CN105755154A - Molecular marker differentiating metastatic squamous cell lung carcinoma from non-metastatic squamous cell lung carcinoma - Google Patents
Molecular marker differentiating metastatic squamous cell lung carcinoma from non-metastatic squamous cell lung carcinoma Download PDFInfo
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Abstract
The invention discloses application of TEX13B gene and an expression product thereof to preparation of diagnostic tools for squamous cell lung carcinoma metastasis. According to the invention, a gene chip and a QPCR experiment prove that the TEX13B gene has difference in the expression of squamous cell lung carcinoma non-metastatic samples and squamous cell lung carcinoma metastatic samples, so that the TEX13B gene is believed to be a molecular marker for diagnosing squamous cell lung carcinoma metastasis. A culture in vitro cell experiment proves that restraining the expression of the TEX13B gene can restrain the adhesion, migration and invasion of lung squamous carcinoma, so that the TEX13B gene is believed to be a drug target for treating squamous cell lung carcinoma metastasis. As a new molecular marker, the TEX13B gene and the expression product thereof have a wide clinical application prospect.
Description
Technical field
The present invention relates to biological technical field, the purposes in the diagnosis shifted more particularly to TEX13B gene, treatment at lung squamous cancer.
Background technology
Pulmonary carcinoma is generally divided into scale cancer, adenocarcinoma, large cell carcinoma and small cell carcinoma 4 type by histologic classification, wherein most commonly seen with scale cancer, accounts for the 40%-70% of whole pulmonary carcinoma.Pulmonary carcinoma is one of current modal malignant tumor, and its mortality rate occupies first of human malignancies.In world's most countries, pulmonary carcinoma accounts for first place in male's common cancer, accounts for the 38.03% of male malignancy death;In women, its mortality rate has exceeded breast carcinoma becomes the first cause of tumor mortality.In recent years, owing to have employed the multidisciplinary synthesis treatment based on operation, the therapeutic effect of pulmonary carcinoma is significantly improved.But, over all, the total 5 years survival rates of patients with lung cancer are still relatively low, are only 8.9%-15%.Tracing it to its cause, when mainly most of patients is medical first, oneself there occurs metastasis, even prognosis best I phase and II phase nonsmall-cell lung cancer, Post operation most of patients still can die from Lung Cancer Recurrence or metastasis.Therefore, inquire into the molecule mechanism of pulmonary carcinoma, find early diagnosis pulmonary carcinoma, predict the biomarker of transfer and the target molecule of therapeutic intervention, diagnosing and treating of pulmonary carcinoma is had great importance.
The transfer of malignant cell is tumor development to the inevitable outcome in late period and the main cause causing death.Up to now, the research of mechanism of tumor metastasis is not yet obtained breakthrough progress.The research of pulmonary carcinoma is such too.Recent studies indicate that, invasion of lung cancer transfer is the multi-step process of extremely complex polygenes regulation and control, relates to the multiple signal path of cell and changes, and the complex biological phenomenon just ultimately formed through multiple stages.Discrete including tumor cell, migrate, secretion enzyme material dissolves surrounding substrate (invasion and attack), interacts with blood vessel or lymphatic endothelial cells, enter and run and plant distal organs in pipe and shift.Research report, by cancer to cancer beside organism MMP-2, MMP-9 expresses all exists the trend being gradually converted into feminine gender by the positive, prompting tumor cell can carry out information exchange by soluble mediators or film binding molecule and Interstitial cell, collaborative generation and adjustment MMP-2, MMP-9, promotes invasion of lung cancer and transfer.In the test of mice, additionally find that heparinase is by promoting the expression of VEGF, activates the hypertrophy of endotheliocyte and the formation of new vessels, promotes lung carcinoma cell metastasis.Research finds that integrin Na can interact with fibronectin (Fibronectin, FN) and plasma glycoprotein (Vitronectin, VN) and promote proliferation of lung cancer cells and transfer.Although lung cancer metastasis has been carried out much research by people, but its metastasis is still unclear.The research metastasis that develops into of high throughput sequencing technologies provides new means.High throughput sequencing technologies is utilized to compare the difference of different phase tumor tissues gene expression profile, the gene that screening tumor development is relevant, it is possible to find neoplasm metastasis mark and Drug therapy target.
