CN107385100B - Application of MCM8 as gastric adenocarcinoma metastasis marker - Google Patents
Application of MCM8 as gastric adenocarcinoma metastasis marker Download PDFInfo
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- CN107385100B CN107385100B CN201710823753.6A CN201710823753A CN107385100B CN 107385100 B CN107385100 B CN 107385100B CN 201710823753 A CN201710823753 A CN 201710823753A CN 107385100 B CN107385100 B CN 107385100B
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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Abstract
The invention discloses MCM8 as a diagnosis and treatment target of gastric adenocarcinoma. Therefore, the MCM8 can be used for developing products for diagnosing gastric adenocarcinoma and medicines for treating gastric adenocarcinoma. The research result of the invention provides a theoretical basis for the clinician to formulate a personalized treatment scheme and can provide a new drug target for the development of gastric adenocarcinoma drugs.
Description
Technical Field
The invention relates to the field of tumor diagnosis and treatment, in particular to a tumor diagnosis method by taking MCM8 abnormality detection as a means; and tumor therapeutic agents which inhibit MCM8 gene or protein.
Background
Gastric cancer is a malignant tumor derived from epithelial cells of the gastric mucosa, mainly adenocarcinoma. Nearly 100 million gastric cancers and 74 million patients died of diseases are newly diagnosed in 2008 globally, and the cancer with the fourth incidence and the second mortality all over the world is a clinically common malignant tumor. Although the global overall incidence of gastric cancer has decreased, nearly two-thirds of cases of gastric cancer are concentrated in economically undeveloped countries and regions, and are highly prevalent in china and other east asia countries. According to the latest version of the Chinese cancer map published in 2014 in the Life Times. Each year, about 3120000 new cases of tumor occur in our country, 8550 cases per day on average, 6 people are diagnosed with cancer every minute, and 5 people die from cancer. According to the survey of the world health organization, China belongs to a high-incidence area of gastric cancer, and the conditions of Liaoning, Shandong, Gansu, Jiangsu, Fujian and other provinces are more serious. Clinically, despite significant advances in the treatment of early stage gastric cancer, often supported by surgical resection plus regional lymph node dissection and postoperative chemoradiotherapy, the long-term survival rate of advanced gastric cancer remains low. Many documents and studies have shown that tumor invasion and metastasis lead to death in most gastric cancer patients and play a key role in the poor prognosis of gastric cancer. Invasion and metastasis of gastric cancer are complex biological processes with multiple gene regulation, multiple factor participation and multiple step progression, and the motility and invasive capability of cancer cells are considered as important conditions for generating metastasis. However, the molecular mechanisms underlying invasion and metastasis of advanced gastric cancer are not clear. Therefore, the search for reliable marker factors for tumor prognosis and the search for molecular targets capable of effectively inhibiting tumor metastasis and invasion and improving tumor prognosis are hot spots of recent research and are also a key point and difficulty in tumor research. Therefore, there is an urgent need to identify important molecules in the advanced stage of gastric cancer, develop new drug action targets, and provide valuable help for tumor treatment and patient prognosis.
Disclosure of Invention
One of the purposes of the invention is to provide a method for realizing the diagnosis of gastric adenocarcinoma metastasis, the prognosis prediction of gastric adenocarcinoma and the prognosis evaluation of gastric adenocarcinoma by detecting the expression difference of MCM8 gene.
The invention also aims to provide a method for treating gastric adenocarcinoma metastasis by inhibiting the expression of MCM8 gene.
The invention also aims to provide a method for screening a gastric adenocarcinoma metastasis treatment drug.
The fourth purpose of the invention is to provide a medicine for treating gastric adenocarcinoma metastasis.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides an application of a reagent for detecting MCM8 in preparation of tools for gastric adenocarcinoma metastasis diagnosis, gastric adenocarcinoma prediction prognosis and gastric adenocarcinoma prognosis evaluation.
Further, the reagent for detecting MCM8 comprises a reagent for detecting the expression level of MCM8 gene.
Further, the reagent for detecting MCM8 includes a reagent capable of quantifying MCM8 gene mRNA, and/or a reagent capable of quantifying MCM8 protein.
The reagent for quantifying MCM8 gene mRNA of the present invention can exert its function based on a known method using nucleic acid molecules: such as PCR, e.g., Southern hybridization, Northern hybridization, dot hybridization, Fluorescence In Situ Hybridization (FISH), DNA microarray, ASO methods, high throughput sequencing platforms, etc. The assay can be performed qualitatively, quantitatively, or semi-quantitatively using the reagent.
