CN105648103B - Purposes of the VSIG10L gene as lung squamous cancer transfer diagnosis and treatment marker - Google Patents
Purposes of the VSIG10L gene as lung squamous cancer transfer diagnosis and treatment marker Download PDFInfo
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Abstract
The invention discloses a kind of gene marker, which is VSIG10L.VSIG10L can be used for judging whether lung squamous cancer occurs to shift or anticipation lung squamous cancer shifts risk.In addition, VSIG10L can be also used for the drug that preparation inhibits lung squamous cancer transfer or prevention lung squamous cancer transfer.The present invention provides new diagnostic method for the clinical lung squamous cancer transfer of diagnosis on a molecular scale, while the gene therapy for lung squamous cancer transfer provides new drug target.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to VSIG10L gene in diagnosis that lung squamous cancer shifts, treatment
Purposes.
Background technique
Lung cancer is to seriously threaten the major disease of human life and health.The World Health Organization (WHO) international cancer in 2011
The 2008 world statistics data results that disease research institution IARC is announced are shown: lung cancer is that morbidity and mortality all rank first
Malignant tumour.Annual whole world neopathy number of cases 160.8 ten thousand, death number 137.7 ten thousand accounts for 12.7% He of whole malignant tumours
18.2%.The statistical result showed that American Cancer Society announces: it is expected that in 2012, the U.S. newly sends out pulmonary carcinosis number of cases and is up to
22.6 ten thousand, because lung cancer death case load is up to 160,000.In China, sampling is looked back by the national third time cause of the death that the Ministry of Public Health is presided over
Investigation result is shown: lung cancer is first of the Death Cause for Malignant Tumors of sample regions, and crude death rate is 30.83/10 ten thousand, wherein male
41.34/10 ten thousand, women 19.84/10 ten thousand；It is the first cancer mortality reason in male, women.With global aging
Accelerate and the factors such as increasing environmental pollution, lung cancer morbidity will continue in rising trend, therefore, study the diagnoses and treatment of lung cancer
Progress demand, must be more more and more intense.
From pathologically, lung cancer can be divided into non-small cell lung cancer, and (85%) NSCLC accounts for about that (SCLC accounts for about with Small Cell Lung Cancer
15%).Wherein, NSCLC is mainly by squamous carcinoma (also known as squamous cell carcinoma or lung squamous cancer), gland cancer (also known as adenocarcinoma of lung) and large cell carcinoma
Composition.Squamous carcinoma and gland cancer respectively account for the 40% of NSCLC.
The morbidity of lung cancer is more hidden, and the patient more than 70% has been developed to middle and advanced stage when medical, loses surgical engine
Meeting.One of the main reason for this is also lung cancer weak curative effect, and the death rate is high.Therefore, early screening, diagnosis become the weight for preventing and treating lung cancer
In it is weight.Currently, clinical used screening lung cancer diagnostic method is mainly the Imaging Technologies such as chest X-ray inspection, CT scan.
Although these image datas play an important role to diagnosis, there is also many limitations, such as false positive rate height, can not detect
Recessive lesion, subclinical lesion and small metastatic lesion.It in addition to this, further include bronchoscopy to the diagnostic method of lung cancer
It looks into, the invasive operations such as aspiration biopsy, time-consuming for such methods, increases patient suffering.Therefore, searching is noninvasive, radiationless, expense is low
Honest and clean, diagnosis is the urgent need of current early diagnosis of cancer quickly and sensibility, the high screening of specificity, diagnostic method.
Summary of the invention
The purpose of the present invention is to provide a kind of molecular markers that can be used for lung squamous cancer transfer diagnosis.Compared to existing lung
The diagnostic method of squamous carcinoma transfer, lung squamous cancer transfer is diagnosed using gene marker has timeliness, specificity and sensitivity,
To make patient that can know disease risks in transfer early stage, for risk height, corresponding prevention and treatment measure is taken.
To achieve the goals above, the present invention adopts the following technical scheme:
The present invention provides the product of detection VSIG10L gene expression answering in the tool of preparation diagnosis lung squamous cancer transfer
With.
