription. Finally, the reaction mixture is briefly centrifuged to precipitate the cDNA, which can be used in subsequent molecular biology experiments, such as PCR amplification.

[0050] 2.3PCR

[0051] The SLK gene primer sequences are as follows:

[0052] Upstream primer: 5'-GGAGAACAAGAGAAAGAGTTGTCCA-3'

[0053] Downstream primer: 5'-CTGCCTTCTGCTGCTGGATG-3'

[0054] The GAPDH gene primer sequences are as follows:

[0055] Upstream primer: 5'-TGGGGAAGGTGAAGGTCGG-3'

[0056] Downstream primer: 5'-CTGGAAGATGGTGATGGGA-3'

[0057] The above primer sequences were provided by Shanghai Sangon Biotechnology Co., Ltd.

[0058] 2.4 Results

[0059] The results are as follows Figure 3 As shown in the figure, compared with the adjacent normal colorectal cancer tissues, the PSI value of SLKExon13 in colorectal cancer tissues was significantly upregulated, and the difference was statistically significant (P<0.05).

[0060] Example 2

[0061] Knockout of SLK exon 13

[0062] 1. Design and synthesis of sgRNA

[0063] sgExon13 sequence: 5'-CCTACAGTGTATAAGCATGC-3'

[0064] The above sgRNA sequences were synthesized by Yunzhou Biotechnology (Guangzhou) Co., Ltd.

[0065] 2. Culture and transfection of colorectal cancer cells

[0066] 2.1 Cell culture

[0067] SW480 cells were cultured in DMEM medium (containing 10% fetal bovine serum, 100 U / ml penicillin, and 100 g / ml streptomycin), and HCT116 cells were cultured in DMEM medium (containing 10% fetal bovine serum, 100 U / ml penicillin, and 100 g / ml streptomycin). Both were cultured continuously in an incubator at 37°C and 5% CO2 saturated humidity.

[0068] 2.2 Transfection of CRISPR / Cas9 gene knockout plasmid (taking a 6 cm diameter dish as an example)

[0069] (1) Before transfection, observe the density and state of the cells under a microscope. The cell confluence should be around 80% during transfection. When plating, the cells should be thoroughly digested and mixed to avoid cell accumulation and growth.

[0070] (2) Pipette 200 μl Buffer was placed in a 1.5 ml EP tube, and 3 μg of plasmid was dissolved in The oscillator vibrates for 10 seconds in the buffer.

[0071] (3) Add 6 μl of transfection reagent ( transfection reagent), shake on a shaker for 1 second, and incubate at room temperature for 10 minutes.

[0072] (4) Add the mixture to a culture dish, mix well, and incubate in an incubator for 24-48 hours.

[0073] 2.3 Screening of stable strains

[0074] (1) Determine the puromycin screening concentration: cells are plated at a certain density in a 12-well plate, and the sensitivity of the cells to puromycin is tested at concentrations of 0, 0.2, 0.5, 1, 1.5, 2, 3, 4, and 5 μg / ml. The lowest concentration at which all cells die after two days is the puromycin screening concentration for the cells, which is 0.2 μg / ml.

[0075] (2) Screening of stable strains: Transfect cells using a small dish. 48 hours after transfection, add 0.6 μg of puromycin to the dish to screen for positive cells. After two days of screening, negative cells gradually die. After one week, the puromycin concentration can be reduced to half. If there is no cell death after adding puromycin, the stable strain screening is complete.

[0076] 3. Detect the knockout efficiency of sgRNA using RT-qPCR

[0077] 3.1 Extract total cell RNA and perform reverse transcription.

[0078] 3.2 The reverse transcribed cDNA was subjected to RT-qPCR.

[0079] 3.3 The RT-qPCR products were subjected to agarose gel electrophoresis and exposed after electrophoresis.

