CN105296622B - The diagnosis and treatment target of LMNB2 gene and its expression product as nasopharyngeal carcinoma - Google Patents
The diagnosis and treatment target of LMNB2 gene and its expression product as nasopharyngeal carcinoma Download PDFInfo
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Abstract
The invention discloses the molecular markers that LMNB2 gene and its expression product can be used as nasopharyngeal carcinoma diagnosis and treatment.May determine that whether subject suffers from nasopharyngeal carcinoma or diagnose subject by the content of LMNB2 gene and its expression product in detection bone tissue whether there is the risk with nasopharyngeal carcinoma.The present invention has found that the expression inhibiting of LMNB2 gene can inhibit nasopharyngeal carcinoma cell to be proliferated by the proliferative conditions of the nasopharyngeal carcinoma cell of research in vitro culture, and by inhibiting the function of LMNB2 albumen that can equally inhibit the proliferation of nasopharyngeal carcinoma cell, the studies above is the result shows that LMNB2 gene and its expression product are a potential drug targets for treating nasopharyngeal carcinoma.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to use of the LMNB2 gene in the diagnosis, treatment of nasopharyngeal carcinoma
On the way.
Background technique
Nasopharyngeal carcinoma is primary in the malignant tumour of mucous membrane of nasopharynx covering epithelium.It is common one of the malignant tumour in China, it is pernicious
Frequency conversion is high, and the Natural Survival time average out to 18.7 months.The ground such as Guangdong, Guangxi, Fujian, the Hunan of China are multiple area, and male is more
Yu Nv.Age of onset is mostly a middle-aged person, also there is teenager patient.(yellow suffers from compared with white people for the cause of disease and ethnic neurological susceptibility
Disease is more), inherent cause and ebv infection etc. it is related, nasopharyngeal carcinoma grade malignancy is higher, and early stage may occur in which cervical lymph node turn
It moves.
The site of pathological change of nasopharyngeal carcinoma is hidden, and the person especially at the top of pharyngeal recess and nasopharynx, early symptom is unobvious, therefore difficult
With early detection, sing misdiagnosis and mistreatment rate is higher.5 years survival rates of nasopharyngeal carcinoma are hovered always 50% or so in recent years, and nasopharyngeal carcinoma is early
Phase makes a definite diagnosis patient's five year survival rate up to 90%, therefore carries out people at highest risk's screening and early diagnosis to nasopharyngeal carcinoma, to carry out
Early treatment is remarkably improved survival.
At present there are many early detection methods of nasopharyngeal carcinoma, including Epstein-Barr virus serologic marker object, nasopharyngeal carcinoma related neoplasms
The detection of marker such as interleukins, tumor necrosis factor, intercellular adhesionmolecule1 and Telomerase etc..Although these indexs
Application alone or in combination be that the diagnosis of nasopharyngeal carcinoma provides useful information, but they lack enough sensitivity with specifically
Property.Therefore a kind of high method for nasopharyngeal carcinoma early diagnosis of sensitivity and specificity is found to be a problem to be solved.
Summary of the invention
In order to make up for the deficiencies of the prior art, it can be used for what nasopharyngeal carcinoma early diagnosed the purpose of the present invention is to provide a kind of
Molecular marker.Compared to the diagnostic method of existing nasopharyngeal carcinoma, using gene marker come diagnosis of nasopharyngeal carcinoma have timeliness,
Specificity and sensitivity, for risk height, are taken corresponding pre- to make patient that can know disease risks in disease early stage
Anti- and remedy measures.
To achieve the goals above, the present invention adopts the following technical scheme:
The present invention provides application of the product of detection LMNB2 gene expression in the tool for preparing diagnosis of nasopharyngeal carcinoma.
Further, it is described detection LMNB2 gene expression product include detect LMNB2 gene mRNA levels product and/
Or the product of detection LMNB2 protein level.