Summary of the invention
It is an object of the invention to provide a kind of molecular marker that can be used for lung squamous cancer transfer diagnosis.Use what gene marker carried out the transfer of Diagnosis of pulmonary scale cancer to have promptness, specificity and susceptiveness, so that patient just can know disease risks in transfer early stage, for risk height, take to prevent accordingly and remedy measures.
To achieve these goals, the present invention adopts the following technical scheme that
The invention provides the application in the instrument preparing the transfer of Diagnosis of pulmonary scale cancer of the product of detection TEX13B gene expression.
Further, the product of described detection TEX13B gene expression includes the product of detection TEX13B gene mRNA levels and/or the product of detection TEX13B protein level.
Further, the product of described detection TEX13B gene expression includes: the product shifted with Diagnosis of pulmonary scale cancer by RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or chip detection TEX13B gene expression.
Further, the product of described RT-PCR Diagnosis of pulmonary scale cancer transfer at least includes the primer of a pair specific amplified TEX13B gene;The product of described real-time quantitative PCR Diagnosis of pulmonary scale cancer transfer at least includes the primer of a pair specific amplified TEX13B gene;The product of described immune detection Diagnosis of pulmonary scale cancer transfer includes: the antibody being combined with TEX13B protein-specific;The product of described in situ hybridization Diagnosis of pulmonary scale cancer transfer includes: with the probe of the nucleic acid array hybridizing of TEX13B gene;The product of described chip Diagnosis of pulmonary scale cancer transfer includes: protein chip and gene chip;Wherein, protein chip includes the antibody being combined with TEX13B protein-specific, and gene chip includes the probe of the nucleic acid array hybridizing with TEX13B gene.
The primer of a pair specific amplified TEX13B gene that the product of described real-time quantitative PCR Diagnosis of pulmonary scale cancer transfer at least includes is such as shown in SEQIDNO.3 and SEQIDNO.4.
The product of described detection TEX13B gene expression can be the reagent of detection TEX13B gene expression, can also be comprise the test kit of described reagent, chip, reagent paper etc., it is also possible to be the high-flux sequence platform using described reagent.
The instrument of described Diagnosis of pulmonary scale cancer transfer includes but not limited to chip, test kit, reagent paper or high-flux sequence platform;High-flux sequence platform is the instrument of a kind of special Diagnosis of pulmonary scale cancer transfer, along with the development of high throughput sequencing technologies, the structure of the gene expression profile of a people will be become and very work easily.By contrasting the gene expression profile of Disease and normal population, it is easy to analyze exception and the disease association of which gene.Therefore, in high-flux sequence, know that the exception of TEX13B gene shifts the relevant purposes falling within TEX13B gene to lung squamous cancer, equally within protection scope of the present invention.
Present invention also offers the instrument of a kind of Diagnosis of pulmonary scale cancer transfer, described instrument includes the reagent of detection TEX13B gene expression;Described reagent includes the primer of detection TEX13B gene mRNA and/or the antibody of probe, detection TEX13B albumen.
Described instrument includes but not limited to chip, test kit, reagent paper or high-flux sequence platform.
Wherein, described chip includes gene chip, protein chip;Described gene chip includes solid phase carrier and is fixed on the oligonucleotide probe of solid phase carrier, and described oligonucleotide probe includes the oligonucleotide probe for TEX13B gene for detecting TEX13B gene transcription level;Described protein chip includes solid phase carrier and is fixed on the specific antibody of TEX13B albumen of solid phase carrier;Described gene chip can be used for detecting the expression of the multiple genes (such as, relevant to lung squamous cancer transfer multiple genes) including TEX13B gene.Described protein chip can be used for detecting the expression of the multiple protein (such as relevant to lung squamous cancer transfer multiple protein) including TEX13B albumen.By being detected by multiple and lung squamous cancer transfer marks simultaneously, it is greatly improved the accuracy rate of lung squamous cancer transfer diagnosis.
Wherein, described test kit includes gene detecting kit and protein immunization detection kit;Described gene detecting kit includes the reagent for detecting TEX13B gene transcription level;Described protein immunization detection kit includes the specific antibody of TEX13B albumen.Further, described reagent includes using reagent required in RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or chip method detection TEX13B gene expression dose process.Preferably, described reagent includes the primer for TEX13B gene and/or probe.Nucleotide sequence information according to TEX13B gene easily designs the primer and probe that may be used for detection TEX13B gene expression dose.
Described reagent paper includes the reagent of detection TEX13B gene expression.
Described high-flux sequence platform includes the reagent of detection TEX13B gene expression.