[0080] 3.3 Statistical analysis

[0081] ImageJ software was used to analyze the grayscale values ​​of the protein bands, and the grayscale values ​​of the target bands were normalized using GAPDH as an internal reference. Data are presented as mean ± standard deviation and statistically analyzed using GraphPad Prism V8.0 software. Differences between the two were analyzed using the t-test, and statistical significance was considered when P < 0.05.

[0082] Specifically, the grayscale values ​​of protein bands are analyzed using ImageJ software. This method can quantify protein expression levels on a gel or membrane. GAPDH is used as an internal reference to correct for differences in sample loading and ensure the accuracy of experimental results. By comparing the grayscale values ​​of the target band with those of the GAPDH band, the expression level of the target protein can be normalized, thereby eliminating nonspecific differences in experimental operations.

[0083] GraphPad Prism V8.0 software was used to perform statistical analysis of the experimental data. This software can handle various types of experimental data and provides a variety of statistical analysis methods. Experimental results are expressed as mean ± standard deviation, which reflects the central tendency and dispersion of the data. A t-test was used to determine whether the difference between two data sets was statistically significant. When the P value was less than 0.05, the difference between the two data sets was considered significant, indicating that the experimental treatment may have had an actual effect on the results, rather than being caused by chance alone.

[0084] Example 3

[0085] Transwell assay to detect the effect of SLK13 exon on cell invasion ability

[0086] 1. Steps:

[0087] 1.1 Remove the frozen Matrigel from the -20°C freezer and store at 4°C overnight to make it liquid.

[0088] 1.2 Take out 200 μl of serum-free cell culture medium, add 50 μl of Matrigel reagent, mix them evenly under low temperature conditions, preferably on ice, then add 100 μl of each, and incubate in a 37°C, carbon dioxide incubator for 5 hours. During this time, observe the liquid frequently. When the liquid turns slightly white, it indicates that it has become solidified.

[0089] Specifically, the Matrigel reagent is mixed with serum-free cell culture medium and then placed under suitable environmental conditions to allow it to solidify. Matrigel is a matrix glue that is often used to simulate the extracellular matrix environment to promote cell attachment, growth and differentiation. In the experiment, after Matrigel is mixed with the culture medium, it is placed under low temperature conditions to prevent it from solidifying prematurely. The mixture is added to a culture plate or culture dish and incubated in an incubator at 37°C and 5% carbon dioxide. These conditions simulate the physiological environment in the human body and are conducive to the solidification of Matrigel and the culture of cells. Observing the color change of the liquid is to confirm whether the Matrigel has solidified correctly.

[0090] 1.3 Digest the transfected cells with trypsin, wash twice with serum-free medium, count the cells, and prepare a cell suspension.

[0091] 1.4 Gently wash the gel once with serum-free culture medium, then suspend 2*104 HCT116 cells in 100 μl DMEM culture medium (6*104 SW480 cells in 100 μl DMEM culture medium), and then inoculate it into the upper chamber of the transwell.

[0092] 1.5 Add 480 μl of the corresponding culture medium and 120 μl of FBS to the lower chamber.

[0093] 1.6 Place in a 37℃ incubator. After culturing for 12 hours, remove the transwell chamber. Repeat 3 samples for each group.

[0094] 1.7 Discard the culture medium from one chamber, wash three times with PBS, fix with 4% paraformaldehyde for 10 min, remove cells from the upper layer that did not pass through the upper chamber with a cotton swab, wash three times with PBS, stain with crystal violet, and observe under a microscope. Repeat the same procedure for the remaining two chambers.

[0095] 2. Results:

[0096] The results of invasion and migration experiments showed that in the scramble group, the average number of migrated SW480 cells was 121 and the number of invaded cells was 100, while the number of migrated HCT116 cells was 153 and the number of invaded cells was 127; in the gRNAE13 group, the average number of migrated SW480 cells was 55 and the number of invaded cells was 41, while the number of migrated HCT116 cells was 132 and the number of invaded cells was 77, and the differences were statistically significant (P<0.05).