Further, the product of the detection LMNB2 gene expression includes: by RT-PCR, real-time quantitative PCR, immune inspection
It surveys, in situ hybridization or chip detection LMNB2 gene expression are with the product of diagnosis of nasopharyngeal carcinoma.
Further, the product with RT-PCR diagnosis of nasopharyngeal carcinoma includes at least drawing for a pair of of specific amplified LMNB2 gene
Object；The product with real-time quantitative PCR diagnosis of nasopharyngeal carcinoma includes at least the primer of a pair of of specific amplified LMNB2 gene；It is described
Product with immune detection diagnosis of nasopharyngeal carcinoma includes: the antibody in conjunction with LMNB2 protein-specific；It is described to be diagnosed in situ hybridization
The product of nasopharyngeal carcinoma includes: the probe with the nucleic acid array hybridizing of LMNB2 gene；The product packet with chip diagnosis of nasopharyngeal carcinoma
It includes: protein chip and genetic chip；Wherein, protein chip includes the antibody in conjunction with LMNB2 protein-specific, genetic chip packet
Include the probe with the nucleic acid array hybridizing of LMNB2 gene.
A pair of of specific amplified LMNB2 gene that the product with real-time quantitative PCR diagnosis of nasopharyngeal carcinoma includes at least draws
Object is as shown in SEQ ID NO.3 and SEQ ID NO.4.
The product of the detection LMNB2 gene expression, which can be the reagent of detection LMNB2 gene expression, be also possible to, includes
Kit, chip, test paper of the reagent etc. are also possible to the high-flux sequence platform using the reagent.
The tool of the diagnosis of nasopharyngeal carcinoma includes but is not limited to chip, kit, test paper or high-flux sequence platform；It is high
Flux microarray dataset is a kind of tool of special diagnosis of nasopharyngeal carcinoma, with the development of high throughput sequencing technologies, to people's
The building of gene expression profile will become very easily work.By comparing the gene expression profile of Disease and normal population,
The exception for being easy to analyze which gene is related to disease.Therefore, the exception and nose of LMNB2 gene are known in high-flux sequence
Pharynx cancer correlation also belongs to the purposes of LMNB2 gene, equally within protection scope of the present invention.
The present invention also provides a kind of tool of diagnosis of nasopharyngeal carcinoma, the tool includes the examination for detecting LMNB2 gene expression
Agent；The reagent includes the primer and/or probe, the antibody for detecting LMNB2 albumen for detecting LMNB2 gene mRNA.
The tool includes but is not limited to chip, kit, test paper or high-flux sequence platform.
Wherein, the chip includes genetic chip, protein-chip；The genetic chip includes solid phase carrier and fixation
In the oligonucleotide probe of solid phase carrier, the oligonucleotide probe includes for detecting being directed to for LMNB2 gene transcription level
The oligonucleotide probe of LMNB2 gene；The protein-chip includes solid phase carrier and the LMNB2 egg for being fixed on solid phase carrier
White specific antibody；The genetic chip can be used for detecting multiple genes including LMNB2 gene (for example, and nasopharynx
The relevant multiple genes of cancer) expression.The protein-chip can be used for detecting multiple eggs including LMNB2 albumen
The expression of white matter (such as multiple protein relevant to nasopharyngeal carcinoma).By the way that multiple markers with nasopharyngeal carcinoma are examined simultaneously
It surveys, is greatly improved the accuracy rate of nasopharyngeal carcinoma diagnosis.
Wherein, the kit includes gene detecting kit and protein immunization detection kit；The genetic test examination
Agent box includes the reagent for detecting LMNB2 gene transcription level；The protein immunization detection kit includes LMNB2 albumen
Specific antibody.Further, the reagent includes using RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or chip
Method detects reagent needed for LMNB2 gene expression dose process.Preference, the reagent include for LMNB2 gene
Primer and/or probe.It is easy to design according to the nucleotide sequence information of LMNB2 gene and can be used for detecting LMNB2 gene table
Up to horizontal primer and probe.
