CN106947820B - Application of VCAN in diagnosis and treatment of colon adenocarcinoma - Google Patents

Abstract

The invention discloses VCAN gene as molecular marker for colon adenocarcinoma diagnosis, the experiment of the invention proves that: compared with normal tissues, the VCAN gene is expressed in a low amount in colon adenocarcinoma tissues. The invention also discloses application of the VCAN gene in preparation of a medicament for treating colon adenocarcinoma. The research result of the invention provides a novel colon adenocarcinoma clinical diagnosis method and simultaneously provides a novel drug target for treating colon adenocarcinoma.

Description

Application of VCAN in the diagnosis and treatment of colon adenocarcinoma

technical field

The present invention relates to the field of tumor diagnosis, treatment, and prognosis prediction, more particularly, the present invention relates to a tumor diagnosis and prognosis prediction method by detecting VCAN abnormality as a means; and a tumor therapeutic agent that activates VCAN gene or protein.

Background technique

Colon adenocarcinoma is one of the most common malignant tumors in humans, and its incidence has gradually increased in recent years, accounting for the second place in all malignant tumors.

The occurrence and development of colon adenocarcinoma is a complex process involving multiple genetic alterations. Its pathogenesis is related to cell division, differentiation, abnormal spatiotemporal expression of gene groups and the interaction of proteins. Identifying oncogenes or tumor suppressor genes related to the pathogenesis of colon adenocarcinoma is of great significance for studying the carcinogenesis mechanism of colon adenocarcinoma and its diagnosis, treatment and prevention.

SUMMARY OF THE INVENTION

One of the objectives of the present invention is to provide a method for diagnosing colon adenocarcinoma by detecting the difference in VCAN gene or protein expression.

The second purpose of the present invention is to provide a method for predicting the prognosis of colon adenocarcinoma by detecting the difference of VCAN gene or protein expression.

The third object of the present invention is to provide a method for treating colon adenocarcinoma by activating VCAN gene or VCAN protein.

The fourth object of the present invention is to provide a method for screening a drug for treating colon adenocarcinoma.

The fifth object of the present invention is to provide a medicament for treating colon adenocarcinoma.

In order to achieve the above object, the present invention adopts the following technical solutions:

The present invention provides the application of a product for detecting VCAN in preparing a diagnostic tool for colon adenocarcinoma.

Further, the product for detecting VCAN includes a product for detecting VCAN gene expression.

Further, the product for detecting VCAN includes a product capable of quantifying VCAN gene mRNA, and/or a product capable of quantifying VCAN protein.

The product of the present invention for quantifying VCAN gene mRNA can perform its function based on known methods using nucleic acid molecules: such as PCR, such as Southern hybridization, Northern hybridization, dot hybridization, fluorescence in situ hybridization (FISH), DNA microarray, ASO method , high-throughput sequencing platforms, etc. Analysis can be performed qualitatively, quantitatively, or semi-quantitatively using this product.

Nucleic acids contained in the above-mentioned products can be obtained by chemical synthesis, or by preparing a gene containing a desired nucleic acid from a biological material, and then amplifying it using primers designed to amplify the desired nucleic acid.

Further, the PCR method is a known method, for example, ARMS (Amplification Refractory Mutation System) method, RT-PCR (reverse transcriptase-PCR) method, nested PCR method and the like. Amplified nucleic acid can be obtained by using dot blot hybridization method, surface plasmon resonance method (SPR method), PCR-RFLP method, in situ RT-PCR method, PCR-SSO (sequence specific oligonucleotide) method, PCR-SSP method method, AMPFLP (Amplifiable Fragment Length Polymorphism) method, MVR-PCR method, and PCR-SSCP (Single Strand Conformation Polymorphism) method.

The product capable of quantifying VCAN gene mRNA includes primers used in real-time quantitative PCR to specifically amplify VCAN gene, and the primer sequences are shown in SEQ ID NO. 1 and SEQ ID NO. 2.

Primers included in the product can be prepared by chemical synthesis, appropriately designed by referring to known information using methods known to those skilled in the art, and prepared by chemical synthesis.