Further, the PCR method is a known method, for example, ARMS (Amplification Refractorymutation System) method, RT-PCR (reverse transcriptase-PCR) method, nested PCR method, or the like. The amplified nucleic acid can be detected by using a dot blot hybridization method, a surface plasmon resonance method (SPR method), a PCR-RFLP method, an in situ RT-PCR method, a PCR-SSO (sequence specific oligonucleotide) method, a PCR-SSP method, an AMPFLP (amplifiable fragment length polymorphism) method, an MVR-PCR method, and a PCR-SSCP (single strand conformation polymorphism) method.
The reagent capable of quantifying the mRNA of the MCM8 gene can be a specific primer of the MCM8 gene or transcript, can also be a specific recognition probe of the MCM8 gene or transcript, or comprises the primer and the probe.
Specific primers for the MCM8 gene or transcript described above include primers for specific amplification of the MCM8 gene used in real-time quantitative PCR. In a specific embodiment of the invention, the primer sequences are shown as SEQ ID NO.1 and SEQ ID NO. 2.
The primer can be prepared by chemical synthesis, appropriately designed by referring to known information using a method known to those skilled in the art, and prepared by chemical synthesis.
The probe may be prepared by chemical synthesis, by appropriately designing with reference to known information using a method known to those skilled in the art, and by chemical synthesis, or may be prepared by preparing a gene containing a desired nucleic acid sequence from a biological material and amplifying it using a primer designed to amplify the desired nucleic acid sequence.
The reagent for quantifying MCM8 protein of the present invention can exert its function based on a known method using an antibody: for example, ELISA, radioimmunoassay, immunohistochemistry, Western blotting, etc. may be included.
The reagent for quantifying MCM8 protein of the invention comprises an antibody or a fragment thereof which specifically binds to MCM8 protein. An antibody or fragment thereof of any structure, size, immunoglobulin class, origin, etc., may be used so long as it binds to the target protein. The antibodies or fragments thereof included in the assay products of the invention may be monoclonal or polyclonal. An antibody fragment refers to a portion of an antibody (partial fragment) or a peptide containing a portion of an antibody that retains the binding activity of the antibody to an antigen. Antibody fragments may include F (ab')2Fab', Fab, single chain fv (scfv), disulfide-bonded fv (dsfv) or polymers thereof, dimerized V regions (diabodies), or CDR-containing peptides. The reagent for quantifying MCM8 protein of the present invention may include an isolated nucleic acid encoding an amino acid sequence of an antibody or encoding an antibody fragment, a vector comprising the nucleic acid, and a cell carrying the vector.
Antibodies can be obtained by methods well known to those skilled in the art. For example, mammalian cell expression vectors that retain all or part of the target protein or incorporate polynucleotides encoding them are prepared as antigens. After immunizing an animal with an antigen, immune cells are obtained from the immunized animal and myeloma cells are fused to obtain hybridomas. The antibody is then collected from the hybridoma culture. Finally, a monoclonal antibody against MCM8 protein can be obtained by subjecting the obtained antibody to antigen-specific purification using MCM8 protein or a portion thereof used as an antigen. Polyclonal antibodies can be prepared as follows: an animal is immunized with the same antigen as above, a blood sample is collected from the immunized animal, serum is separated from the blood, and then antigen-specific purification is performed on the serum using the above antigen. The antibody fragment 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, proteins or peptides may be fluorescently labeled as follows: the protein or peptide is washed with phosphate buffer, a dye prepared with DMSO, a buffer, or the like is added, and the solution is mixed and left at room temperature for 10 minutes. In addition, labeling may be carried out using commercially available labeling kits, such as biotin labeling kit, e.g., biotin labeling kit-NH 2, biotin labeling kit-SH (Dojindo laboratories); alkaline phosphatase labeling kits such as alkaline phosphatase labeling kit-NH 2, alkaline phosphatase labeling kit-sh (dojindo laboratories); peroxidase labeling kits such as peroxidase labeling kit-NH 2, peroxidase labeling kit-NH 2(Dojindo Laboratories); phycobiliprotein labeling kits such as phycobiliprotein labeling kit-NH 2, phycobiliprotein labeling kit-SH, B-phycoerythrin labeling kit-NH 2, B-phycoerythrin labeling kit-SH, R-phycoerythrin labeling kit-NH 2, R-phycoerythrin labeling kit SH (dojindo laboratories); fluorescent labeling kits such as fluorescein labeling kit-NH 2, HiLyte Fluor (TM)555 labeling kit-NH 2, HiLyte Fluor (TM)647 labeling kit-NH 2(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, a suitable instrument can be used to detect the labeled antibody or fragment thereof.