The test paper includes the reagent for detecting LMNB2 gene expression.
The high-flux sequence platform includes the reagent for detecting LMNB2 gene expression.
It can be DNA, RNA, DNA-RNA chimera, PNA or other with the probe of the nucleic acid array hybridizing of LMNB2 gene
Derivative.There is no limit for the length of the probe, as long as completing specific hybrid, being specifically bound with purpose nucleotide sequence,
Any length is ok.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the probe
Length can be grown to 60,80,100,150,300 base-pairs or longer or even whole genes.Since different probe lengths is to miscellaneous
Efficiency, signal specificity is handed over to have different influences, the length of the probe is typically at least 14 base-pairs, and longest does not surpass generally
30 base-pairs are crossed, the length complementary with purpose nucleotide sequence is best with 15-25 base-pair.The probe self-complementary sequence
Column are most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.
Further, the specific antibody of the LMNB2 albumen includes monoclonal antibody, polyclonal antibody.The LMNB2 egg
White specific antibody include complete antibody molecule, antibody any segment or modification (for example, chimeric antibody, scFv,
Fab, F (ab ') 2, Fv etc..As long as the segment can retain the binding ability with LMNB2 albumen.For protein level
Antibody preparation when well known to a person skilled in the art and the present invention may use any method to prepare the antibody.
In specific embodiments of the present invention, it is described detection LMNB2 gene mRNA primer include SEQ ID NO.3 and
Primer pair shown in SEQ ID NO.4.
The present invention also provides the inhibitor of LMNB2 gene and/or its expression product in the drug for preparing treatment nasopharyngeal carcinoma
In application.The inhibitor includes the reagent for inhibiting LMNB2 gene expression, and/or the examination for inhibiting LMNB2 gene expression product
Agent.
Further, the reagent for inhibiting LMNB2 gene expression includes the reagent of suppressor transcription, suppressor translation
Reagent；The reagent for inhibiting LMNB2 gene expression product includes the reagent for inhibiting LMNB2 gene mRNA, inhibits LMNB2 egg
White reagent.The reagent for inhibiting LMNB2 gene mRNA includes the reagent for inhibiting mRNA stability, inhibits mRNA translation activity
Reagent.The reagent for inhibiting LMNB2 albumen includes the reagent for inhibiting LMNB2 protein stability, inhibits LMNB2 protein active
Reagent, inhibit LMNB2 protein function reagent.
Further, the reagent for inhibiting LMNB2 gene mRNA includes the double stranded RNA for being directed to LMNB2 gene mRNA；Suppression
The reagent of LMNB2 protein function processed includes the tumor vaccine of LMNB2 antigen protein, the antibody for inhibiting LMNB2 protein function.It is described
Antibody can be polyclonal antibody or monoclonal antibody.
In specific embodiments of the present invention, the double stranded RNA for LMNB2 gene mRNA is siRNA.
In order to ensure LMNB2 gene can be rejected efficiently or silencing, it is special that siRNA is devised according to the mRNA sequence of LMNB2 gene
Property segment.The design of siRNA is according to general design principle (Elbashir et.al 2001, the Schwarz et.al delivered
2003, Khvorova et.al 2003, Reynolds et.al 2004, Hsieh et.al 2004, Ui-Tei et.al
2004), pass through online tool complete design, the online tool are as follows: siRNASelectionProgram of Whitehead
Institute (BingbingYuan et.al 2004, http://jura.wi.mit.edu/bioc/siRNAext/) and
BLOCK-iTTM RNAi Designer ofINVITROGEN(winner of the 2004 Frost& Sullivan
Excellence in Research Award, https: //rnaidesigner.invitrogen.com/sirna/).In order to
It further increases the validity of siRNA segment, the siRNA piece of screening is designed for the advantages of comprehensive two Photographing On-line tools
It is disconnected.Finally, siRNA sequence is filtered by sequence analysis (NCBI BLAST), to improve the specificity of siRNA segment and subtract
The undershooting-effect of few RNAi interference.