Further, the product of the detection VSIG10L gene expression includes the production for detecting VSIG10L gene mRNA levels
Product, and/or the product for detecting VSIG10L protein level.
Further, the product of the detection VSIG10L gene expression includes: by RT-PCR, real-time quantitative PCR, is immunized
Detection, in situ hybridization or chip detection VSIG10L gene expression are to diagnose the product that lung squamous cancer shifts.
Further, the product with RT-PCR diagnosis lung squamous cancer transfer includes at least a pair of of specific amplified VSIG10L base
The primer of cause；The product with real-time quantitative PCR diagnosis lung squamous cancer transfer includes at least a pair of of specific amplified VSIG10L gene
Primer；The product with immune detection diagnosis lung squamous cancer transfer includes: the antibody in conjunction with VSIG10L protein-specific；
The product in situ hybridization diagnosis lung squamous cancer transfer includes: the probe with the nucleic acid array hybridizing of VSIG10L gene；It is described
Product with chip diagnosis lung squamous cancer transfer includes: protein chip and genetic chip；Wherein, protein chip includes and VSIG10L
The antibody that protein-specific combines, genetic chip includes the probe with the nucleic acid array hybridizing of VSIG10L gene.
A pair of of specific amplified VSIG10L base that the product with real-time quantitative PCR diagnosis lung squamous cancer transfer includes at least
The primer of cause is as shown in SEQ ID NO.3 and SEQ ID NO.4.
The product of the detection VSIG10L gene expression can be the reagent of detection VSIG10L gene expression, be also possible to
Kit, chip, test paper comprising the reagent etc. are also possible to the high-flux sequence platform using the reagent.
The tool of the diagnosis lung squamous cancer transfer includes but is not limited to that chip, kit, test paper or high-flux sequence are flat
Platform；High-flux sequence platform is a kind of tool of special diagnosis lung squamous cancer transfer, right with the development of high throughput sequencing technologies
The building of the gene expression profile of one people will become very easily work.By the gene for comparing Disease and normal population
Express spectra, the exception for being easy to analyze which gene are related to disease.Therefore, VSIG10L gene is known in high-flux sequence
The exception purposes for also belonging to VSIG10L gene related to lung squamous cancer transfer, equally within protection scope of the present invention.
The present invention also provides a kind of tool of diagnosis lung squamous cancer transfer, the tool includes detection VSIG10L gene table
The reagent reached；The reagent include detect VSIG10L gene mRNA primer and/or probe, detection VSIG10L albumen it is anti-
Body.
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 the needle for detecting VSIG10L gene transcription level
To the oligonucleotide probe of VSIG10L gene；The protein-chip includes solid phase carrier and is fixed on solid phase carrier
The specific antibody of VSIG10L albumen；The genetic chip can be used for detecting multiple genes including VSIG10L gene
The expression of (for example, multiple genes relevant to lung squamous cancer transfer).The protein-chip can be used for detecting
The expression of multiple protein (such as multiple protein relevant to lung squamous cancer transfer) including VSIG10L albumen.Pass through
Multiple markers with lung squamous cancer transfer are detected simultaneously, are greatly improved the accuracy rate of lung squamous cancer transfer diagnosis.
Wherein, the kit includes gene detecting kit and protein immunization detection kit；The genetic test examination
Agent box includes the reagent for detecting VSIG10L gene transcription level；The protein immunization detection kit includes VSIG10L egg
White specific antibody.Further, the reagent include using RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or
Chip method detects reagent needed for VSIG10L gene expression dose process.Preference, the reagent include being directed to
The primer and/or probe of VSIG10L gene.Being easy to design according to the nucleotide sequence information of VSIG10L gene can be used for
Detect the primer and probe of VSIG10L gene expression dose.
The test paper includes the reagent for detecting VSIG10L gene expression.
The high-flux sequence platform includes the reagent for detecting VSIG10L gene expression.