Can be DNA, RNA, DNA-RNA chimera, PNA or other derivant with the probe of the nucleic acid array hybridizing of TEX13B gene.The length of described probe does not limit, as long as completing specific hybrid and purpose nucleotide sequence is specific binding, any length can.The length of described probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the length of described probe can be grown to 60,80,100,150,300 base pairs or longer, even whole gene.Owing to hybridization efficiency, signal specificity are had different impacts, the length of described probe be typically at least 14 base pairs by different probe length, the longest it is usually no more than 30 base pairs, best with 15-25 base pair with the length of purpose nucleotide sequence complementary.Described probe self-complementary sequences is most preferably less than 4 base pairs, in order to avoid affecting hybridization efficiency.
Further, the specific antibody of described TEX13B albumen includes monoclonal antibody, polyclonal antibody.The specific antibody of described TEX13B albumen include complete antibody molecule, any fragment of antibody or modify (such as, chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc..As long as described fragment can retain and the binding ability of TEX13B albumen.Well known to a person skilled in the art during for the preparation of the antibody of protein level, and the present invention can use any method to prepare described antibody.
In specific embodiments of the present invention, the primer of described detection TEX13B gene mRNA includes the primer pair shown in SEQIDNO.3 and SEQIDNO.4.
Present invention also offers the application in the medicine of preparation treatment lung squamous cancer transfer of the inhibitor of TEX13B gene and/or its expression product.Described inhibitor includes the reagent suppressing TEX13B gene expression and/or the reagent suppressing TEX13B gene expression product.
Further, the described reagent suppressing TEX13B gene expression includes the reagent of reagent that suppressor gene transcribes, suppressor gene translation;The described reagent suppressing TEX13B gene expression product includes suppressing the reagent of TEX13B gene mRNA, suppressing the reagent of TEX13B albumen.The described reagent suppressing TEX13B gene mRNA includes suppressing the reagent of mRNA stability, suppressing the reagent of mRNA translation activity.The described reagent suppressing TEX13B albumen includes suppressing the reagent of TEX13B protein stability, suppressing the reagent of the reagent of TEX13B protein active, suppression TEX13B protein function.
Further, it is suppressed that the reagent of TEX13B gene mRNA includes the double stranded RNA for TEX13B gene mRNA;The reagent suppressing TEX13B protein function includes the tumor vaccine of TEX13B antigen protein, suppresses the antibody of TEX13B protein function.Described antibody can be polyclonal antibody, or monoclonal antibody.
In specific embodiments of the present invention, the described double stranded RNA for TEX13B gene mRNA is siRNA.Can efficiently be rejected in order to ensure TEX13B gene or reticent, the siRNA specific fragment according to the mRNA sequential design of TEX13B gene.nullThe design of siRNA is according to the general design principle (Elbashiret.al2001 delivered,Schwarzet.al2003,Khvorovaet.al2003,Reynoldset.al2004,Hsiehet.al2004,Ui-Teiet.al2004),By online tool complete design,This online tool is: siRNASelectionProgramofWhiteheadInstitute (BingbingYuanet.al2004,Http:// jura.wi.mit.edu/bioc/siRNAext/) and BLOCK-iTTMRNAiDesignerofINVITROGEN (winnerofthe2004Frost&SullivanExcellenceinResearchAward,https://rnaidesigner.invitrogen.com/sirna/).In order to improve the effectiveness of siRNA segment further, the advantage of comprehensive two Photographing On-line instruments designs the siRNA segment for screening.Finally, filter siRNA sequence by sequence analysis (NCBIBLAST), to improve the specificity of siRNA segment and to reduce the effect of missing the target of RNAi interference.
Preferably, the sequence of described siRNA is such as shown in SEQIDNO.9 and SEQIDNO.10.
Present invention also offers a kind of pharmaceutical composition for treating lung squamous cancer transfer, described pharmaceutical composition includes the inhibitor of TEX13B gene recited above and/or its expression product.
The pharmaceutical composition of the present invention also includes pharmaceutically acceptable carrier, and wherein this carrier can be the mixing of more than excipient, diluent, thickening agent, filler, bonding agent, disintegrating agent, lubricant, oils and fats or the base of non-oils and fats, surfactant, suspending agent, gellant, adjuvant, preservative, antioxidant, stabilizer, coloring agent or spice either or both of which.
The pharmaceutical composition of the present invention can be used for manufacturing the medicament for the treatment of lung squamous cancer transfer.