The nucleic acids described above may also include probes, which may be prepared by chemical synthesis, appropriately designed by reference to known information using methods known to those skilled in the art, and prepared by chemical synthesis, or may be prepared by chemical synthesis from Biomaterials are prepared by preparing a gene containing the desired nucleic acid sequence and amplifying it using primers designed to amplify the desired nucleic acid sequence.

The product of the present invention for quantifying VCAN protein can perform its function based on known methods using antibodies: for example, ELISA, radioimmunoassay, immunohistochemistry, Western blotting and the like can be included.

The products for quantifying VCAN protein of the present invention include antibodies or fragments thereof that specifically bind to VCAN protein. An antibody or fragment thereof of any structure, size, immunoglobulin class, origin, etc. can be used, so long as it binds the target protein. The antibodies or fragments thereof included in the assay products of the present invention may be monoclonal or polyclonal. An antibody fragment refers to a part of an antibody (partial fragment) or a peptide containing a part of an antibody that retains the antigen-binding activity of the antibody. Antibody fragments can include F(ab') ₂ , Fab', Fab, single-chain Fv (scFv), disulfide-bonded Fv (dsFv) or polymers thereof, dimerized V regions (diabodies), or contain CDR peptides. Products of the invention for quantification of VCAN proteins may include an isolated nucleic acid encoding an antibody or an amino acid sequence encoding an antibody fragment, a vector comprising the nucleic acid, and cells carrying the vector.

Antibodies can be obtained by methods known to those skilled in the art. For example, mammalian cell expression vectors that retain polypeptides that retain all or part of the target protein or incorporate polynucleotides encoding them are prepared as antigens. After immunizing an animal with the antigen, immune cells are obtained from the immunized animal and fused with myeloma cells to obtain hybridomas. Antibodies are then collected from hybridoma cultures. Finally, a monoclonal antibody against the VCAN protein can be obtained by subjecting the obtained antibody to antigen-specific purification using the VCAN protein or part thereof used as an antigen. Polyclonal antibodies can be prepared by immunizing animals with the same antigens as above, collecting blood samples from the immunized animals, isolating serum from the blood, and subjecting the serum to antigen-specific purification using the antigens described above. Antibody fragments can be obtained by treating the obtained antibody with an enzyme or by using sequence information of the obtained antibody.

Binding of the label to the antibody or fragment thereof can be carried out by methods generally known in the art. For example, a protein or peptide can be fluorescently labeled by washing the protein or peptide with phosphate buffer, adding a dye prepared with DMSO, buffer, etc., then mixing the solution and leaving it at room temperature for 10 minutes. In addition, commercial labeling kits can be used, such as biotin labeling kits, such as biotin labeling kit-NH2, biotin labeling kit-SH (Dojindo Laboratories); alkaline phosphatase labeling kits such as alkaline phosphate Enzyme Labeling Kit-NH2, Alkaline Phosphatase Labeling Kit-SH (Dojindo Laboratories); Peroxidase Labeling Kits such as Peroxidase Labeling Kit-NH2, Peroxidase Labeling Kit-NH2 (Dojindo Laboratories); phycobiliprotein labeling kits such as phycobiliprotein labeling kit-NH2, phycobiliprotein labeling kit-SH, B-phycoerythrin labeling kit-NH2, B-phycoerythrin labeling kit-SH, R-Phycoerythrin Labeling Kit-NH2, R-Phycoerythrin Labeling Kit SH (Dojindo Laboratories); Fluorescent Labeling Kits such as Fluorescein Labeling Kit-NH2, HiLyte Fluor(TM) 555 Labeling Kit-NH2, HiLyte Fluor(TM) 647 Labeling Kit - NH2 (Dojindo Laboratories); and DyLight 547 and DyLight647 (Techno Chemical Corp.), Zenon(TM), Alexa Fluor(TM) Antibody Labeling Kit, Qdot(TM) Antibody Labeling Kit (Invitrogen Corporation) and EZ-Marker Protein Labeling Kit (Funakoshi Corporation). For proper labeling, the labeled antibody or fragment thereof can be detected using a suitable instrument.