Preferably, the sequence of the siRNA is as shown in SEQ ID NO.7 and SEQ ID NO.8.
The present invention also provides a kind of for treating the pharmaceutical composition of nasopharyngeal carcinoma, and described pharmaceutical composition includes institute above
The inhibitor of the LMNB2 gene and/or its expression product stated.
Pharmaceutical composition of the invention further includes pharmaceutically acceptable carrier, and wherein the carrier can be excipient, dilution
Agent, thickener, filler, bonding agent, disintegrating agent, lubricant, grease or non-grease base, surfactant, suspending agent, glue
Mixing more than solidifying agent, adjuvant, preservative, antioxidant, stabilizer, colorant or fragrance either or both of them.
Pharmaceutical composition of the invention can be used for manufacturing the medicament for the treatment of nasopharyngeal carcinoma.
Pharmaceutical composition of the invention, wherein the mammal can be human patients.
Pharmaceutical composition of the invention can for example be given in a manner of one of oral, injection, smearing or patch to the people
In class patient.
Pharmaceutical composition of the invention can also can be with the drug combination of other treatment nasopharyngeal carcinoma, a variety of Drug combinations
The success rate for the treatment of is mentioned significantly.
In the context of the present invention, " LMNB2 gene " includes any function etc. of LMNB2 gene and LMNB2 gene
The polynucleotides of jljl.LMNB2 gene includes and LMNB2 gene in the public GenBank GeneBank in the current world
(NC_000019.10) DNA sequence dna has 70% or more homology, and encodes the DNA sequence dna of identical function protein；
Preferably, the coded sequence of LMNB2 gene includes following any DNA molecular:
(1) DNA sequence dna shown in SEQ ID NO.1 in sequence table；
(2) under strict conditions with 1) DNA sequence dna that limits hybridizes and encodes the DNA sequence dna of identical function protein；
(3) DNA sequence dna limited with (1) or (2) has 70%, preferably, 90% or more homology, and encodes identical function
The DNA molecular of energy protein.
In specific embodiments of the present invention, the coded sequence of the LMNB2 gene is shown in SEQ ID NO.1
DNA sequence dna.
In the context of the present invention, LMNB2 gene expression product includes the part of LMNB2 albumen and LMNB2 albumen
Peptide.The partial peptide of the LMNB2 albumen contains functional domain relevant to nasopharyngeal carcinoma.
" LMNB2 albumen " includes any functional equivalent of LMNB2 albumen and LMNB2 albumen.The functional equivalent
Including LMNB2 albumen conservative variation protein or its active fragment or its reactive derivative, allelic variant, natural mutation
Body, induced mutants, can be with the encoded protein of DNA of the DNA hybridization of LMNB2 under high or low stringent condition.
Preferably, LMNB2 albumen is the protein with following amino acid sequences:
(1) protein that the amino acid sequence shown in SEQ ID NO.2 in sequence table forms；
(2) amino acid sequence shown in SEQ ID NO.2 by the substitution of one or several amino acid residues and/or is lacked
Lose and/or addition and with the ammonia with the same function as shown in SEQ ID NO.2 of amino acid sequence shown in SEQ ID NO.2
Protein derived from base acid sequence.The number for the amino acid for replacing, lacking or adding is usually 1-50, preferably 1-30
It is a, more preferably 1-20, most preferably 1-10.
(3) there is at least 80% homology (also known as sequence identity) with amino acid sequence shown in SEQ ID NO.2,
It is highly preferred that the homology with amino acid sequence at least about 90% to 95% shown in SEQ ID NO.2, be often 96%, 97%,
98%, the polypeptide that the amino acid sequence of 99% homology is constituted.
In specific embodiments of the present invention, the LMNB2 albumen is with amino acid sequence shown in SEQ ID NO.2
The protein of column.
It is known that, conventionally, the modification of one or more amino acid will not influence the function of protein in a protein.