The pharmaceutical composition of the present invention, wherein this mammal can be human patients.
The pharmaceutical composition of the present invention can such as to be administered orally, to inject, to smear or one of them mode of paster gives to this human patients's body.
The pharmaceutical composition of the present invention also can with the drug combination of other treatment lung squamous cancer transfer, and multi-medicament is used in combination the success rate that can significantly mention treatment.
In the context of the present invention, " TEX13B gene " includes the polynucleotide of any function equivalent of TEX13B gene and TEX13B gene.TEX13B gene includes having more than 70% homology and coding identical function protein DNA sequence with TEX13B gene (NC_000023.11) DNA sequence in international common core sequence databank GeneBank at present;
Preferably, the coded sequence of TEX13B gene includes any DNA molecular following:
(1) DNA sequence shown in SEQ ID NO.1;
(2) under strict conditions with 1) the DNA sequence hybridization that limits and coding identical function protein DNA sequence;
(3) DNA sequence limited with (1) or (2) has 70%, preferably, more than 90% homology, and coding identical function protein DNA molecule.
In specific embodiments of the present invention, the coded sequence of described TEX13B gene is the DNA sequence shown in SEQIDNO.1.
In the context of the present invention, TEX13B gene expression product includes TEX13B albumen and the partial peptide of TEX13B albumen.The partial peptide of described TEX13B albumen contains the functional domain relevant to lung squamous cancer transfer.
" TEX13B albumen " includes any function equivalent of TEX13B albumen and TEX13B albumen.Described function equivalent includes TEX13B albumen conservative variation's protein or its active fragment, or its reactive derivative, allelic variant, natural mutation, induced mutants, can with the protein coded by the DNA of the DNA hybridization of TEX13B under high or low stringent condition.
Preferably, TEX13B albumen is the protein with following amino acid sequences:
(1) protein that the aminoacid sequence shown in SEQ ID NO.2 forms;
(2) aminoacid sequence shown in SEQIDNO.2 had the protein that the aminoacid sequence shown in SEQIDNO.2 of identical function is derivative through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and with the aminoacid sequence shown in SEQIDNO.2.The amino acid whose number replacing, lack or adding is generally 1-50, it is preferred that 1-30, and more preferably 1-20,1-10 is individual best.
(3) with the aminoacid sequence shown in SEQIDNO.2, there is at least 80% homology (being also called sequence iden), more preferably, with the aminoacid sequence shown in SEQIDNO.2 at least about 90% to 95% homology, be often 96%, 97%, 98%, 99% homology aminoacid sequence constitute polypeptide.
In specific embodiments of the present invention, described TEX13B albumen is the protein with the aminoacid sequence shown in SEQIDNO.2.
It is known that, conventionally, in a protein, one or more amino acid whose modifications are without influence on the function of protein.Those skilled in the art can approve the aminoacid of change single amino acids or little percentage ratio or be conservative modifications to indivedual interpolations of aminoacid sequence, disappearance, insertion, replacement, and wherein the generation that changes of protein has the protein of identity function.It is well known in the art for providing intimate amino acid whose Conservative substitution tables.
By adding the fusion protein that the example of the protein of an aminoacid or multiple Modification of amino acid residues is TEX13B albumen.Peptide or protein with TEX13B protein fusion is not limited, as long as the fusion protein of gained retains the biologic activity of TEX13B albumen.
The TEX13B albumen of the present invention also includes the non-conservative modification to the aminoacid sequence shown in SEQIDNO.2, as long as the protein through modifying remains able to retain the biologic activity of TEX13B albumen.In this type of modifying protein, the amino acid number of sudden change is usually 10 or less, for instance 6 or less, for instance 3 or less.
In the context of the present invention, " transfer of Diagnosis of pulmonary scale cancer " both include judging whether experimenter has suffered from lung squamous cancer transfer, also include judging experimenter and whether exist suffer from the risk that lung squamous cancer shifts.
In the context of the present invention, " treatment lung squamous cancer shifts " changes from the state of disease and divides, it is possible to include the healing completely of the alleviation of disease, disease.
Advantages of the present invention and beneficial effect:
Present invention firstly discovers that TEX13B gene expression is relevant to lung squamous cancer transfer, by detecting the expression of TEX13B in experimenter's tissue, may determine that whether experimenter suffers from lung squamous cancer transfer or judge whether experimenter exists the risk suffering from lung squamous cancer transfer, thus instructing clinicist to provide prevention scheme or therapeutic scheme to experimenter.