As a sample of the test product according to the present invention, eg a tissue sample or fluid obtained from a biopsy subject can be used. The sample is not particularly limited as long as it is suitable for the assay of the present invention; for example, it may include tissue, blood, plasma, serum, lymph, urine, serous cavity fluid, spinal fluid, synovial fluid, aqueous humor, tears, Saliva, or a fraction or processed material thereof.

In specific embodiments of the invention, the sample is from a subject's tissue.

Further, the product of the quantitative VCAN gene or VCAN protein may be a reagent for detecting VCAN gene or VCAN protein, a kit, chip, test paper, etc. comprising the reagent, or a high-throughput method using the reagent Sequencing Platform.

The present invention also provides a tool for diagnosing colon adenocarcinoma, which can detect the expression level of VCAN gene.

Further, the tools include reagents capable of quantifying VCAN gene mRNA, and/or reagents capable of quantifying VCAN protein.

Further, the reagent capable of quantifying VCAN gene mRNA is a primer used in real-time quantitative PCR to specifically amplify the VCAN gene, and the primer sequences are shown in SEQ ID NO.1 and SEQ ID NO.2.

Further, the tools for diagnosing colon adenocarcinoma include but are not limited to chips, kits, test strips, or high-throughput sequencing platforms; high-throughput sequencing platforms are a special tool for diagnosing colon adenocarcinoma, and with the With the development of sequencing technology, the construction of a person's gene expression profile will become a very convenient task. By comparing the gene expression profiles of disease patients and normal people, it is easy to analyze which gene abnormality is related to the disease. Therefore, it is also within the protection scope of the present invention to know that the abnormality of VCAN gene is related to colon adenocarcinoma in high-throughput sequencing.

The number of amino acids recognized by the anti-VCAN antibody or its fragment used in the detection product and diagnostic tool of the present invention is not particularly limited, as long as the antibody can bind to VCAN.

The present invention also provides a method for diagnosing colon adenocarcinoma, the method comprising the steps of:

(1) Obtain a sample from the subject;

(2) Detecting the expression level of VCAN gene or protein in the subject sample;

(3) Correlating the measured expression level of VCAN gene or protein with whether the subject is diseased or not.

(4) Compared with the control, the expression level of VCAN gene or protein is reduced, the subject is judged to have colon adenocarcinoma or has a risk of colon adenocarcinoma, or the colon adenocarcinoma patient is judged to have recurrence, or Colon adenocarcinoma patients were judged to have a poor prognosis.

The present invention also provides a method for treating colon adenocarcinoma, which comprises activating VCAN gene or VCAN protein.

Further, the method includes promoting the expression of VCAN gene, or promoting the expression of VCAN protein or enhancing the activity of VCAN protein.

The present invention also provides a screening method for tumor drugs, which can be determined by measuring the expression level of VCAN gene or VCAN protein at a certain period after adding test drugs to cancer cells or after administering test drugs to tumor model animals The effect of improving tumor prognosis. More specifically, when the expression level of VCAN gene or VCAN protein increases after addition or administration of a test drug, or returns to a normal level, the drug can be selected as a therapeutic drug for improving tumor prognosis.

The present invention also provides a medicament for treating colon adenocarcinoma, the medicament comprising an activator of VCAN.

The inventive activator of VCAN is not limited, as long as the activator can promote or enhance the expression or activity of VCAN or substances involved in the upstream or downstream pathway of VCAN, and is an effective drug for treating tumors.

The present invention also provides the application of the above-mentioned activator in preparing a medicament for treating colon adenocarcinoma.

Further, the activator includes VCAN gene, VCAN protein, promoting miRNA, promoting transcription regulator, or promoting targeted small molecule compound.

On the one hand, the activator of the present invention can be used to supplement the deletion or deficiency of endogenous VCAN protein, by increasing the expression of VCAN protein, thereby treating colon adenocarcinoma caused by VCAN protein deficiency. On the other hand, it can be used to enhance the activity of VCAN protein to treat colon adenocarcinoma.