The obtaining of the sample for detecting the expression level of MCM8 gene according to the invention is a routine technique in the field, and preferably can be obtained by a method which is non-invasive or minimally invasive.
The sample may be (but is not limited to): peripheral blood, bone marrow, lymph nodes, peritoneal lavage fluid, parietal cells or gastric juice. In a specific embodiment of the invention, the sample is from a tissue of a subject.
It is well known to those skilled in the art that cells from tumor tissue are shed into body fluids, these shed cells are called circulating tumor cells, which have the same properties as the tumor cells in tumor tissue, and thus the detection of the properties of the circulating tumor cells in body fluids, especially in blood, can represent the properties of the tumor tissue. In the present invention, the detection of MCM8 gene expression in tumor tissues can be used for diagnosing gastric adenocarcinoma metastasis, and the detection of MCM8 gene expression in circulating tumor cells can be easily obtained and used for diagnosing gastric adenocarcinoma metastasis.
The invention also provides a tool for gastric adenocarcinoma metastasis diagnosis, gastric adenocarcinoma prediction prognosis and gastric adenocarcinoma prognosis evaluation, wherein the tool can detect the MCM8 gene expression level.
Further, the tool comprises a reagent capable of quantifying MCM8 gene mRNA, and/or a reagent capable of quantifying MCM8 protein.
Typically, the reagents are present in suitable containers. Each of the primers or probes can be adjusted to at least one desired amount of concentration using a diluent such as deionized water and dispensed into a container.
Further, the reagent capable of quantifying the mRNA of the MCM8 gene comprises a primer for specifically amplifying the MCM8 gene used in real-time quantitative PCR, and the sequence of the primer is shown as SEQ ID NO.1 and SEQ ID NO. 2.
Further, the tools for gastric adenocarcinoma metastasis diagnosis, gastric adenocarcinoma prognosis prediction and gastric adenocarcinoma prognosis evaluation include, but are not limited to, a chip, a kit, a test paper or a high throughput sequencing platform; the high-throughput sequencing platform is a special tool, and with the development of high-throughput sequencing technology, the construction of a gene expression profile of a person becomes very convenient work. By comparing the gene expression profiles of patients with diseases and normal people, the abnormality of which gene is related to the disease can be easily analyzed. Therefore, the knowledge that the abnormality of MCM8 gene is related to gastric adenocarcinoma metastasis in high-throughput sequencing also belongs to the novel application of MCM8, and is also within the protection scope of the invention.
The kit of the present invention may further comprise a reagent for extracting nucleic acid, a reagent for PCR, a reagent for staining or developing color, and the like. For example, such agents include, but are not limited to: an extraction solution, an amplification solution, a hybridization solution, a color development solution, a washing solution, and the like.
In addition, the kit can also comprise instructions and the like for describing a method for detecting gastric adenocarcinoma metastasis.
The kit of the present invention may contain a plurality of different reagents suitable for practical use (e.g., for different detection methods), and is not limited to the reagents listed so far, and any reagent that determines metastasis of gastric adenocarcinoma based on the detection of MCM8 gene or transcript is included in the scope of the present invention.
The invention also provides a method for diagnosing gastric adenocarcinoma metastasis, predicting gastric adenocarcinoma prognosis or evaluating gastric adenocarcinoma prognosis, which comprises the following steps:
(1) obtaining a sample from a subject;
(2) detecting the level of MCM8 gene expression in a subject sample;
(3) correlating the measured MCM8 gene expression level with a disease association of the subject.
(4) The statistically elevated level of MCM8 gene expression compared to normal controls indicates that the subject is judged to have metastasized gastric adenocarcinoma, or that the subject is predicted to have a poor prognosis of metastasis of gastric adenocarcinoma, or that the subject being assessed for metastasis of gastric adenocarcinoma has relapsed.
The invention also provides a method of treating gastric adenocarcinoma metastasis, the method comprising inhibiting MCM8 gene or MCM8 protein.
Further, the methods comprise inhibiting the expression of MCM8 gene, or inhibiting the expression of MCM8 gene or inhibiting the activity of MCM8 protein.
The present invention also provides a screening method of tumor drug, which can determine the effect of tumor drug on improving tumor prognosis by measuring the expression level of MCM8 gene or MCM8 protein after adding test drug to cancer cells or at a certain period after administering test drug to tumor model animals. More specifically, when the expression level of MCM8 gene or MCM8 protein decreases or returns to normal level after addition or administration of a test drug, the drug may be selected as a therapeutic drug for improving tumor prognosis.