Present invention finds a kind of new molecular marked compound-TEX13B gene, compare traditional detection means, gene diagnosis more in time, more special, sensitiveer, it is possible to realize lung squamous cancer transfer early diagnosis, thus reduce lung squamous cancer transfer mortality rate.
Accompanying drawing explanation
Fig. 1 shows the expression utilizing genechip detection TEX13B gene in lung squamous cell carcinoma cancers;
Fig. 2 show utilize Westernblot detect TEX13B albumen expression in lung squamous cell carcinoma cancers;
Fig. 3 shows and utilizes QPCR to detect the expression of TEX13B gene in Lung Squamous Carcinoma Cells;
Fig. 4 show utilize QPCR detect the siRNA impact on TEX13B gene expression;
Fig. 5 show utilize Westernblot detect the siRNA impact on TEX13B protein expression;
Fig. 6 display interference TEX13B gene expression impact on Lung Squamous Carcinoma Cells adhesive capacity.
Specific embodiment
Below in conjunction with drawings and Examples, the present invention is further detailed explanation.Following example are merely to illustrate the present invention rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in embodiment, generally conventionally condition, such as Sambrook et al., molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to manufacturer it is proposed that condition.
The differential expression of embodiment 1TEX13B gene
1, sample acquisition: it is the excision specimen of From Lung Squamous Carcinoma Patients that 40 example lung squamous cell carcinoma cancers (include 20 examples and have transfer sample and 20 examples without transfer sample) above-mentioned sample.The acquirement of above-mentioned all specimen is each through the agreement of committee of organizational ethics.The clinical data of tissue samples includes: sex, age, tumor size, pathological grading (Edmonson), whether shifts, whether recur.
2, the acquisition of lung squamous cancer transfer tissue RNA
Use Trizol one-step method to extract lung squamous cancer transfer total tissue RNA, read 260nm and the 280nm absorbance (A) located by NanodropND-1000 and measure the purity of RNA solution.Through 1% denaturing formaldehyde agarose gel electrophoresis, observe under ultraviolet transmission light, the integrity of detection RNA.
3, gene chip hybridization and scanning
After the linearized amplification of total serum IgE, cy3-UTP labelling, the cRNAs after fluorescent labeling adopts RNEASYMiniKit purification, with the RNAFragmentationReagents of Amhion, the cRNAs that labelling is good is carried out fragmentation process.Adopt people's full genome chip of expression spectrum (4x44K gene) of U.S. Agilent company, 65 DEG C of hybridization 17h in chip hybridization stove, then eluting, dyeing, finally use AgilentDNAMicroarrayScanner scanner scanning.
4, chip data processes and analyzes
Chip after hybridization reads after data point through chip scanner, imports data to analyze software, for gene more than 2.0 or less than 0.5 of the natural logrithm absolute value of two groups of ratios as difference expression gene.
5, statistical procedures
Adopting SPSS13.0 statistical software to carry out data analysis, group difference compares employing one factor analysis of variance method, and P < 0.05 difference has significant.
6, result
Result shows (as shown in Figure 1), compared with the lung squamous cell carcinoma cancers that transfer does not occur, has occurred the mRNA level in-site of TEX13B gene in the lung squamous cell carcinoma cancers of transfer to dramatically increase, and difference has statistical significance (P < 0.05).
The differential expression of embodiment 2TEX13B albumen
1, object of study is with embodiment 1.
2, tissue total protein is extracted
The operation of protein extraction is carried out according to the description of EpiQuik tissue/cell total protein extraction test kit.
3, Westernblot detection
The protein quantification of extraction is carried out SDS-PAGE electrophoresis, carries out transferring film afterwards, closing, primary antibodie are hatched, two anti-hatch, develop the color.
4, statistical procedures
Use ImageJ software to be analyzed the gray value of protein band, with β-actin for internal reference, the gray value of TEX13B protein band is normalized.Result data is all represent in the way of mean+SD, adopts SPSS13.0 statistical software to carry out statistical analysis, and difference between the two adopts t inspection, it is believed that when P < has statistical significance when 0.05.
5, result
Result is as in figure 2 it is shown, compared with the lung squamous cell carcinoma cancers that transfer does not occur, occurred the expression of TEX13B albumen in the lung squamous cell carcinoma cancers of transfer to dramatically increase, and difference has statistical significance (P < 0.05).
