CN107034270B - CLIC3 as diagnosis and treatment target of lung adenocarcinoma - Google Patents
CLIC3 as diagnosis and treatment target of lung adenocarcinoma Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57423—Specifically defined cancers of lung
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Abstract
The invention discloses a CLIC3 gene as a diagnosis and treatment target of lung adenocarcinoma. The invention can judge whether the subject suffers from the lung adenocarcinoma or diagnose whether the subject has the risk of suffering from the lung adenocarcinoma by detecting the content of the CLIC3 gene and the expression product thereof in the lung tissue of the subject, or judge the responsiveness of the patient to the drug treatment, or judge the relapse and the prognosis condition of the patient. In addition, the CLIC3 gene and the expression product thereof are proved to be used as the drug target for treating the lung adenocarcinoma by researching the proliferation index of the lung adenocarcinoma cells cultured in vitro.
Description
Technical Field
The invention relates to the fields of tumor diagnosis, treatment and prognosis prediction, in particular to a tumor diagnosis and prognosis prediction method by taking CLIC3 abnormality detection as a means; and a tumor therapeutic agent which activates CLIC3 gene or protein.
Background
Lung cancer is a serious disease that is seriously threatening to human life and health. The 2008 global statistics results published by World Health Organization (WHO) international agency for research on cancer IARC 2011 show: lung cancer is a malignant tumor with high morbidity and mortality. The number of new cases is 160.8 ten thousand and the number of death cases is 137.7 ten thousand worldwide each year, accounting for 12.7% and 18.2% of all malignant tumors. The statistical results published by the american cancer society show that: it is expected that in 2012, the number of new lung cancer cases in the united states will reach 22.6 ten thousand, and the number of deaths due to lung cancer will reach 16 ten thousand. In China, the third national cause of death review sample survey conducted by the ministry of health shows that: the lung cancer is the first cause of death of malignant tumors in a sample area, and the gross mortality rate is 30.83/10 ten thousand, wherein 41.34/10 ten thousand are used for men and 19.84/10 ten thousand are used for women; the first cause of cancer death in both men and women. With the acceleration of global aging and the aggravation of environmental pollution, the incidence of lung cancer will continue to rise, and therefore, the development and demand for research on lung cancer diagnosis and treatment will be increasingly strong.
Tumor markers are biologically active substances produced by abnormal expression of cancer genes or other tumor-associated genes and their products in tumor cells or tissues or shed from cancer tissues themselves, and are expressed to some extent or produced in minimal amounts in normal tissues or benign diseases. It reflects the process of cancer generation and development, can be detected in tissues, body fluids and excreta of tumor patients, and is widely applied to the diagnosis of tumors, the monitoring of recurrence, metastasis, prognosis, the prediction of curative effect and the like. The only markers currently used in the clinical auxiliary diagnosis of lung cancer are protein markers, such as CEA, CA125, CYFRA21-1, and although the above protein markers are currently used in the auxiliary diagnosis of lung cancer, the diagnosis efficiency is low and the clinical requirement is not met. Therefore, the development of new diagnostic markers and diagnostic methods has been elusive.
The early diagnosis of tumors at the molecular level, especially at the gene level, has become a trend in the field of tumor diagnosis, and in the diagnosis of lung cancer, the application numbers are: 201510220102.9, 201510233085.2, 201510243857, 201610202285.6, 201610200867, 201610200574.2 and 201610200855.8 patent documents all disclose gene markers that can be used for lung cancer diagnosis. The present application is based on the prior art to find new biomarkers that can be used for lung cancer diagnosis.
Disclosure of Invention
One of the objects of the present invention is to provide a method for diagnosing lung adenocarcinoma by detecting the difference in expression of CLIC3 gene or protein.
The invention also aims to provide a method for predicting the prognosis of lung adenocarcinoma by detecting the difference of CLIC3 gene or protein expression.
It is a further object of the present invention to provide a method for treating lung adenocarcinoma by activating either the CLIC3 gene or the CLIC3 protein.
The fourth purpose of the invention is to provide a method for screening a medicament for treating lung adenocarcinoma.