Those skilled in the art can approve the amino acid for changing single amino acids or small percentage or to amino acid sequence it is individual add,
Missing, insertion, replacement are conservative modifications, and wherein the change of protein generates the protein with identity function.Function phase is provided
As the Conservative substitution tables of amino acid be well known in the art.
Example by one amino acid of addition or the protein of more amino acid modification is the fusion of LMNB2 albumen
Albumen.For the peptide or protein with LMNB2 protein fusion, there is no limit as long as resulting fusion protein retains LMNB2 egg
White biological activity.
LMNB2 albumen of the invention also includes the non-conservative modification to amino acid sequence shown in SEQ ID NO.2, as long as
Protein by modification still is able to retain the biological activity of LMNB2 albumen.It is mutated in such modification protein
Amino acid number be usually 10 perhaps less such as 6 perhaps less such as 3 or less.
In the context of the present invention, " diagnosis of nasopharyngeal carcinoma " both include judge subject whether suffered from nasopharyngeal carcinoma or
Including judging that subject whether there is the risk with nasopharyngeal carcinoma.
In the context of the present invention, " treatment nasopharyngeal carcinoma " divides from the state change of disease, may include the slow of disease
The complete healing of solution, disease.
The advantages of the present invention:
Present invention firstly discovers that LMNB2 gene expression is related to nasopharyngeal carcinoma, pass through detection subject's nasopharyngeal epithelium tissue
The expression of middle LMNB2, it can be determined that whether subject suffers from nasopharyngeal carcinoma or judge that subject whether there is with nasopharyngeal carcinoma
Risk, so that clinician be instructed to provide prevention scheme or therapeutic scheme to subject.
Present invention finds a kind of new molecular marked compound-LMNB2 genes, compared to traditional detection means, gene diagnosis
More in time, more special, sensitiveer, it can be realized the early diagnosis of nasopharyngeal carcinoma, to reduce the death rate of nasopharyngeal carcinoma.
Detailed description of the invention
Fig. 1, which is shown, utilizes expression of the genechip detection LMNB2 gene in nasopharyngeal epithelium tissue；
Fig. 2 shows the expression using Western blot detection LMNB2 albumen in nasopharyngeal epithelium tissue；
Fig. 3 shows the jamming effectiveness using QPCR detection siRNA to LMNB2 gene；
Fig. 4 shows the influence using Western blot detection siRNA to LMNB2 protein expression；
The influence that Fig. 5 display inhibits LMNB2 protein function to be proliferated nasopharyngeal carcinoma cell.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this
It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip
Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.
The differential expression of 1 LMNB2 gene of embodiment
1, research object:
Patient 40 of nasopharyngeal carcinoma are diagnosed as, patient 40 for being diagnosed as nasopharyngeal carcinoma chronic inflammation (serve as normal nose
Swallow epithelial tissue control), it is utilized respectively microdissection technology and obtains tissues of nasopharyngeal carcinoma and normal nasopharyngeal epithelial tissue.
2, the acquisition of the RNA of tissues of nasopharyngeal carcinoma and normal nasopharyngeal epithelial tissue
The total serum IgE that tissues of nasopharyngeal carcinoma and normal nasopharyngeal epithelial tissue are extracted using Trizol one-step method, passes through Nanodrop
ND-1000 reads the purity of absorbance value (A) the measurement RNA solution at 260nm and 280nm.It is solidifying through 1% denaturing formaldehyde agarose
Gel electrophoresis is observed under ultraviolet transmission light, detects the integrality of RNA.
3, gene chip hybridization and scanning
After the linearized amplification of total serum IgE, cy3-UTP is marked, and the cRNAs after fluorescent marker uses RNEASY Mini Kit
Purifying, carries out fragmentation processing to the cRNAs marked with the RNA Fragmentation Reagents of Amhion.Using beauty
People's full genome chip of expression spectrum (4x44K gene) of Agilent company of state, 65 DEG C of hybridization 17h, are then washed in chip hybridization furnace
De-, dyeing, finally uses Agilent DNA MicroarrayScanner scanner scanning.