The expression in Lung Squamous Carcinoma Cells system of the embodiment 3TEX13B gene
1, cell is cultivated
Lung Squamous Carcinoma Cells strain NCI-H520, NCI-H596, NCI-H2170, NCI-H226 are cultivated in DMEM culture medium and 10% hyclone, human squamous lung cancer strain BEAS-2B is cultivated in BEGM culture fluid and 10% hyclone, is placed in 37 DEG C, 5%CO2In incubator.
2、QPCR
2.1 cell total rnas extract: utilizing the RNA of QINGEN company to extract test kit and carry out the extraction of cell total rna, instruction carries out to specifications.
2.2 reverse transcriptions
The Reverse Transcriptase kit utilizing TAKARA company carries out the reverse transcription of RNA.
2.3QPCR
(1) design of primers
According to the coded sequence design QPCR amplimer of TEX13B gene and GAPDH gene in Genbank, Shanghai Sangon Biological Engineering Technology And Service Co., Ltd synthesize.Concrete primer sequence is as follows:
TEX13B gene:
Forward primer is 5 '-CCATGTCTGTCTTCTCTG-3 ' (SEQIDNO.3);
Reverse primer is 5 '-TATGAGTATGTGAATGAGGTAG-3 ' (SEQIDNO.4),
GAPDH gene:
Forward primer is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQIDNO.5);
Reverse primer is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQIDNO.6).
(2) PCR reaction system is prepared according to table 1:
Wherein, SYBRGreen polymerase chain reaction system is purchased from Invitrogen company.
Table 1PCR reaction system
Reagent | Volume |
Forward primer | 1μl |
Reverse primer | 1μl |
SYBR Green polymerase chain reaction system | 12.5μl |
Template | 2μl |
Deionized water | Supply 25 μ l |
(3) PCR reaction condition: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 40s) * 42 circulations.Using SYBRGreen as fluorescent marker, in the enterprising performing PCR reaction of LightCycler quantitative real time PCR Instrument, determining purpose band by melt curve analysis analysis and electrophoresis, Δ Δ CT method carries out relative quantification.
2.4 statistical methods
Experiment all completes for 3 times according to repetition, result data is all represent in the way of mean+SD, SPSS13.0 statistical software is adopted to carry out statistical analysis, difference between interference TEX13B gene expression group and matched group adopts t inspection, it is believed that when P < has statistical significance when 0.05.
2.5 results
As it is shown on figure 3, compared with human squamous lung cancer strain BEAS-2B, TEX13B gene expression significantly raised (P < 0.05) in Lung Squamous Carcinoma Cells strain NCI-H520, NCI-H596, NCI-H2170, NCI-H226.
Embodiment 4 suppresses TEX13B gene expression
1, siRNA design synthesis
SiRNA sequence for TEX13B:
SiRNA1-TEX13B:
Positive-sense strand is 5 '-AUUUUCUCGAUGAUGAAGGCC-3 ' (SEQIDNO.7);
Antisense strand is 5 '-CCUUCAUCAUCGAGAAAAUGG-3 ' (SEQIDNO.8),
SiRNA2-TEX13B:
Positive-sense strand is 5 '-UAUAUUCUCGAAGUAGAACUC-3 ' (SEQIDNO.9);
Antisense strand is 5 '-GUUCUACUUCGAGAAUAUAUC-3 ' (SEQIDNO.10),
SiRNA3-TEX13B:
Positive-sense strand is 5 '-UAUGAGUAUGUGAAUGAGGUA-3 ' (SEQIDNO.11);
Antisense strand is 5 '-CCUCAUUCACAUACUCAUACC-3 ' (SEQIDNO.12)
Above siRNA and negative control siRNA-NC provides by Shanghai JiMa pharmacy Technology Co., Ltd.
2, lung squamous cancer shifts cultivation and the transfection of cell
Cultivating NCI-H520 cell, step is with embodiment 3.
By NCI-H520 cell by 1 × 104/ hole is inoculated in 24 porocyte culture plates, at 37 DEG C, 5%CO2In incubator, cell cultivates 24h, transfection transfects according to the description of lipofectamine 2000 (purchased from Invitrogen company), experiment is divided into negative control group and experimental group (20nM), wherein, the sequence of negative control group siRNA and TEX13B gene is without homology, concentration is 20nM/ hole, transfects respectively simultaneously.
3, the jamming effectiveness of QPCR experiment detection siRNA is utilized.