The fifth purpose of the invention is to provide a medicament for treating lung adenocarcinoma.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides an application of a product for detecting CLIC3 in preparing a lung adenocarcinoma diagnostic tool.
Further, the product for detecting the CLIC3 comprises a product for detecting the expression level of the CLIC3 gene.
Still further, the product for detecting CLIC3 comprises a product capable of quantifying CLIC3 gene mRNA, and/or a product capable of quantifying CLIC3 protein.
The product of the present invention for quantifying CLIC3 gene mRNA can exert its function based on known methods 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 product can be used to conduct the assay qualitatively, quantitatively, or semi-quantitatively.
The nucleic acid 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 a primer designed to amplify the desired nucleic acid.
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 product capable of quantifying CLIC3 gene mRNA comprises a primer for specifically amplifying CLIC3 gene used in real-time quantitative PCR, and the primer sequence is shown as SEQ ID NO.1 and SEQ ID NO. 2.
The primers included in the product 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 above-mentioned nucleic acids may further include a probe which 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, or can be prepared by preparing a gene containing a desired nucleic acid sequence from a biological material and amplifying it using a primer designed for amplifying the desired nucleic acid sequence.
The product of the invention for quantifying the CLIC3 protein can exert its function based on known methods using antibodies: for example, ELISA, radioimmunoassay, immunohistochemistry, Western blotting, etc. may be included.
The product for quantifying CLIC3 protein comprises an antibody or fragment thereof specifically binding to CLIC3 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 product of the invention for quantifying CLIC3 protein can include an isolated nucleic acid encoding an amino acid sequence of an antibody or encoding a fragment of an antibody, a vector comprising the nucleic acid, and a cell harboring 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 CLIC3 protein can be obtained by subjecting the obtained antibody to antigen-specific purification using CLIC3 protein or a part 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.
As a sample of the detection product according to the present invention, a tissue sample or fluid obtained from a biopsy subject, for example, 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 comprise tissue, blood, plasma, serum, lymph, urine, serosal cavity fluid, spinal fluid, synovial fluid, aqueous humor, tears, saliva, or fractions or treated materials thereof.
In a specific embodiment of the invention, the sample is from a tissue of a subject.
Further, the product for quantifying the CLIC3 gene or the CLIC3 protein can be a reagent for detecting the CLIC3 gene or the CLIC3 protein, a kit, a chip, a test paper and the like containing the reagent, and can also be a high-throughput sequencing platform using the reagent.
The invention also provides a tool for diagnosing lung adenocarcinoma, which can detect the expression level of the CLIC3 gene.
Further, the means comprise an agent capable of quantifying the mRNA of the CLIC3 gene, and/or an agent capable of quantifying the CLIC3 protein.
Further, the reagent capable of quantifying the mRNA of the CLIC3 gene is a primer for specifically amplifying the CLIC3 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 means for diagnosing lung adenocarcinoma includes, but is not limited to, a chip, a kit, a strip, or a high throughput sequencing platform; the high-throughput sequencing platform is a special tool for diagnosing lung adenocarcinoma, and with the development of a 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 application of CLIC3 in high-throughput sequencing to the association of CLIC3 gene abnormality and lung adenocarcinoma is also included in the protection scope of the present invention.
The number of amino acids recognized by the anti-CLIC 3 antibody or a fragment thereof used in the detection product, diagnostic tool, or the like of the present invention is not particularly limited as long as the antibody can bind to CLIC 3.
The present invention also provides a method for diagnosing lung adenocarcinoma, comprising the steps of:
(1) obtaining a sample from a subject;
(2) detecting the expression level of CLIC3 gene or protein in a sample from the subject;
(3) correlating the measured expression level of CLIC3 gene or protein with the presence or absence of disease in the subject.
(4) When the expression level of CLIC3 gene or protein is decreased compared with that of the control, the subject is judged to have lung adenocarcinoma, or the patient with lung adenocarcinoma is judged to have recurrence, or the patient with lung adenocarcinoma is judged to have poor prognosis.
The invention also provides a method of treatment of lung adenocarcinoma, said method comprising activating the CLIC3 gene or CLIC3 protein.