4, chip data processing and analysis
Chip after hybridization imports data to analysis software, for two groups of ratios after chip scanner reads data point
Natural logrithm absolute value be greater than 2.0 or the gene less than 0.5 as difference expression gene.
5, statistical procedures
Data analysis is carried out using 13.0 statistical software of SPSS, group difference compares using one-way analysis of variance method, P <
0.05 difference has significant.
6, result
(as shown in Figure 1) as the result is shown, compared with normal tissue, the mRNA level in-site of LMNB2 gene is aobvious in tissues of nasopharyngeal carcinoma
It writes and increases, difference has statistical significance (P < 0.05).
The differential expression of 2 LMNB2 albumen of embodiment
1, research object is the same as embodiment 1.
2, tissue total protein is extracted
The operation of protein extraction is carried out according to the specification of EpiQuik tissue/cell total protein extraction kit.
3, Western blot is detected
The protein quantification of extraction is subjected to SDS-PAGE electrophoresis, carry out later transferring film, closing, primary antibody is incubated for, secondary antibody is incubated for,
Colour developing.
4, statistical procedures
The gray value of protein band is analyzed using Image J software, using β-actin as internal reference, by LMNB2 albumen
The gray value of band is normalized.Result data is indicated in a manner of mean+SD, is used
SPSS13.0 statistical software is next for statistical analysis, and difference between the two is examined using t, it is believed that has system as P < 0.05
Meter learns meaning.
5, result
As a result as shown in Fig. 2, compared with normal tissue, the expression of LMNB2 albumen is dramatically increased in tissues of nasopharyngeal carcinoma,
Difference has statistical significance (P < 0.05).
Embodiment 3 inhibits LMNB2 gene expression
1, siRNA design synthesis
For the siRNA sequence of LMNB2:
SiRNA1-LMNB2:
Positive-sense strand is 5 '-UGUAAACGGUGAGCUAUUGCC-3 ' (SEQ ID NO.7)；
Antisense strand is 5 '-CAAUAGCUCACCGUUUACACC-3 ' (SEQ ID NO.8),
SiRNA2-LMNB2:
Positive-sense strand is 5 '-AAUUCUCUAGAAAUGUAUCAA-3 ' (SEQ ID NO.9)；
Antisense strand is 5 '-GAUACAUUUCUAGAGAAUUUC-3 ' (SEQ ID NO.10),
SiRNA3-LMNB2:
Positive-sense strand is 5 '-UUAGUGUUUCCAAAGACUCAA-3 ' (SEQ ID NO.11)；
Antisense strand is 5 '-GAGUCUUUGGAAACACUAAGA-3 ' (SEQ ID NO.12)
Negative control siRNA sequence (siRNA-NC):
Positive-sense strand is 5 '-CGUACGCGGAAUACUUCGA-3 ' (SEQ ID NO.13)；
Antisense strand is 5 '-UCGAAGUAUUCCGCGUACG-3 ' (SEQ ID NO.14).
2, the culture and transfection of nasopharyngeal carcinoma cell
It is single that human nasopharynx's cancer cell line HONE-1 is incubated at 10% fetal calf serum of addition, 100 μ g/ml streptomysins and 100
(Hyclone is purchased from) in the RPMI-1640 culture medium of position/ml (units/ml) penicillin, and the nasopharyngeal carcinoma of logarithmic growth phase presses 1
×104/ hole is inoculated into 24 porocyte culture plates, in 37 DEG C, 5%CO2Cell culture for 24 hours, is transfected according to liposome in incubator
The specification transfection of transfection reagent 2000 (be purchased from Invitrogen company), experiment is divided into, negative control group and experimental group
(20nM), wherein for the sequence of negative control group siRNA and LMNB2 gene without homology, concentration is the hole 20nM/, while being turned respectively
Dye.