The invention also provides a medicament for treating gastric adenocarcinoma metastasis, which comprises an inhibitor of MCM 8.
The inhibitor of MCM8 of the present invention is not limited as long as the inhibitor is capable of inhibiting the expression or activity of MCM8 or a substance involved in the upstream or downstream pathway of MCM8, and is a drug effective for treating tumor metastasis.
The invention also provides application of the inhibitor in preparing a medicament for treating gastric adenocarcinoma metastasis.
Further, the inhibitor comprises an interfering RNA aiming at MCM8 gene expression, or a negative regulation miRNA, a negative regulation type transcription regulation factor or a repression type targeting small molecule compound.
The inhibitors of the present invention may be used by formulating pharmaceutical compositions in any manner known in the art. Such compositions comprise the active ingredient in admixture with one or more pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients, depending on the mode of administration and the dosage form envisaged. Therapeutically inert inorganic or organic carriers known to those skilled in the art include, but are not limited to, lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyols such as polyethylene glycol, water, sucrose, ethanol, glycerol and the like, various preservatives, lubricants, dispersants, flavoring agents. Moisturizers, antioxidants, sweeteners, colorants, stabilizers, salts, buffers and the like may also be added as needed to aid in the stability of the formulation or to aid in the enhancement of the activity or its bioavailability or to produce an acceptable mouthfeel or odor upon oral administration, formulations which may be used in such compositions may be in the form of their original compounds as such, or optionally in the form of their pharmaceutically acceptable salts, and the inhibitors of the present invention may be administered alone or in various combinations, as well as in combination with other therapeutic agents. The compositions so formulated may be administered in any suitable manner known to those skilled in the art, as desired. In using the pharmaceutical compositions, a safe and effective amount of an inhibitor of the present invention is administered to a human, wherein the safe and effective amount is typically at least about 100 micrograms per kilogram of body weight for oral administration. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
The medicine of the present invention may be prepared into various preparation forms. Including, but not limited to, tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories for transdermal, mucosal, nasal, buccal, sublingual or oral use.
The route of administration of the drug of the present invention is not limited as long as it exerts the desired therapeutic or prophylactic effect, and includes, but is not limited to, intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesicular, intramuscular, intratracheal, subcutaneous, transdermal, transpleural, topical, inhalation, transmucosal, dermal, gastrointestinal, intraarticular, intraventricular, rectal, vaginal, intracranial, intraurethral, intrahepatic, intratumoral. In some cases, the administration may be systemic. In some cases topical administration.
The dose of the drug of the present invention is not limited as long as the desired therapeutic effect or prophylactic effect is obtained, and can be appropriately determined depending on the symptoms, sex, age, and the like. The dose of the therapeutic agent or prophylactic agent of the present invention can be determined using, for example, the therapeutic effect or prophylactic effect on a disease as an index.
The "MCM 8 gene" (NC _000020.11(5950652..6000941))) sequences of the present invention can be queried in the NCBI database.
In the context of the present invention, "diagnosis of gastric adenocarcinoma metastasis" includes determining whether a subject has developed gastric adenocarcinoma metastasis, determining whether a subject is at risk for gastric adenocarcinoma metastasis, and determining whether a patient with gastric adenocarcinoma metastasis has relapsed metastasis.
As used herein, "treatment" encompasses treatment-related diseases or disease states in a mammal, such as a human, having the associated disease or disorder, and includes:
(1) preventing the occurrence of a disease or condition in a mammal, particularly when the mammal is susceptible to said disease condition but has not been diagnosed as having such a disease condition;
(2) inhibiting a disease or disease state, i.e., preventing its occurrence; or
(3) Alleviating the disease or condition, i.e., causing regression of the disease or condition.
The term "treatment" generally refers to the treatment of a human or animal (e.g., as applied by a veterinarian) wherein some desired therapeutic effect is achieved, e.g., inhibiting the progression of a condition (including slowing the progression, stopping the progression), ameliorating the condition, and curing the condition. Treatment as a prophylactic measure (e.g., prophylaxis) is also included. The use of a patient who has not yet developed a condition but who is at risk of developing the condition is also encompassed by the term "treatment".
The invention has the advantages and beneficial effects that:
the invention discovers and verifies the close correlation between the MCM8 gene expression and the gastric adenocarcinoma tissue metastasis for the first time, and has the advantages of large verified sample amount and accurate result. The proposal of the correlation provides a new way for diagnosing and treating the metastasis of the gastric adenocarcinoma.