[0097] The ExonSkipDB database provides the isoform abundance and PSI values ​​of individual exons of the SLK gene in 33 cancer tissues and 31 normal tissues from The Cancer Genome Atlas (TCGA).

[0098] The clinically relevant alternative splicing of SLK1 exon 3 in 33 cancer tissues (TCGA) and 31 normal tissues (GTEx) was systematically analyzed using the OncoSplicing database.

[0099] In summary: The present invention achieves early diagnosis of colorectal cancer by detecting the expression of exon 13 of the SLK gene. The early symptoms of colorectal cancer are not obvious, and most patients are already in the middle or late stages when diagnosed, missing the best time for treatment. The present invention uses specific primers to amplify and detect exon 13 of the SLK gene, which can accurately identify the colorectal cancer-related molecular marker SLK-LmRNA, and can detect abnormal expression in the early stages of colorectal cancer, thereby buying precious treatment time for patients, significantly improving the sensitivity and specificity of diagnosis, and effectively avoiding missed diagnosis and misdiagnosis.

[0100] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A kit for detecting colorectal cancer, characterized in that: The kit contains a primer pair for specifically amplifying exon 13 of the SLK gene, and the sequences of the primer pair are: upstream primer: 5'-GGAGAACAAGAGAAAGAGTTGTCCA-3', downstream primer: 5'-CTGCCTTCTGCTGCTGGATG-3'.

2. A kit for detecting colorectal cancer according to claim 1, characterized in that: The kit also includes a reverse transcription reagent for reverse transcribing the extracted total RNA into cDNA.

3. A method for accurate diagnosis and treatment of colorectal cancer based on the SLK gene, comprising a kit for detecting colorectal cancer according to any one of claims 1 to 2, characterized in that: The following steps are involved: Extracting total RNA from the sample to be tested; Use the reverse transcription reagent in the kit to reverse transcribe the total RNA into cDNA; PCR amplification was performed using the primer pairs in the kit to obtain the amplified product; The amplified product was subjected to agarose gel electrophoresis to observe whether there was a band at 229 bp in the gel. If there was a band, it indicated that SLK-L mRNA was present in the sample to be tested, and there was a risk of colorectal cancer.

4. The method for accurate diagnosis and treatment of colorectal cancer based on the SLK gene according to claim 3, characterized in that: The method further comprises the step of detecting using a nucleic acid probe specific for exon 13 of the SLK gene, wherein the probe is coupled with a detectable group selected from a chromophore, a chemiluminescent group, a fluorophore or an isotope.

5. The method for accurate diagnosis and treatment of colorectal cancer based on the SLK gene according to claim 3, characterized in that: The sample to be tested is a human tissue or blood sample.

6. The method for accurate diagnosis and treatment of colorectal cancer based on the SLK gene according to claim 3, characterized in that: The method also includes a step of quantitatively analyzing the test results to evaluate the severity of colorectal cancer by detecting the expression level of SLK-L mRNA.

7. A precise diagnosis and treatment method for colorectal cancer based on the SLK gene, characterized in that: The following steps are also included: Design and synthesize sgRNA targeting exon 13 of the SLK gene; The sgRNA and CRISPR / Cas9 gene knockout plasmid were co-transfected into colorectal cancer cells; Use puromycin to screen stable strains; Total cellular RNA was extracted and reverse transcribed; RT-qPCR was performed on the reverse transcribed cDNA to detect the knockout efficiency of sgRNA; The Transwell experiment was used to detect changes in the invasive ability of colorectal cancer cells after knocking out exon 13 of the SLK gene. If the invasive ability is reduced, it indicates that the knockout is effective and has the potential to treat colorectal cancer.

8. The method for accurate diagnosis and treatment of colorectal cancer based on the SLK gene according to claim 7, characterized in that: The sequence of the sgRNA is: 5'-CCTACAGTGTATAAGCATGC-3', and the screening concentration of puromycin is 0.2 μg / mL.

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