2, the jamming effectiveness of detection siRNA is tested using QPCR.
2.1 extraction cell total rnas are operated using conventional method.
2.2 reverse transcription
The reverse transcription of RNA is carried out using the Reverse Transcriptase kit of TAKARA company.
2.3QPCR
(1) design of primers
QPCR amplimer is designed according to the coded sequence of LMNB2 gene and GAPDH gene in Genbank, is given birth to by Shanghai
The synthesis of work biotechnology Services Co., Ltd.Specific primer sequence is as follows:
LMNB2 gene:
Forward primer is 5 '-ATGCGTGAGAATGAGAAT-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-CCTGTTGGTGGAAAAGAT-3 ' (SEQ ID NO.4),
GAPDH gene:
Forward primer is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQ ID NO.5)；
Reverse primer is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.6).
(2) PCR reaction system is prepared according to table 1:
Wherein, SYBR Green polymerase chain reaction system is purchased from Invitrogen company.
1 PCR reaction system of table
| 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.With SYBR Green work
For fluorescent marker, PCR reaction is carried out on Light Cycler fluorescence quantitative PCR instrument, passes through melt curve analysis analysis and electrophoresis
Determine that purpose band, Δ Δ CT method carry out relative quantification.
2.4 statistical method
Experiment is completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD,
Using SPSS13.0 statistical software come for statistical analysis, the difference between LMNB2 gene expression panel and control group is interfered to adopt
It is examined with t, it is believed that there is statistical significance as P < 0.05.
2.5 result
As a result as shown in figure 3, compared with siRNA2-LMNB2, siRNA3-LMNB2, siRNA1-LMNB2 can be more effective
Inhibition LMNB2 gene expression, difference have statistical significance (P < 0.05), carry out subsequent reality using siRNA1-LMNB2
It tests.
3, the jamming effectiveness of Western blot experiment detection siRNA1-LMNB2
Step is the same as embodiment 2.
As a result as shown in figure 4, transfecting LMNB2 albumen in the cell of siRNA1-LMNB2 compared with transfecting siRNA-NC group
Content be substantially reduced, difference have statistical significance (P < 0.05).
Measurement of the expression of embodiment 4LMNB2 gene to nasopharyngeal carcinoma cell proliferative capacity
It is proliferated using Cell Counting kit-8 (cck-8) kit for detecting nasopharyngeal carcinoma cell
1, step
The culture and transfection of nasopharyngeal carcinoma cell are carried out according to the method for preceding embodiment, cell is divided into two experimental groups:
Group 1: transfection siRNA-NC groups of cells；
Group 2: transfection siRNA-LMNB2 groups of cells.
After transfection for 24 hours, with 2.5 × 105/ ml density is inoculated in 96 porocyte culture plates, and each experimental group design three is multiple
Hole, every 100 μ l of hole, is placed in 37 DEG C, 5%CO2It is incubated in incubator, after cell is adherent for 24 hours, respectively in the culture of required detection
10 μ l CK-8 solution are added in every hole in hole, continue to be incubated for 1h in cell incubator, measure each hole absorbance value (OD at 450nm
Value)
2, statistical method
Experiment is completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD,
Using SPSS13.0 statistical software come for statistical analysis, difference between the two is examined using t, it is believed that is had as P < 0.05
It is statistically significant.
3, result
The results are shown in Table 2, and compared with transfecting siRNA-NC group, transfection siRNA-LMNB2 groups of cells cell Proliferation is slow,
Difference has statistical significance (P < 0.05).It is above-mentioned the experimental results showed that, LMNB2 gene expression promotes the increasing of nasopharyngeal carcinoma cell
It grows.