With the probe of the nucleic acid array hybridizing of VSIG10L gene can be DNA, RNA, DNA-RNA chimera, PNA or its
Its derivative.There is no limit as long as complete specific hybrid and purpose nucleotide sequence specificity knot for the length of the probe
It closes, 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 grow to 60,80,100,150,300 base-pairs or longer or even whole genes.Due to different probe lengths pair
Hybridization efficiency, signal specificity have different influences, and the length of the probe is typically at least 14 base-pairs, and longest is generally not
More than 30 base-pairs, the length complementary with purpose nucleotide sequence are best with 15-25 base-pair.The probe self-complementary
Sequence is most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.
Further, the specific antibody of the VSIG10L albumen includes monoclonal antibody, polyclonal antibody.It is described
The specific antibody of VSIG10L albumen includes complete antibody molecule, any segment of antibody or modification (for example, inosculating antibody
Body, scFv, Fab, F (ab ') 2, Fv etc..As long as the segment can retain the binding ability with VSIG10L albumen.For
Well known to a person skilled in the art and the present invention may use any method to prepare when the preparation of the antibody of protein level
The antibody.
In specific embodiments of the present invention, the primer of the detection VSIG10L gene mRNA includes SEQ ID NO.3
With primer pair shown in SEQ ID NO.4.
The present invention also provides the inhibitor of VSIG10L gene and/or its expression product in preparation treatment lung squamous cancer transfer
Drug in application.The inhibitor includes the reagent, and/or inhibition VSIG10L gene table for inhibiting VSIG10L gene expression
Up to the reagent of product.
Further, the reagent for inhibiting VSIG10L gene expression includes the reagent of suppressor transcription, suppressor turns over
The reagent translated；The reagent for inhibiting VSIG10L gene expression product includes the reagent for inhibiting VSIG10L gene mRNA, inhibits
The reagent of VSIG10L albumen.The reagent for inhibiting VSIG10L gene mRNA includes the reagent for inhibiting mRNA stability, inhibits
MRNA translates active reagent.It is described inhibit VSIG10L albumen reagent include inhibit VSIG10L protein stability reagent,
Inhibit the reagent of VSIG10L protein active, inhibit the reagent of VSIG10L protein function.
Further, the reagent for inhibiting VSIG10L gene mRNA includes the double-strand ribose core for being directed to VSIG10L gene mRNA
Acid；The reagent of VSIG10L protein function is inhibited to include the tumor vaccine of VSIG10L antigen protein, inhibit VSIG10L protein function
Antibody.The antibody can be polyclonal antibody or monoclonal antibody.
In specific embodiments of the present invention, the double stranded RNA for VSIG10L gene mRNA is
siRNA.In order to ensure VSIG10L gene can be rejected efficiently or silencing, devised according to the mRNA sequence of VSIG10L gene
SiRNA specific fragment.SiRNA design according to delivered general design principle (Elbashir et.al 2001,
Schwarz et.al 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/
) and BLOCK-iTTM RNAi Designer ofINVITROGEN (winner of the 2004Frost& siRNAext/
Sullivan Excellence in Research Award, https: //rnaidesigner.invitrogen.com/
sirna/).In order to be designed for the advantages of further increasing the validity of siRNA segment, integrate two Photographing On-line tools
The siRNA segment of screening.Finally, siRNA sequence is filtered by sequence analysis (NCBI BLAST), to improve siRNA piece
Disconnected specificity simultaneously reduces the undershooting-effect that RNAi is interfered.
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 lung squamous cancer transfer, and described pharmaceutical composition includes upper
The inhibitor of VSIG10L gene and/or its expression product described in face.
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 lung squamous cancer transfer.
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.
The drug combination that pharmaceutical composition of the invention can also be shifted with other treatment lung squamous cancer, a variety of Drug combinations
The success rate for the treatment of can be mentioned significantly.
In the context of the present invention, " VSIG10L gene " includes any of VSIG10L gene and VSIG10L gene
The polynucleotides of functional equivalent.VSIG10L gene include in the public GenBank GeneBank in the current world
VSIG10L gene (NC_000019.10) DNA sequence dna has 70% or more homology, and encodes the DNA of identical function protein
Sequence；
Preferably, the coded sequence of VSIG10L 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 VSIG10L gene is shown in SEQ ID NO.1
DNA sequence dna.