Step is with embodiment 3.
Result is as shown in Figure 4, compared with siRNA1-TEX13B, siRNA3-TEX13B, siRNA2-TEX13B can the more effective expression suppressing TEX13B gene, difference has a statistical significance (P < 0.05), uses siRNA2-TEX13B to carry out follow-up experiment.
4, the jamming effectiveness of Westernblot experiment detection siRNA2-TEX13B
Step is with embodiment 2.
Result is as it is shown in figure 5, compared with transfection siRNA-NC group, in the cell of transfection siRNA2-TEX13B, the content of TEX13B albumen substantially reduces, and difference has statistical significance (P < 0.05).
Embodiment 5 studies the TEX13B gene expression impact on Lung Squamous Carcinoma Cells adhesive capacity
1, cell is cultivated with transfection with embodiment 4.
2, cell adhesion experiments
0.25% trypsinization is used to become cell suspension in the NCI-H520 cell of transfection 48h, with 5 × 104Individual/ml is inoculated in 96 porocyte culture plates, and after every hole 0.1ml, 60min, 37 DEG C of PBS wash away the cell not adhered to, and mtt assay surveys each hole 490nm wavelength light absorption value.The relative populations adhering to living cells is represented by absorbance value size.
3, result
As shown in Figure 6, compared with siRNA-NC group, siRNA2-TEX13B group absorbance value is remarkably decreased (P < 0.05) to result.Above-mentioned test result indicate that suppresses TEX13B expression can significantly inhibit NCI-H520 cell adhesion ability, shows that TEX13B is conducive to NCI-H520 cell adhesion simultaneously.
Embodiment 6 studies the TEX13B gene expression impact on Lung Squamous Carcinoma Cells migration, invasive ability
1, cell is cultivated with transfection with embodiment 4.
2, experiment is migrated
The NCI-H520 cell of transfection 48h uses trypsinization and counts, and takes 105Individual cell is placed in 1.5mLEP pipe, adds 200 μ L serum-free medium re-suspended cells, adds in transwell cell, and bottom chamber adds the DMEM culture medium of 10% hyclone, puts into 37 DEG C, 5%CO2Incubator cultivates 24h.Take transwell cell, wipe the cell of the inside with cotton swab, and wash the inside remaining cell with PBS gently off.Take 8 random field under microscope after fixing dyeing to count.
3, Matrigel
The NCI-H520 cell of transfection 48h uses trypsinization and counts, and takes 105Individual cell is placed in 1.5mLEP pipe, adds 200 μ L serum-free medium re-suspended cells, adds in the transwell cell of paving matrigel, and bottom chamber adds the DMEM culture medium of 10%FBS, puts into 37 DEG C, 5%CO2Incubator cultivates 24h.Take transwell cell, wipe the cell of the inside with cotton swab, and wash the inside remaining cell with PBS gently off.Take 8 random field under microscope after fixing dyeing to count.
4, result
Migrating experiment: compared with siRNA-NC group, siRNA2-TEX13B group is through the Leukopenia 54% of transwell cell basement membrane.
Matrigel: compared with siRNA-NC group, siRNA2-TEX13B group is through the Leukopenia 61% of the transwell cell basement membrane that spread matrigel.
Above-mentioned test result indicate that, it is suppressed that TEX13B expresses can significantly inhibit NCI-H520 cell migration, invasive ability, shows that TEX13B gene expression is conducive to migration and the invasion and attack of NCI-H520 cell simultaneously.
In embodiment 7 Lung Squamous Carcinoma Cells antibody and experiment
1, cell is cultivated with embodiment 4.
2, experiment is migrated
Cell is divided into two process groups,
Experimental group 1 (matched group): add unrelated monoclonal antibody (1:50) in NCI-H520 cell;
Experimental group 2:NCI-H520 cell adds anti-human TEX13B monoclonal antibody (1:50).
By NCI-H520 cell at 37 DEG C, 5%CO2After the incubator effect of hatching 24 hours, use trypsinization and count, taking 105Individual cell is placed in 1.5mLEP pipe, adds 200 μ L serum-free medium re-suspended cells, adds in transwell cell, and bottom chamber adds the DMEM culture medium of 10% hyclone, puts into 37 DEG C, 5%CO2Incubator cultivates 24h.Take transwell cell, wipe the cell of the inside with cotton swab, and wash the inside remaining cell with PBS gently off.Take 8 random field under microscope after fixing dyeing to count.