Further, the method comprises promoting the expression of the CLIC3 gene, or promoting the expression of the CLIC3 protein or enhancing the activity of the CLIC3 protein.
The invention also provides a screening method of tumor drugs, which can measure the effect of the tumor drugs on improving tumor prognosis by measuring the expression level of the CLIC3 gene or the CLIC3 protein at a certain period after adding test drugs to cancer cells or after administering the test drugs to tumor model animals. More specifically, when the expression level of the CLIC3 gene or the CLIC3 protein is increased or restored to a normal level after the addition or administration of a test drug, the drug can be selected as a therapeutic drug for improving the prognosis of tumor.
The invention also provides a medicament for treating lung adenocarcinoma, which comprises an activator of CLIC 3.
The activator of CLIC3 of the invention is not limited as long as the activator can promote or enhance the expression or activity of CLIC3 or a substance involved in the pathway upstream or downstream of CLIC3, and is a drug effective for treating tumors.
The invention also provides application of the activator in preparing a medicament for treating lung adenocarcinoma.
Further, the activator comprises a CLIC3 gene, a CLIC3 protein, a promoting miRNA, a promoting transcriptional regulator, or a promoting targeted small molecule compound.
The activator can be used for supplementing the deletion or deficiency of endogenous CLIC3 protein and treating lung adenocarcinoma caused by CLIC3 protein deficiency by increasing the expression of CLIC3 protein. On the other hand, the protein can be used for enhancing the activity of the CLIC3 protein, thereby treating lung adenocarcinoma.
The medicament of the present invention may be administered alone or together with other medicaments as a medicine. Other drugs that can be administered together with the inventive drug are not limited as long as it does not impair the effect of the inventive therapeutic or prophylactic drug, and preferably, drugs for treating or preventing tumors may include, for example, alkylating agents such as ifosfamide, cyclophosphamide, dacarbazine, temozolomide, nimustine, busulfan, procarbazine, melphalan, and ramustine; antimetabolites such as enocitabine, capecitabine, carmofur, cladribine, gemcitabine, cytarabine octadecylphosphate (cytarabine ocfosfate), tegafur-uracil, tegafur-gimeracil oteracil potassium, doxifluridine, hydroxyurea, fluorouracil, fludarabine, pemetrexed, pentostatin, mercaptopurine, and methotrexate; plant alkaloids such as irinotecan, etoposide, sobuzolff, docetaxel, nogitecan, paclitaxel, vinorelbine, vindesine, and vinblastine; anticancer antibiotics such as actinomycin D, aclarubicin, amrubicin, idarubicin, epirubicin, netostatin stimalamer, daunorubicin, doxorubicin, pirarubicin, bleomycin, pellomycin, mitomycin C, and mitoxantrone; platinum-based drugs such as oxaliplatin, carboplatin, cisplatin, and nedaplatin; hormonal agents such as anastrozole, exemestane, estramustine, ethinylestradiol, chlormadinone, goserelin, tamoxifen, dexamethasone, toremifene, bicalutamide, flutamide, prednisolone, fosfestrol, mitotane, methyltestosterone, medroxyprogesterone, meindroxane, leuprorelin, and letrozole; biological response modifiers such as interferon alpha, interferon beta, interferon gamma, interleukin, ubenimex, dry BCG, and lentinan; and molecularly targeted drugs such as imatinib (imatinib), gefitinib (gefitinib), gemumab, ozomicin, tamibarotene, trastuzumab, tretinoin, bortezomib (bortezomib), and rituximab, and the like.
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.
In the context of the present invention, "diagnosing lung adenocarcinoma" includes determining whether a subject has had lung adenocarcinoma, determining whether a subject is at risk for having lung adenocarcinoma, determining whether a lung adenocarcinoma patient has relapsed and metastasized, determining the responsiveness of a lung adenocarcinoma patient to drug treatment, or determining the prognosis of a lung adenocarcinoma patient.
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 discloses a molecular marker for diagnosing lung adenocarcinoma, which can be used for judging the early stage of the occurrence of the lung adenocarcinoma and provides the survival rate of patients.