The invention develops a reagent or a kit suitable for gastric adenocarcinoma metastasis detection, and the detection sensitivity is good.
Drawings
FIG. 1 shows the detection of differential expression of MCM8 gene in metastatic gastric adenocarcinoma tissues and normal control tissues using QPCR;
FIG. 2 shows that the differential expression of MCM8 protein in metastatic gastric adenocarcinoma tissues and normal control tissues is detected by Western blot experiment;
FIG. 3 shows the inhibition rate of MCM8 gene expression detected by Western blot experiment.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations.
EXAMPLE 1 differential expression of MCM8 Gene
1. Sample collection
50 cases of metastatic gastric adenocarcinoma tissues and paracancerous normal tissues thereof are collected, and 45 cases of non-metastatic gastric adenocarcinoma tissues and paracancerous normal tissues thereof are collected. Tissue samples were sampled with the assistance of a pathologist, and the sample collection criteria were as follows: (1) primary gastric adenocarcinoma without other diseases, and patients do not undergo radiotherapy and chemotherapy before operation; (2) the sample is diagnosed as gastric adenocarcinoma by a pathology department, and tumor cells are not detected in paracancerous normal tissues; (3) in order to avoid unnecessary cross contamination, two sets of equipment are adopted during sampling, firstly, a paracancer normal tissue sample which is separated from cancer tissues by at least 5cm and has no obvious lesion is observed by naked eyes, then, a full-layer gastric adenocarcinoma tissue sample is taken, the sample is immediately subpackaged and put into an EP tube added with 1ml of RNAlater after being collected, the temperature is kept overnight at 4 ℃, and then, the paracancer normal tissue sample is placed in a refrigerator at 70 ℃ below zero for long-term storage. And numbering and labeling on each EP tube. And simultaneously making remarks of related information on the sample record book.
2. RNA (ribonucleic acid) for extracting paired gastric adenocarcinoma and tissue sample beside gastric adenocarcinoma
RNA extraction kit (Trans Zol) from Beijing Quanji Biotech Ltd is usedTMUp PlusRNA Kit) tissue sample RNA was extracted. The method comprises the following specific steps:
(1) weighing the ultra-low temperature frozen sample, quickly transferring to a mortar precooled with liquid nitrogen, sufficiently grinding with a pestle until the sample is ground into powder, transferring the powder-ground sample to a centrifuge tube, and adding 1ml of Trans Zol into every 50-100mg of the sampleTMHomogenizing Up with homogenizer, or repeatedly blowing and sucking with gun. The mixture was allowed to stand at room temperature for 5 minutes.
(2) 1ml of Trans Zol is usedTMUp, 0.2ml of chloroform was added thereto, followed by vigorous shaking for 30 seconds and incubation at room temperature for 3 minutes.
(3) Centrifugation at 10000 Xg for 15 minutes at 4 ℃. The sample now separated into three layers, a colorless aqueous phase (upper layer), an intermediate layer, and a pink organic phase (lower layer). RNA was in the colorless aqueous phase and 500. mu.l of the colorless aqueous layer was pipetted.
(4) The aspirated 500. mu.l of colorless aqueous phase was transferred to a new centrifuge tube, 500. mu.l of absolute ethanol was added, and the mixture was gently inverted and mixed. (centrifugation can be carried out at room temperature thereafter)
(5) Mu.l of the resulting solution was added to the column together with the pellet, centrifuged at 12000 Xg for 30 seconds at room temperature, and the effluent discarded (which could be done in several portions if the volume was larger than the column capacity).
(6) Add 500. mu.l of CB9, centrifuge at 12000 Xg at RT for 30 seconds and discard the effluent.
(7) Repeating the step (6) once.
(8) Adding 500 μ l WB9 (checking whether absolute ethanol is added before use), centrifuging at 12000 Xg for 30 s at room temperature, and discarding the effluent
(9) Repeating the step (8) once.
(10) Centrifuging at 12000 Xg room temperature for 2min, completely removing residual ethanol, standing at room temperature for several min, and completely air drying the column.
(11) The column was placed in an RNase-free Tube (kit prepared), and 30. mu.l of RNase-free Water was added to the center of the column, and the column was allowed to stand at room temperature for 1 minute.
(12) The RNA was eluted by centrifugation at 12000 Xg for 1 minute at room temperature.
(13) The RNA was stored in a freezer at-80 ℃.
3. Determination of concentration and purity of extracted RNA
RNA concentration and purity were determined using an ND-1000 instrument from Nano Drop, USA.