3, Matrigel
Cell is divided into two process groups:
Experimental group 1 (matched group): add unrelated monoclonal antibody (1:50) in NCI-H520 cell;
Experimental group 2:NCI-H520 cell adds anti-human TEX13B monoclonal antibody (1:50).
By NCI-H520 cell at 37 DEG C, 5%CO2After the incubator effect of hatching 24 hours, use trypsinization and count, taking 105Individual cell is placed in 1.5mLEP pipe, adds 200 μ L serum-free medium re-suspended cells, adds in the transwell cell of paving matrigel, and bottom chamber adds the DMEM culture medium of 10%FBS, puts into 37 DEG C, 5%CO2Incubator cultivates 24h.Take transwell cell, wipe the cell of the inside with cotton swab, and wash the inside remaining cell with PBS gently off.Take 8 random field under microscope after fixing dyeing to count.
4, result
Migrating experiment: compared with matched group, the groups of cells of anti-human TEX13B monoclonal antibody passes the Leukopenia of transwell cell basement membrane 68%.
Matrigel: compared with matched group, the groups of cells traverse of anti-human TEX13B monoclonal antibody had spread the Leukopenia of the transwell cell basement membrane of matrigel 72%.
Above-mentioned test result indicate that, it is suppressed that TEX13B protein function can significantly inhibit NCI-H520 cell migration, invasive ability, shows that TEX13B albumen is conducive to migration and the invasion and attack of NCI-H520 cell simultaneously.
The explanation of above-described embodiment is only intended to understand the method for the present invention and core concept thereof.It should be pointed out that, for the person of ordinary skill of the art, under the premise without departing from the principles of the invention, it is also possible to the present invention carries out some improvement and modification, these improve and modify also by the protection domain falling into the claims in the present invention.
Claims (10)
1. the product of detection TEX13B gene expression application in the instrument preparing the transfer of Diagnosis of pulmonary scale cancer.
2. application according to claim 1, it is characterised in that described product includes: the product shifted with Diagnosis of pulmonary scale cancer by RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization, chip or high-flux sequence detection of platform TEX13B gene expression;The product of described RT-PCR Diagnosis of pulmonary scale cancer transfer at least includes the primer of a pair specific amplified TEX13B gene;The product of described real-time quantitative PCR Diagnosis of pulmonary scale cancer transfer at least includes the primer of a pair specific amplified TEX13B gene;The product of described immune detection Diagnosis of pulmonary scale cancer transfer includes: the antibody being combined with TEX13B protein-specific;The product of described in situ hybridization Diagnosis of pulmonary scale cancer transfer includes: with the probe of the nucleic acid array hybridizing of TEX13B gene;The product of described chip Diagnosis of pulmonary scale cancer transfer includes: protein chip and gene chip;Wherein, protein chip includes the antibody being combined with TEX13B protein-specific, and gene chip includes the probe of the nucleic acid array hybridizing with TEX13B gene.
3. application according to claim 2, it is characterised in that the primer of a pair specific amplified TEX13B gene that the product of described real-time quantitative PCR Diagnosis of pulmonary scale cancer transfer at least includes is such as shown in SEQIDNO.3 and SEQIDNO.4.
4. the instrument of a Diagnosis of pulmonary scale cancer transfer, it is characterised in that described instrument includes the reagent of detection TEX13B gene expression;Described reagent includes the primer of detection TEX13B gene mRNA and/or the antibody of probe, detection TEX13B albumen.
5. instrument according to claim 4, it is characterised in that the primer of described detection TEX13B gene mRNA includes the primer pair shown in SEQIDNO.3 and SEQIDNO.4.
The application in the medicine of preparation treatment lung squamous cancer transfer of the inhibitor of 6.TEX13B gene and/or its expression product.
7. application according to claim 6, it is characterised in that described inhibitor includes the reagent suppressing TEX13B gene expression and/or the reagent suppressing TEX13B gene expression product.
8. application according to claim 7, it is characterised in that the reagent of described suppression TEX13B gene expression product includes the antibody suppressing siRNA and/or the TEX13B albumen for TEX13B gene.
9. application according to claim 8, it is characterised in that the described siRNA sequence for TEX13B gene is such as shown in SEQIDNO.9 and SEQIDNO.10.
10. the pharmaceutical composition being used for treating lung squamous cancer transfer, it is characterised in that described pharmaceutical composition includes the inhibitor according to any one of claim 6-9.
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