In the context of the present invention, VSIG10L gene expression product includes VSIG10L albumen and VSIG10L albumen
Partial peptide.The partial peptide of the VSIG10L albumen contains functional domain relevant to lung squamous cancer transfer.
" VSIG10L albumen " includes any functional equivalent of VSIG10L albumen and VSIG10L albumen.The function
Equivalent includes VSIG10L albumen conservative variation protein or its active fragment or its reactive derivative, allelic variant,
Natural mutation, induced mutants, can be with the encoded egg of DNA of the DNA hybridization of VSIG10L under high or low stringent condition
White matter.
Preferably, VSIG10L 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, Chang Wei 96%, 97%,
98%, the polypeptide that the amino acid sequence of 99% homology is constituted.
In specific embodiments of the present invention, the VSIG10L albumen is with amino acid shown in SEQ ID NO.2
The protein of sequence.
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 melting for VSIG10L albumen
Hop protein.For the peptide or protein with VSIG10L protein fusion, there is no limit as long as resulting fusion protein retains
The biological activity of VSIG10L albumen.
VSIG10L albumen of the invention also includes the non-conservative modification to amino acid sequence shown in SEQ ID NO.2, only
It still to be able to retain the biological activity of VSIG10L albumen by the protein of modification.In such modification protein
The amino acid number of mutation is usually 10 perhaps less such as 6 perhaps less such as 3 or less.
In the context of the present invention, " transfer of diagnosis lung squamous cancer " both includes judging whether subject has suffered from lung squamous cancer
Transfer also includes the risk that judges subject and whether there is with lung squamous cancer transfer.
In the context of the present invention, " transfer for the treatment of lung squamous cancer " divides from the state change of disease, may include disease
Alleviation, disease complete healing.
The advantages of the present invention:
Present invention firstly discovers that VSIG10L gene expression is to lung squamous cancer transfer related, by detection subject's tissue
The expression of VSIG10L, it can be determined that whether subject suffers from lung squamous cancer transfer or judge that subject whether there is with lung squama
The risk of metastasis of cancer, so that clinician be instructed to provide prevention scheme or therapeutic scheme to subject.
Present invention finds a kind of new molecular marked compound-VSIG10L genes, and compared to traditional detection means, gene is examined
It is disconnected more timely, more special, sensitiveer, it can be realized the early diagnosis of lung squamous cancer transfer, to reduce the death of lung squamous cancer transfer
Rate.
Detailed description of the invention
Fig. 1, which is shown, utilizes expression of the genechip detection VSIG10L gene in lung squamous cell carcinoma cancers；
Fig. 2 shows the expression using Western blot detection VSIG10L albumen in lung squamous cell carcinoma cancers；
Fig. 3 shows the expression using VSIG10L gene in QPCR detection Lung Squamous Carcinoma Cells；
Fig. 4 shows the influence using QPCR detection siRNA to VSIG10L gene expression；
Fig. 5 shows the influence using Western blot detection siRNA to VSIG10L protein expression.
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 VSIG10L gene of embodiment
1, sample acquisition: the above-mentioned sample of 40 lung squamous cell carcinoma cancers (including 20 have transfer sample and 20 without transfer sample)
For the operation Operated Specimens of Lung Squamous Carcinoma Patients.The acquirement of above-mentioned all samples passes through the agreement of the committee, organizational ethics.Tissue
Whether the clinical data of sample includes: gender, age, tumor size, pathological grading (Edmonson), transfer, whether recurrence etc..