Using an ultraviolet spectrophotometer Nano Drop 1000 to double click a Nano Drop icon on a computer screen and entering a system menu; after selecting a nucleic acid measurement option, adding 2 mu l of double distilled water into the sample adding hole according to system prompt; clicking to determine to initialize the system; add 2. mu.l of double distilled water to the wells, select RNA items, and click Blank to measure system background. And (4) wiping the double distilled water with a piece of rear lens wiping paper, then dropping the RNA sample to be detected, and clicking Measure. Reading the value and recording the RNA concentration (ng/. mu.l), wherein the care of the A value 260/280 is necessary, and if the value is between 1.8 and 2.0, the RNA sample quality is better. The RNA concentration of each sample was labeled completely and then frozen in a freezer at-70 ℃.
4. Extracted RNA integrity assay
Agarose gel detection of RNA samples the procedure:
1) electrophoresis tank and glue makingCleaning of the tool: cleaning with detergent (generally soaking overnight), washing with water, and washing with 3% H2O2Filling the electrophoresis tank, standing at room temperature for 10min, washing with 0.1% (V/V) DEPC water, and air drying for use.
2) Preparing glue: 0.5g of agarose powder was weighed into an Erlenmeyer flask containing 45ml of DEPC water, and heated to completely dissolve the agarose. After a little cooling, 5ml of 10 XTAE electrophoresis buffer and a final concentration of 0.5. mu.g/ml ethidium bromide were added. Then pouring gel into the gel groove, inserting a comb, horizontally placing for use after solidification.
3) Sample adding: the sample is mixed with 6 Xelectrophoresis buffer solution and loaded into the gel sample application hole.
4) Electrophoresis: and opening the electrophoresis apparatus, and stabilizing the voltage of 80V electrophoresis.
5) After electrophoresis (about 30 minutes), the gel was stored by UV light and photographed with a gel imaging system.
Integrity determination by agarose gel electrophoresis detection, if can clearly see 28s rRNA, 18s R RNA, 5srRNA three bands, and 28s rRNA brightness should be 18s rRNA two times. The completeness of the extracted total RNA is better, and the RNA quality meets the requirement.
5. Design and preparation of primers
The primer sequence for detecting MCM8 gene expression by qRT-PCR and the primer sequence for amplifying internal reference GAPDH by qRT-PCR are designed and synthesized by biological engineering (Shanghai) corporation, and the sequence information is recorded into NCBI software design primer by searching UCSC database, and is confirmed to be correct in blast in the gene bank.
MCM8 gene primer:
an upstream primer: 5'-ATACCAGATATAGCAACT-3' (SEQ ID NO. 1);
downstream primer 5'-CATCATTAGACAATCCTT-3' (SEQ ID NO.2),
GAPDH gene primers:
an upstream primer: 5'-GGGAGCCAAAAGGGTCA-3' (SEQ ID NO. 3);
a downstream primer: 5'-GAGTCCTTCCACGATACCAA-3' (SEQ ID NO. 4).
6. Real-time fluorescent quantitative cDNA reverse transcription detection step
Mu.g of total RNA was reverse transcribed with reverse transcription buffer to synthesize cDNA. A25-mu-l reaction system is adopted, 1 mu g of total RNA is taken from each sample as template RNA, and the following components are respectively added into a PCR tube: DEPC water, 5 Xreverse transcription buffer, 10mmol/L dNTP, 0.1mmol/L DTT, 30. mu. mmol/L Oligo dT, 200U/. mu. L M-MLV, template RNA. Incubate at 42 ℃ for 1h, 72 ℃ for 10min, and centrifuge briefly.
7、PCR
(1) A Bio-RAD real-time fluorescence quantitative PCR instrument was used to prepare a reaction system as shown in Table 1.
TABLE 1 PCR reaction System
(2) The following qPCR reaction parameters were used: pre-denaturation at 95 deg.C for 10min, then denaturation at 95 deg.C for 15s, annealing at 57 deg.C, and extension for 1min for 40 cycles; then, the fluorescence signal was collected and the product dissolution curve was prepared, and 3 replicates of each sample were averaged. By using 2-△△CtAnd (3) analyzing the expression level of the MCM8 by a relative quantification method, wherein Ct is the intensity value of a fluorescence signal detected in a reaction system by a thermal cycler. The calculation method comprises the following steps: delta Ct ═ (Ct target gene-Ct reference gene) tumor tissue experimental group- (Ct target gene-Ct reference gene) normal tissue control group, 2-△△CtThe expression of the target gene in the experimental group is shown as the fold change relative to the control group, and the analysis of the experimental data is performed by the Bio-RAD analysis software.