The medicament of the present invention can be administered as a medicine alone or in combination with other medicaments. Other drugs that can be administered together with the drug of the present invention are not limited as long as it does not impair the effect of the therapeutic or prophylactic drug of the present invention, preferably, the drug for treating or preventing tumors may include, for example, alkylation agents, such as ifosfamide, cyclophosphamide, dacarbazine, temozolomide, nimustine, busulfan, procarbazine, melphalan, and ramustine; antimetabolites, such as enoxaine , capecitabine, carmofur, cladribine, gemcitabine, cytarabine, cytarabine ocfosfate, tegafur, tegafur-uracil, tegafur Gimeracil oteracil potassium, deoxyfluridine, hydroxyurea, fluorouracil, fludarabine, pemetrexed, pentostatin, mercaptopurine, and methotrexate; plant alkaloids such as iritinib Pharmacokinetics, Etoposide, Sobuzoxan, Docetaxel, Nogitecan, Paclitaxel, Vinorelbine, Vindesine, and Vinblastine; Anticancer Antibiotics such as Actinomycin D, Arubicin, Ammonium rubicin, idarubicin, epirubicin, netstatin stimalamer, daunorubicin, doxorubicin, pirarubicin, bleomycin, pelomycin, mitomycin C , and mitoxantrone; platinum-based drugs such as oxaliplatin, carboplatin, cisplatin, and nedaplatin; hormonal drugs such as anastrozole, exemestane, estramustine, ethinyl estradiol , clodgestrol, goserelin, tamoxifen, dexamethasone, toremifene, bicalutamide, flutamide, prednisolone, fostrol, mitotane, methyltestosterone, Medroxyprogesterone, methandrosane, leuprorelin, and letrozole; biological response modifiers such as interferon alpha, interferon beta, interferon gamma, interleukin, ubenimex, dried BCG, and lentinan; and molecularly targeted drugs such as imatinib, gefitinib, gemtuzumab, ozogamicin, tamibarotene, trastuzumab, tretinoin, bortezomib (bortezomib), and rituximab.

The medicament of the present invention can be prepared into various dosage forms as required. Including, but not limited to, tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories for transdermal, mucosal, nasal, buccal, sublingual or oral use.

The administration route of the medicament of the present invention is not limited as long as it can exert the desired therapeutic effect or preventive effect, including but not limited to intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesicular, Intramuscular, intratracheal, subcutaneous, through skin, through pleura, topical, inhalation, through mucosa, skin, gastrointestinal, intraarticular, intraventricular, rectal, vaginal, intracranial, intraurethral, intrahepatic, intratumoral. In some cases, systemic administration is possible. In some cases it is administered topically.

The dose of the drug of the present invention is not limited, as long as the desired therapeutic effect or preventive effect is obtained, and can be appropriately determined according to symptoms, sex, age, and the like. The dose of the therapeutic drug or prophylactic drug of the present invention can be determined using, for example, the therapeutic effect on the disease or the preventive effect as an index.

In the context of the present invention, "diagnosing colon adenocarcinoma" includes judging whether a subject has had colon adenocarcinoma, judging whether the subject is at risk for colon adenocarcinoma, judging whether a patient with colon adenocarcinoma has recurred and metastasized , to determine the response of patients with colon adenocarcinoma to drug treatment, or to determine the prognosis of patients with colon adenocarcinoma.

"Treatment" as used herein encompasses treating a related disease or disease state in a mammal, such as a human, afflicted with a related disease or disorder and includes:

(1) preventing the occurrence of a disease or disease state in a mammal, especially when the mammal is susceptible to the disease state but has not been diagnosed with the disease state;

(2) inhibits the disease or disease state, i.e. prevents its occurrence; or

(3) Alleviation of the disease or disease state, even if the disease or disease state resolves.

The term "treatment" generally refers to the treatment of humans or animals (eg, as applied by a veterinarian), wherein some desired therapeutic effect can be achieved, eg, inhibition of progression of a disorder (including slowing down the rate of progression, arresting progression), amelioration of the disorder, and cure disease. Treatment as a preventive measure (eg, prophylaxis) is also included. Use in patients who have not yet developed the disorder but are at risk of developing the disorder is also included in the term "treatment".