The therapeutic agent of the present invention comprising an activator of the CLIC3 gene or protein is useful as a novel therapeutic agent for lung adenocarcinoma.
Drawings
FIG. 1 shows a statistical graph of the detection of differential expression of CLIC3 gene at the mRNA level using QPCR;
FIG. 2 shows a statistical chart of the detection of differential expression of CLIC3 gene at the protein level using immunoblotting;
FIG. 3 shows a statistical chart for detecting overexpression of CLIC3 gene at mRNA level using QPCR;
FIG. 4 shows a statistical chart for detecting the overexpression of CLIC3 gene at the protein level using immunoblotting;
FIG. 5 shows a statistical plot of the effect of CLIC3 gene overexpression on lung adenocarcinoma cell proliferation.
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 screening for differentially expressed genes
1. Material taking:
cancer tissue and paracarcinoma tissue were surgically removed from 8 patients with primary lung adenocarcinoma as experimental specimens. All cancer tissues were pathologically confirmed post-operatively as lung adenocarcinoma. All patients with primary lung adenocarcinoma do not undergo radiotherapy and chemotherapy before operation, and all clinical data of all cases are complete.
2. Tissue RNA acquisition
Total RNA was extracted from tissue samples, the concentration and purity of the extracted RNA was determined using a Nanodrop2000, RNA integrity was determined by agarose gel electrophoresis, and RIN was determined by Agilent 2100. The total amount of RNA required for single library construction is 5ug, the concentration is more than or equal to 200 ng/mu L, and the OD260/280 is between 1.8 and 2.2.
3. Removal of rRNA
A part (> 24%) of long non-coding RNA in cells is short of a traditional poly A tail, so that more comprehensive lncRNA information can be obtained by constructing a library in a way of removing rRNA.
4. Fragmented RNA
The Illumina platform is used for sequencing short sequence fragments, mRNA and lncRNA obtained by removing rRNA are complete RNA sequences, the average length can reach several kb, and random interruption is needed. The RNA can be randomly fragmented into small fragments of about 200bp by using metal ions.
5. Reverse Synthesis of cDNA
When double-strand synthesis is performed by reverse-transcribing a single-strand cDNA using mRNA as a template with a random primer by reverse transcriptase, dUTP is used instead of dTTP in dNTPs reagents so that the base in the second strand of the cDNA contains A/U/C/G.
6. Connection adapter
The double-stranded cDNA structure is sticky-ended, and is made blunt-ended by adding End Repair Mix, followed by an A base at the 3' End for ligation to a Y-shaped adaptor.
7. UNG enzyme digestion of cDNA double strand
Before PCR amplification, the second strand of cDNA was digested with UNG enzyme, so that only the first strand of cDNA was contained in the library.
8. Sequencing on Illumina Hiseq4000
Enriching the library, and amplifying 15 cycles by PCR;
band of interest was recovered from 2% Agarose gel (verified Low Range Ultra Agarose);
TBS380(PicoGreen) is quantified and mixed according to the data proportion;
performing bridge PCR amplification on cBot to generate clusters;
hiseq4000 sequencing platform, 2 × 150bp sequencing.
9. Raw sequencing data filtering
Trimming off low-quality bases (with the quality value of less than 20) at the tail ends (5 'end and 3' end) of the sequence;
removing reads with the N content ratio exceeding 10%;
10. differential mRNA expression analysis
Analysis software: cuffdiff (http:// Cufflinks. cbcb. umd. edu /)
Cuffdiff is a tool used for calculating differential expression in a Cufflinks suite, and Cuffdiff uses the result of Tophat comparison to call Cufflinks to calculate the expression quantity of each gene/transcript. The software is usually run with default parameters, while the parameters are appropriately adjusted according to the actual conditions, such as the amount of sequencing data and the genome condition.
Significantly different mRNA screening conditions: p-value <0.05, and the difference between the count averages of the two groups is greater than 10.
11. Results
The sequencing result shows that: compared with the paracarcinoma tissues, 1321 genes are differentially expressed in lung adenocarcinoma tissues, 587 are up-regulated and 734 are down-regulated.