2 nasopharyngeal carcinoma cell OD value of table
| Experimental group | OD value (optical density) |
| siRNA-NC | 0.1734±0.004 |
| siRNA-LMNB2 | 0.0982±0.005 |
In 5 nasopharyngeal carcinoma cell antibody of embodiment and test
1, step:
Nasopharyngeal carcinoma cell is inoculated in 96 porocyte culture plates, every hole 2x103A cells/well/200 μ l, cell are adherent
After be handled as follows:
Experimental group 1 (control group): unrelated monoclonal antibody (1:50) is added in nasopharyngeal carcinoma cell；
Experimental group 3: anti-human LMNB2 monoclonal antibody (1:50) is added in nasopharyngeal carcinoma cell.
By cell in 37 DEG C, 5%CO2After incubator is incubated for 24 hours, it is added3H-TdR (1 hole μ Ci/), it is small to be further cultured for 24
When, cell is collected, liquid scintillation solution is added, β calculating instrument detects cpm value.
2, statistical method
Experiment is completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD,
Using SPSS13.0 statistical software come for statistical analysis, difference between the two is examined using t, it is believed that is had as P < 0.05
It is statistically significant.
3, result
As a result as shown in figure 5, compared to control group, the groups of cells cells proliferation slowed down of anti-human LMNB2 monoclonal antibody is added.It is above-mentioned
Nasopharyngeal carcinoma cell can be inhibited to be proliferated the experimental results showed that inhibiting the function of LMNB2 albumen.
The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this
For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention
And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.
Claims (9)
1. detecting application of the product of LMNB2 gene expression in the tool for preparing diagnosis of nasopharyngeal carcinoma.
2. application according to claim 1, which is characterized in that the product include: by RT-PCR, real-time quantitative PCR,
Immune detection, in situ hybridization, chip or high-flux sequence detection of platform LMNB2 gene expression are with the product of diagnosis of nasopharyngeal carcinoma；Institute
State the primer that a pair of of specific amplified LMNB2 gene is included at least with the product of RT-PCR diagnosis of nasopharyngeal carcinoma；It is described to use real-time quantitative
The product of PCR diagnosis of nasopharyngeal carcinoma includes at least the primer of a pair of of specific amplified LMNB2 gene；It is described to diagnose nasopharynx with immune detection
The product of cancer includes: the antibody in conjunction with LMNB2 protein-specific；The product in situ hybridization diagnosis of nasopharyngeal carcinoma includes:
With the probe of the nucleic acid array hybridizing of LMNB2 gene；The product with chip diagnosis of nasopharyngeal carcinoma includes: protein chip and gene
Chip；Wherein, protein chip includes the antibody in conjunction with LMNB2 protein-specific, and genetic chip includes the core with LMNB2 gene
The probe of acid sequence hybridization.
3. application according to claim 2, which is characterized in that the product with real-time quantitative PCR diagnosis of nasopharyngeal carcinoma is extremely
The primer for a pair of of the specific amplified LMNB2 gene for including less is as shown in SEQ ID NO.3 and SEQ ID NO.4.
4. application according to claim 1, the tool includes the reagent for detecting LMNB2 gene expression；The reagent packet
Include the primer and/or probe, the antibody for detecting LMNB2 albumen of detection LMNB2 gene mRNA.
5. application according to claim 4, which is characterized in that the primer of the detection LMNB2 gene mRNA includes SEQ
Primer pair shown in ID NO.3 and SEQ ID NO.4.
6.LMNB2 application of the inhibitor of gene and/or its expression product in the drug of preparation treatment nasopharyngeal carcinoma.
7. application according to claim 6, which is characterized in that the inhibitor includes the examination for inhibiting LMNB2 gene expression
Agent, and/or the reagent for inhibiting LMNB2 gene expression product.
8. application according to claim 7, which is characterized in that it is described inhibit LMNB2 gene expression product reagent include
Inhibit the antibody of the siRNA, and/or LMNB2 albumen for LMNB2 gene.
9. application according to claim 8, which is characterized in that such as SEQ ID of the siRNA sequence for LMNB2 gene
Shown in NO.7 and SEQ ID NO.8.
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