Advantages and beneficial effects of the present invention:

The present invention discovers a molecular marker for diagnosing colon adenocarcinoma, and the molecular marker can be used as a judgment in the early stage of colon adenocarcinoma, and the survival rate of the patient is provided.

The therapeutic drug comprising the activator of VCAN gene or protein of the present invention can be used as a therapeutic drug for new colon adenocarcinoma.

Description of drawings

Figure 1 shows the differential expression of VCAN gene in colon adenocarcinoma tissue and normal tissue detected by QPCR;

Figure 2 shows the differential expression of VCAN gene in colon adenocarcinoma tissue and normal tissue detected by Western blot assay;

Figure 3 shows the overexpression efficiency of VCAN gene detected by Western blot assay;

Figure 4 shows the effect of VCAN gene expression on the proliferation of colon adenocarcinoma cells detected by MTT assay;

Figure 5 shows the effect of VCAN gene expression on apoptosis of colon adenocarcinoma cells detected by flow cytometry.

specific implementation

The present invention will be described in further detail below with reference to the accompanying drawings and embodiments. The following examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental method of unreceipted specific conditions in the embodiment, usually according to conventional conditions, such as people such as Sambrook, molecular cloning: the conditions described in the laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggestion conditions of.

Example 1 Screening of differentially expressed genes

1. Experimental materials

The tissue specimens of 10 patients with colon cancer who underwent surgical treatment and complete medical history in the hospital were selected and diagnosed as colon adenocarcinoma by the pathology department. There were 5 males and 5 females; according to the American Joint Committee on Cancer (AJCC, 2009) ) staging criteria for colorectal cancer: 6 cases of stage I-II and 4 cases of stage III-IV. Histological grading: 3 cases were well differentiated, 4 cases were moderately differentiated, and 3 cases were poorly differentiated. All patients received radiotherapy, chemotherapy and biological agents before surgery. The normal control tissue was normal distal colonic mucosal tissue in 10 cases, all from healthy colonoscopy subjects.

2. RNA extraction, cDNA synthesis

The total RNA of tissue cells was extracted according to the instructions of Trizol, and then the mRNA was reverse transcribed into cDNA according to the instructions of the reverse transcription kit.

3. Biotin-labeled cRNA hybridization

Using cDNA as a template, biotin-labeled cRNA was synthesized by in vitro transcription using the MessageAmpTM II-Biotin Arna Amplification Kit (Ambion Company), and after purification, 5× fragmentation buffer was added according to the single-round chip amplification procedure of Affymetrix Company’s eukaryotic expression profile. Fragmented cRNA with a size distribution of 35-200 nt; after the preparation of the target, the hybridization solution was prepared using the Eukaryotic Hybridization Control Kit (Affymetrix Company), and the chip injected with the hybridization solution was placed in a hybridization furnace in a balanced manner, and rotated at 45 °C and 60 r/min. Hybridize for 16h (Hybridization Oven 640, Affymetrix Company), and then complete the washing and staining of the chip in the washing workstation (Fluidics Station 450, Affymetrix Company) provided by Affymetrix Company;

4. Scan and analyze

Scanner 3000(Affymetrix公司)扫描仪扫描图像。扫描图像首先用

Operating Software Version1.4(GCOS 1.4,Affymetrix公司)软件进行图像到信号值的转换,转换成原始数据文件。然后使用软件dChip 2006中的invariant setNormalization方法和Model-base ExpressionIndex模型对GCOS输出结果进行进一步的数据分析。根据在各芯片中检测到的P值，当数据集的检测值Call值(Absolute Call,AbsCall)为不存在A(Absent)或者临界值M(Marginal)时,被视为不表达,只有Abs Call值为存在P(present)的数据集才被用于进一步的分析。application

Images were scanned with a Scanner 3000 (Affymetrix, Inc.) scanner. Scan the image first with

Operating Software Version 1.4 (GCOS 1.4, Affymetrix Company) software converts images to signal values and converts them into raw data files. Further data analysis was then performed on the GCOS output using the invariant setNormalization method and the Model-base ExpressionIndex model in the software dChip 2006. According to the P value detected in each chip, when the detected value Call value (Absolute Call, AbsCall) of the dataset is the absence of A (Absent) or the critical value M (Marginal), it is regarded as not expressed, only Abs Call Only datasets with a value of P(present) were used for further analysis.