2, the acquisition of lung squamous cancer transfer tissue RNA
Lung squamous cancer is extracted using Trizol one-step method and shifts total tissue RNA, and 260nm is read by Nanodrop ND-1000
With the purity of absorbance value (A) the measurement RNA solution at 280nm.Through 1% denaturing formaldehyde agarose gel electrophoresis, ultraviolet transmission
It is observed under 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 (4x 44K gene) of Agilent company, state, 65 DEG C of hybridization 17h in chip hybridization furnace, then
Elution, dyeing, finally use 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 the lung squamous cell carcinoma cancers not shifted, the lung squamous cancer group that has shifted
The mRNA level in-site for knitting middle VSIG10L gene dramatically increases, and difference has statistical significance (P < 0.05).
The differential expression of 2 VSIG10L 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 VSIG10L egg
The gray value of informal voucher 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 the lung squamous cell carcinoma cancers not shifted, in the lung squamous cell carcinoma cancers that have shifted
The expression of VSIG10L albumen dramatically increases, and difference has statistical significance (P < 0.05).
Expression of the 3 VSIG10L gene of embodiment in Lung Squamous Carcinoma Cells system
1, cell culture
By Lung Squamous Carcinoma Cells strain NCI-H520, NCI-H596, NCI-H2170, NCI-H226 in DMEM culture medium and 10%
It is cultivated in fetal calf serum, human squamous lung cancer strain BEAS-2B is cultivated in BEGM culture solution and 10% fetal calf serum, is placed in 37
DEG C, 5%CO2In incubator.
2、QPCR
2.1 cell total rnas extract: carrying out the extraction of cell total rna using the RNA extracts kit of QINGEN company, press
Book instruction as directed carries out.
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 VSIG10L gene and GAPDH gene in Genbank, by Shanghai
The synthesis of Sheng Gong biotechnology Services Co., Ltd.Specific primer sequence is as follows:
VSIG10L gene:
Forward primer is 5 '-CCCTGGTTCTGAAGTATT-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-GGTCTTAACAGTGAAGGA-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
(3) PCR reaction condition: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 40s) * 45 circulations.Using SYBR Green as
Fluorescent marker carries out PCR reaction on Light Cycler fluorescence quantitative PCR instrument, true by melt curve analysis analysis and electrophoresis
Determine purpose band, Δ Δ CT method carries 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 difference for statistical analysis, between interference VSIG10L gene expression panel and control group
It is examined using t, it is believed that there is statistical significance as P < 0.05.
2.5 result
As shown in figure 3, compared with human squamous lung cancer strain BEAS-2B, Lung Squamous Carcinoma Cells strain NCI-H520, NCI-H596,
VSIG10L gene expression apparent increase (P < 0.05) in NCI-H2170, NCI-H226.
Embodiment 4 inhibits VSIG10L gene expression
1, siRNA design synthesis
For the siRNA sequence of VSIG10L:
SiRNA1-VSIG10L:
Positive-sense strand is 5 '-AAAUAUCAGGAAAUACUUCAG-3 ' (SEQ ID NO.7)；
Antisense strand is 5 '-GAAGUAUUUCCUGAUAUUUCG-3 ' (SEQ ID NO.8),
SiRNA2-VSIG10L:
Positive-sense strand is 5 '-AGAGUUUAAGAUCCAUAUCAU-3 ' (SEQ ID NO.9)；
Antisense strand is 5 '-GAUAUGGAUCUUAAACUCUCU-3 ' (SEQ ID NO.10),
SiRNA3-VSIG10L:
Positive-sense strand is 5 '-UUUUUCUCAGGAGUCUUUCCA-3 ' (SEQ ID NO.11)；
Antisense strand is 5 '-GAAAGACUCCUGAGAAAAAGA-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 lung squamous cancer metastatic cells
NCI-H520 cell is cultivated, step is the same as embodiment 3.
NCI-H520 cell is pressed 1 × 104/ hole is inoculated into 24 porocyte culture plates, in 37 DEG C, 5%CO2In incubator
For 24 hours, transfection is transfected according to the specification of lipofectamine 2000 (being purchased from Invitrogen company), experiment for cell culture
Be divided into negative control group and experimental group (20nM), wherein the sequence of negative control group siRNA and VSIG10L gene without homology,
Concentration is the hole 20nM/, while being transfected respectively.