5. Statistical analysis statistical software SPSS19.0 is used for data analysis, and paired T test is used to judge whether the expression of MCM8 in gastric adenocarcinoma tissue and para-carcinoma tissue sample has statistically significant difference, and P <0.05 is statistically significant difference.
6. Results
The results are shown in figure 1, in the metastatic group, compared with the stomach adenocarcinoma tissue and the paracarcinoma tissue, the expression level of MCM8 gene mRNA is increased, and the difference has statistical significance; in contrast, in the non-metastatic group, compared with the para-carcinoma tissue, the expression level of the MCM8 gene mRNA is not changed statistically, and the same expression level of the MCM8 gene mRNA is detected in the para-carcinoma tissue of the metastatic group and the non-metastatic group. The above results indicate that MCM8 gene is involved in metastasis of gastric adenocarcinoma tissue.
Example 2 differential expression of MCM8 protein
1. Extraction of Total protein from tissue samples of example 1
The protein extraction procedure was performed according to the instructions of the EpiQuik tissue/cell total protein extraction kit.
2、Western blot
β -actin is used as an internal reference, 50 mu G of total protein is subjected to SDS-PAGE, then is electrically transferred to a PVDF membrane, is lightly shaken and sealed for 1h at room temperature by 1 xTBST containing 5 percent of skimmed milk powder, rabbit anti-human MCM8 monoclonal antibody (diluted 1: 800) and mouse anti-human β -actin polyclonal antibody (diluted 1: 3000) are respectively added, the mixture is kept overnight at 4 ℃, the membrane is washed for 4 times by 1 xTBST, goat anti-rabbit and goat anti-mouse Ig G (diluted 1: 2000) are added, the incubation is carried out for 1h at room temperature, the membrane is washed for 4 times by 1 xTBST, then the mixture is placed in a Super Signal chemiluminescence reagent for reaction for 2min, an X-ray film is exposed in a dark room, and the development and fixation are carried out.
3. Statistical treatment
The grey scale values of the protein bands are analyzed by using Image J software, β -actin is used as an internal reference, the grey scale values of the MCM8 protein bands are subjected to normalization processing, the result data are expressed in a mode of mean value +/-standard deviation, statistical analysis is carried out by using SPSS13.0 statistical software, and the difference between the two is considered to have statistical significance when P is less than 0.05 by using a t test.
4. Results
The results are shown in fig. 2, in the metastatic group, compared with the stomach adenocarcinoma tissue and the para-carcinoma tissue, the protein level of MCM8 is increased, and the difference is statistically significant; in the non-metastatic group, compared with the para-cancer tissue, the MCM8 protein level is not changed statistically, and the MCM8 protein level in the para-cancer tissue of the metastatic group and the para-cancer tissue of the non-metastatic group is detected to be the same. The above results indicate that MCM8 protein is involved in metastasis of gastric adenocarcinoma tissue.
Example 3 interference with MCM8 Gene expression
1. Interfering RNA design Synthesis
siRNA was designed and synthesized by Shanghai Jima pharmaceutical technology, Inc. according to the MCM8 gene sequence. The Shanghai Jima pharmaceutical technology Co., Ltd simultaneously provided negative control siRNA (siRNA-NC) having no sequence homology with MCM8 gene.
siRNA-MCM8：
The sense strand is 5'-CACAGTTTTTGCTTTCAACAAAG-3' (SEQ ID NO. 5);
the antisense strand is 5'-TGGATCGATTCATACCATATAAA-3' (SEQ ID NO.6),
2. culture of human gastric adenocarcinoma cells
Human gastric adenocarcinoma SGC7901 cell is cultured in RPMI1640 culture medium containing 10% fetal calf serum, penicillin 100units/ml and streptomycin 100 μ g/ml at 37 deg.C and 5% CO2The culture medium is changed for 1 time every 24 hours, and the culture medium is subcultured for 1 time in 48 hours. Cells in logarithmic growth phase were taken for subsequent experiments.
3. Cell transfection
Liposome Lipofectamine2000 was used as the transfection reagent. Experiment was divided into 2 groups: negative control group (transfection siRNA-NC); experimental group (transfection siRNA-MCM 8). SGC7901 cells in the logarithmic growth phase were taken and seeded in 6-well cell culture plates. After 24h, the coverage rate of the cell culture plate is about 70-80%. The transfection protocol was performed according to Lipofectamine2000 instructions.
4. Western blot experiment for detecting interference efficiency of siRNA-MCM8
The procedure is as in example 2.