Example 2 validation of Large samples of the selected differentially expressed genes
Based on the results of early high-throughput transcriptome deep sequencing, we selected the CLIC3 gene for validation based on the size of P value.
1. Sample collection
45 cases of lung adenocarcinoma tissue and corresponding paraneoplastic tissue were collected according to the method of example 1.
2. Validation at mRNA level
2.1 extraction of tissue RNA
The procedure is as in example 1.
2.2 reverse transcription
Primescript 1 was used for reverse transcriptionstThe strand cDNA synthesis kit comprises the following operation steps:
(1) the following reaction liquids were added to the microcentrifuge tube, as shown in table 1:
TABLE 1 reaction liquid
(2) Incubating at 70 deg.C for 5min, and rapidly cooling to 4 deg.C;
adding the following reaction reagents into a microcentrifuge tube to prepare a reaction system:
TABLE 2 preparation of the reaction System
Gently shaking, rapidly centrifuging, reacting at 42 deg.C for 1h, stopping reaction at 70 deg.C for 10min, cooling at 4 deg.C, and storing at-20 deg.C.
Using SYBP Premix Ex TapTMThe kit II is carried out in an Eppendorf Real-time PCR analyzer, and the specific operation is as follows:
(1) the following PCR reaction solutions were prepared on ice:
TABLE 3 preparation of PCR reaction solution
The primer sequences were designed as follows:
CLIC3 gene:
5’-CTGCCCATCCTGCTCTAT-3’(SEQ ID NO.1)；
5’-CAGCGTCTCCTCCAGAAA-3’(SEQ ID NO.2)
β-actin：
5’-GTGGGGCGCCCCAGGCACCA-3’(SEQ ID NO.3)；
5’-CTCCTTAATGTCACGCACGATTT-3’(SEQ ID NO.4)
(2) and the computer is used for executing the following programs: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15 s. Annealing at 59 ℃ for 20s, and extension at 72 ℃ for 20s, for 40 cycles.
The result is obtained by a relative quantitative method using formula 2-△△ctAnd (4) calculating. The experiment was repeated 3 times.
△ct＝ct(A)-ct(β-actin)
Δ ct (experimental group) — Δ ct (control group)
The results are shown in figure 1, and the mRNA level of CLIC3 gene was significantly reduced in lung adenocarcinoma tissue compared to paracarcinoma tissue, with the difference having statistical significance (P < 0.05).
3. Verification at protein level
Each histone was extracted according to the RIPA protein lysate kit instructions, and the protein concentration in the sample was detected using the BCA protein concentration assay kit. The change of the CLIC3 protein is detected by a conventional Western-blot method, each group of experiments are repeated for 3 times, beta-actin is taken as an internal reference, the absorbance quantitative analysis of CLIC3 protein bands is carried out, and the expression quantity is represented by the ratio of CLIC3 protein/beta-actin absorbance.
The results are shown in figure 2, with significant reduction in the levels of CLIC3 protein in lung adenocarcinoma tissue compared to paracarcinoma tissue, with a statistical significance for the difference (P < 0.05).
Example 3 CLIC3 Gene overexpression
1. Plasmid construction
Amplification primers were designed based on the coding sequence of the CLIC3 gene, the design of which is well known to those skilled in the art. The coding sequence of the full-length CLIC3 gene was amplified from a cDNA library of adult fetal brain (Clontech, cat # 638831), the above cDNA sequence was inserted into the eukaryotic cell expression vector pcDNA3.1, and the obtained recombinant vector pcDNA3.1-CLIC3 was ligated for subsequent experiments.
2. Culture and transfection of lung adenocarcinoma cells
2.1 cell culture
Culturing the lung adenocarcinoma cell strain A549 in RPMI1640 culture medium and 10% fetal calf serum.
2.2 transfection of cells
(1) The day before transfection, 0.5-2 x 105Each tumor cell was suspended in 500. mu.l of antibiotic-free medium and inoculated into a 24-well plate.