5. Screening of differentially expressed genes in tissues

Screening of differentially expressed genes was performed using the Significant Analysis of Microarray Software (SAM) algorithm.

6. Results

A total of 485 differentially expressed genes were screened by the chip. Compared with normal tissues, 289 genes were up-regulated and 196 genes were down-regulated in colon adenocarcinoma tissues.

Example 2 Validation of differentially expressed genes in large samples

1. Research objects

The tissue specimens of 50 patients with colon cancer who underwent surgical treatment and complete medical history in the hospital were selected and diagnosed as colon adenocarcinoma by the pathology department. There were 20 males and 30 females; according to the American Joint Committee on Cancer (AJCC, 2009) ) developed colorectal cancer staging criteria: 15 cases of stage I-II and 35 cases of stage III-IV. Histological grading: 17 cases were well differentiated, 15 cases were moderately differentiated, and 18 cases were poorly differentiated. All patients received radiotherapy, chemotherapy and biological agents before surgery. The normal control tissues were 40 cases of normal distal colonic mucosa, all from healthy colonoscopy subjects.

2. RNA extraction and cDNA synthesis

RNA extraction and cDNA synthesis were performed according to the method of Example 1.

3. QPCR

The primers were designed by the primer design software Primer 5.0, and synthesized by Dalian Bao Biological Company and Shanghai Yingjun Company. The primer sequences used for VCAN gene and internal reference gene are as follows:

VCAN gene primer sequences

5'-GGAGAAGACTGTGTTGTA-3' (SEQ ID NO. 1),

5'-CGTATAGGTGAGATGGTAAT-3' (SEQ ID NO. 2);

GAPDH gene primer sequences

5'-AAGGTCGGAGTCAACGGATTTG-3' (SEQ ID NO. 3),

5'-CCATGGGTGGAATCATATTGGAA-3' (SEQ ID NO. 4).

A QPCR reaction was performed using 2.0 μl of cDNA. The amplification program was: 95°C for 5 min, (95°C for 5s, 60°C for 60s)*45 cycles. Using SYBR Green as a fluorescent marker, the PCR reaction was performed on a Light Cycler fluorescence real-time quantitative PCR instrument, and the ΔΔCT method was used for relative quantification.

4. Western blot detection

Total cell protein was extracted. The extracted total protein was quantified using BCA protein concentration kit. 50 μg of total protein was taken from each sample, electrophoresed on 12% SDS-PAGE for 1.5 h, and then transferred to membrane. Blocked with 5% nonfat milk powder, the primary antibody was incubated at 4 °C overnight, and the secondary antibody was incubated at 37 °C for 2 h. Electrochemical luminescence ECL luminescent liquid color development, gel imaging system exposure color;

5. Statistical processing

The gray value of the protein band was analyzed using Image J software, and the gray value of the VCAN protein band was normalized with β-actin as the internal reference. The result data are expressed in the way of mean ± standard deviation. SPSS13.0 statistical software is used for statistical analysis. The difference between the two is tested by t test, and it is considered to be statistically significant when P<0.05.

6. Results

(1) QPCR results

As shown in Figure 1, compared with normal tissue, the mRNA level of VCAN gene in colon adenocarcinoma tissue was significantly decreased, and the difference was statistically significant (P<0.05).

(2) Western blot results

As shown in Figure 2, compared with normal tissue, the level of VCAN protein in colon adenocarcinoma tissue was significantly decreased, and the difference was statistically significant (P<0.05).