5. Results
As shown in FIG. 3, compared with the negative control group (transfected siRNA-NC), the expression level of MCM8 protein in the cells of the experimental group (transfected siRNA-MCM8) is significantly reduced, and the difference is statistically significant (P < 0.05).
Example 4 Effect of MCM8 Gene expression on invasion and migration Capacity of gastric adenocarcinoma cells
1. Detection of gastric adenocarcinoma cell migration ability in vitro
48h after transfection, each group of transfected SGC7901 cells was prepared into single cell suspension by using double-antibody-free RPMI1640 culture solution, and the amount of the single cell suspension was 106The cells were placed in a Transwell upper chamber at a density of 400. mu.l, and 600. mu.l of a conditioned medium of mouse fibroblast cell line NIH3T3 was placed in the lower chamber at 37 ℃ in 5% CO2And (3) in an incubator, after 24h of culture, wiping off cells in an upper chamber by using a wet cotton swab, fixing for 15min by using absolute ethyl alcohol, carrying out HE staining, observing under a light mirror, randomly selecting 5 fields from the upper field, the lower field, the left field, the right field and the middle field, counting the transmembrane cells, and taking an average value to represent the in vitro migration capacity of the SGC7901 cells by using the number of the transmembrane cells.
As a result: the number of transmembrane cells (174.2 +/-13.6) of SGC7901 cells in the experimental group is obviously reduced compared with that of negative control groups (368.5 +/-8.9), and the difference is not statistically significant (P is more than 0.05). The experimental result shows that the inhibition of the expression of the MCM8 gene can inhibit the migration of gastric adenocarcinoma cells.
2. Detection of in vitro invasion capacity of gastric adenocarcinoma cells
Diluting ECM matrix glue with serum-free RP-MI1640 culture solution at a ratio of 1: 7, uniformly coating 30 μ l of ECM matrix glue on the upper chamber surface of the bottom membrane of the Tran-swell chamber, and irradiating overnight with ultraviolet rays to naturally polymerize the ECM matrix glue into gel. Preparing single cell suspension from SGC7901 cells of each group with double-antibody-free RPMI1640 culture solution, and adding 10% of the single cell suspension5Each 400. mu.l was added to the upper chamber of a Transwell and the rest was subjected to the same migration experiment, and the number of transmembrane cells was used as an indicator of the in vitro invasion ability of SGC7901 cells.
As a result: the number of transmembrane cells (32.8 +/-4.5) of SGC7901 cells in the experimental group is obviously reduced compared with that of negative control groups (91.6 +/-3.8). The experimental result shows that the inhibition of the expression of the MCM8 gene can inhibit the invasion of gastric adenocarcinoma cells.
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.
Sequence listing
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Claims (7)
1. Application of a reagent for detecting MCM8 in preparation of a tool for gastric adenocarcinoma metastasis diagnosis.
2. The use of claim 1, wherein the reagent for detecting MCM8 comprises a reagent for detecting the expression level of MCM8 gene.
3. The use according to claim 1 or 2, characterized in that the reagent for detecting MCM8 comprises a reagent capable of quantifying MCM8 gene mRNA, and/or a reagent capable of quantifying MCM8 protein.
4. The use according to claim 3, wherein the reagent capable of quantifying MCM8 gene mRNA comprises primers for specific amplification of MCM8 gene used in real-time quantitative PCR; the reagent capable of quantifying MCM8 protein includes an antibody that specifically binds to MCM8 protein.
5. The use of claim 1, wherein the means comprises a kit, a chip, a strip, a high throughput sequencing platform.
Use of an inhibitor of MCM8 in the manufacture of a medicament for the treatment of gastric adenocarcinoma metastasis.
7. The use as claimed in claim 6 wherein the inhibitor comprises an inhibitor capable of inhibiting the expression or activity of MCM8 or a substance involved in a pathway upstream or downstream of MCM 8.
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| Title |
|---|
| Akiko Ono MD 等.Overexpression of heat shock protein 27 in squamous cell carcinoma of the uterine cervix: a proteomic analysis using archival formalin-fixed, paraffin-embedded tissues.《Human Pathology》.2009,第40卷第41-49页. * |
| 张磊 等.MCM5和E2F-1在胃癌组织中的表达及临床病理意义.《中国现代普通外科进展》.2009,第12卷(第11期),第943-946页. * |
| 许天祥 等.MCM5在胃癌中的表达及其临床意义.《内蒙古医学杂志》.2014,第46卷(第8期),第904-907页. * |
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