(2) Cell density should reach 80% -90% on day of transfection, and the following complex a was prepared: diluting 1 μ g plasmid DNA in serum-free medium, and mixing gently; and (3) a compound B: mu.l of Lipofectamine2000 was diluted in serum-free medium and mixed well.
(3) Mix complex a and B, mix gently, incubate at room temperature.
(4) Adding 100 μ l liposome complex into tumor cells, mixing gently, and placing the cells at 37 deg.C containing 5% CO2Incubate the incubator for 5-7 hours.
(5) 1ml of growth medium containing 2 times the normal serum and antibiotic concentrations was added and the cells were cultured for an additional 18-24 hours.
3. Detection of overexpression of pcDNA3.1-CLIC3 Using QPCR assay
3.1 extraction of cellular Total RNA Using conventional methods.
3.2 reverse transcription
The procedure is as in example 2.
3.3 QPCR
The procedure is as in example 2.
3.4 results
As shown in FIG. 3, pcDNA3.1-CLIC3 was successfully overexpressed, and the difference was statistically significant (P < 0.05).
3. Western blot experiment for detecting overexpression condition of pcDNA3.1-CLIC3
The procedure is as in example 2.
As shown in FIG. 4, the content of CLIC3 protein in the cells transfected with pcDNA3.1-CLIC3 was significantly increased compared to that in the pcDNA3.1-transfected group, and the difference was statistically significant (P < 0.05).
Example 4 measurement of the proliferative Capacity of Lung adenocarcinoma cells by expression of CLIC3 Gene
1. The method comprises the following steps:
24 hours after transfection, lung adenocarcinoma cells A549 were seeded into 96-well cell culture plates at 2 x 10 per well3Individual cells/well/200 μ l, cells were grouped as follows:
experimental group 1 (control group): transfecting a lung adenocarcinoma cell with pcDNA3.1;
experimental group 2: the lung adenocarcinoma cells were transfected with pcDNA3.1-CLIC 3.
Cells were incubated at 37 ℃ with 5% CO2After incubation of the incubator for another 24 hours, the cell proliferation rate was measured according to the instructions of the Brd U cell proliferation kit (Chemicon International).
2. Statistical method
The experiments were performed in 3 replicates, the results were expressed as mean ± sd, and were statistically analyzed using SPSS13.0 statistical software, with the difference between the two using the t-test, and considered statistically significant when P < 0.05.
3. Results
The results are shown in fig. 5, where the cell proliferation was reduced compared to experimental group 1, and the difference was statistically significant (P < 0.05). The experimental results show that CLIC3 expression can inhibit lung adenocarcinoma cell proliferation.
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
<110> Hospital of Hebei medical university (tumor hospital of Hebei province)
<120> CLIC3 as diagnosis and treatment target of lung adenocarcinoma
<160>4
<170>PatentIn version 3.5
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Claims (1)
- The application of an activator of CLIC3 in preparing a medicament for treating lung adenocarcinoma is characterized in that the activator is pcDNA3.1 overexpression vector of CLIC3 gene.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104818334A (en) * | 2015-06-02 | 2015-08-05 | 北京泱深生物信息技术有限公司 | Tiny RNA related to lung adenocarcinoma metastasis |
| CN105624325A (en) * | 2016-03-31 | 2016-06-01 | 北京泱深生物信息技术有限公司 | Marker for diagnosing and treating lung adenocarcinoma |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104818334A (en) * | 2015-06-02 | 2015-08-05 | 北京泱深生物信息技术有限公司 | Tiny RNA related to lung adenocarcinoma metastasis |
| CN105624325A (en) * | 2016-03-31 | 2016-06-01 | 北京泱深生物信息技术有限公司 | Marker for diagnosing and treating lung adenocarcinoma |
Non-Patent Citations (2)
| Title |
|---|
| Ion Channel Gene Expression in Lung Adenocarcinoma:;Jae-Hong Ko et al;《PLOS ONE》;20140123;第9卷(第1期);第1-12页 * |
| 唾液腺黏液表皮样癌中CLIC3的表达及意义;王智明等;《中国口腔颌面外科杂志》;20160531;第14卷(第3期);全文 * |
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