Example 3 The effect of VCAN gene expression on the proliferation ability of colon adenocarcinoma cells

1. Construction of VCAN gene recombinant plasmid

(1) Amplify the coding sequence of the VCAN gene;

(2) Design amplification primers;

(3) Connect the amplified VCAN gene into the expression vector pcDNA3.0 to construct the pcDNA3.0-VCAN recombinant expression vector.

1.2 Culture and transfection of colon adenocarcinoma cells

HT-29 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, culture parameters: 37°C, 5% CO ₂ saturated humidity. Lipofectamine 2000 was used as transfection reagent. The experiment was divided into two groups: negative control group (transfected with pcDNA3.0); experimental group (transfected with pcDNA3.0-VCAN). HT-29 cells in logarithmic growth phase were taken and seeded in 6-well cell culture plates. After 24h, the coverage of the cell culture plate was about 70%-80%. The transfection method was carried out according to the instructions of Lipofectamine2000.

1.3 Western blot assay to detect the overexpression efficiency of pcDNA3.0-VCAN

The steps are the same as in Example 2.

1.4 Results

As shown in Figure 3, compared with the negative control group (transfected with pcDNA3.0), the expression of VCAN protein in the cells of the experimental group (transfected with pcDNA3.0-VCAN) was significantly increased, and the difference was statistically significant (P<0.05). ).

2. MTT assay to analyze cell proliferation activity

Collect the cells of each group in the logarithmic growth phase, adjust the cell density to 1*10 ⁴ /ml, inoculate in a 96-well plate, 100 μL per well, and place in an incubator at 37°C with a volume fraction of 5% CO ₂ and a saturated humidity cultivated within. MTT detection was carried out after 24, 48, and 72 h respectively: add 10 μl (5%) of MTT solution to each well, and culture in an incubator at 37° C. with a volume fraction of 5% CO ₂ and saturated humidity. After culturing for 3 h, carefully remove the supernatant, add 150 μL of DMSO to each well, and place on a horizontal shaker for 30 min. A microplate reader was used to measure the A value at a wavelength of 490 nm.

3. Experimental results

The results are shown in Figure 4. Compared with the cells of the negative control group (transfected with pcDNA3.0), the cells in the experimental group (transfected with pcDNA3.0-VCAN) proliferated slowly, and the difference was statistically significant (P<0.05). The above experimental results showed that VCAN gene expression inhibited the proliferation of colon adenocarcinoma cells.

Example 4 Effect of VCAN gene expression on apoptosis of colon adenocarcinoma cells

1. Steps: Collect the cells of each group 24 hours after transfection, wash twice with ice phosphate buffer, adjust the cell concentration to 10 ⁴ /ml with 1x Binding Buffer, add 5 μL of AnnexinV diluent and mix well, then add 2.5 μL of iodine propidium, and mix. Place on ice box for 10 min in the dark, add 400 μL of 1x Binding Buffer, and detect the apoptosis ratio on the machine.

2. Experimental results

The results are shown in Figure 5. Compared with the cells of the negative control group (transfected with pcDNA3.0), the apoptosis rate of the experimental group (transfected with pcDNA3.0-VCAN) increased, and the difference was statistically significant (P<0.05). . The above experimental results indicated that VCAN gene expression promoted the apoptosis of colon adenocarcinoma cells.

The description of the above embodiment is only for understanding the method and the core idea of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can also be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

SEQUENCE LISTING

<110> Beijing Yangshen Bio-Information Technology Co., Ltd.

<120> Use of VCAN in the diagnosis and treatment of colon adenocarcinoma

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 18

<212> DNA

<213> Artificial sequences

<400> 1

ggagaagact gtgttgta 18

<210> 2

<211> 20

<212> DNA

<213> Artificial sequences

<400> 2

cgtataggtg agatggtaat 20

<210> 3

<211> 22

<212> DNA

<213> Artificial sequences

<400> 3

aaggtcggag tcaacggatt tg 22

<210> 4

<211> 23

<212> DNA

<213> Artificial sequences

<400> 4

ccatgggtgg aatcatattg gaa 23

Claims (1)

1. Application of a reagent for promoting the overexpression of VCAN gene in the preparation of a medicament for the treatment of colon adenocarcinoma.

Images