3, the jamming effectiveness of detection siRNA is tested using QPCR.
Step is the same as embodiment 3.
As a result as shown in figure 4, compared with siRNA1-VSIG10L, siRNA3-VSIG10L, siRNA2-VSIG10L can
More effectively inhibit VSIG10L gene expression, difference have statistical significance (P < 0.05), using siRNA2-VSIG10L into
The subsequent experiment of row.
4, the jamming effectiveness of Western blot experiment detection siRNA2-VSIG10L
Step is the same as embodiment 2.
As a result as shown in figure 5, transfecting VSIG10L in the cell of siRNA2-VSIG10L compared with transfecting siRNA-NC group
The content of albumen is substantially reduced, and difference has statistical significance (P < 0.05).
Embodiment 5 studies influence of the VSIG10L gene expression to Lung Squamous Carcinoma Cells adhesive capacity
1, cell culture and transfection are the same as embodiment 4.
2, cell adhesion experiments
The NCI-H520 cell for transfecting 48h is digested to cell suspension using 0.25% pancreatin, with 5 × 104A/ml inoculation
After 96 porocyte culture plates, every hole 0.1ml, 60min, 37 DEG C of PBS wash away nonadherent cell, and mtt assay surveys each hole 490nm wave
Long absorbance value.The relative populations of adherency living cells are represented with absorbance value size.
3, result
SiRNA2-VSIG10L group relative optical density number is that 0.148 ± 0.056, siRNA-NC group relative optical density number is
1.527±0.132.Compared with siRNA-NC group, siRNA2-VSIG10L group absorbance value is remarkably decreased (P < 0.05).Above-mentioned reality
It tests the result shows that inhibiting VSIG10L expression that can significantly inhibit NCI-H520 cell adherence ability, while showing that VSIG10L is advantageous
In NCI-H520 cell adherence.
Embodiment 6 studies VSIG10L gene expression to Lung Squamous Carcinoma Cells migration, the influence of invasive ability
1, cell culture and transfection are the same as embodiment 4.
2, migration experiment
The NCI-H520 cell of transfection 48h is digested and is counted using pancreatin, takes 105A cell is placed in 1.5mL EP pipe,
200 μ L serum free mediums are added, cell is resuspended, be added in the cell transwell, 10% fetal calf serum is added in bottom chamber
DMEM culture medium is put into 37 DEG C, 5%CO2Incubator culture is for 24 hours.The cell transwell is taken, the cell of the inside is wiped with cotton swab, and
The inside remaining cell is gently washed off with PBS.8 random fields are taken to be counted under microscope after fixed dyeing.
3, Matrigel
The NCI-H520 cell of transfection 48h is digested and is counted using pancreatin, takes 105A cell is placed in 1.5mL EP pipe,
200 μ L serum free mediums are added, cell is resuspended, is added in the cell transwell by paving matrigel, bottom chamber is added
The DMEM culture medium of 10%FBS, is put into 37 DEG C, 5%CO2Incubator culture is for 24 hours.The cell transwell is taken, wipes the inside with cotton swab
Cell, and gently wash off with PBS the inside remaining cell.8 random fields are taken to be counted under microscope after fixed dyeing.
4, result
Migration experiment: compared with siRNA-NC group, siRNA2-VSIG10L group is thin across the cell transwell basilar memebrane
Born of the same parents reduce about 78%.
Matrigel: compared with siRNA-NC group, siRNA2-VSIG10L group was across having spread matrigel
The Leukopenia of the cell transwell basilar memebrane 72%.
It is above-mentioned the experimental results showed that, inhibit VSIG10L expression can significantly inhibit NCI-H520 cell migration, invasion energy
Power, while showing that VSIG10L gene expression is conducive to the migration and invasion of NCI-H520 cell.
In 7 Lung Squamous Carcinoma Cells antibody of embodiment and test
1, cell culture is the same as embodiment 4.
2, migration experiment
Cell is divided into two processing groups,
Experimental group 1 (control group): unrelated monoclonal antibody (1:50) is added in NCI-H520 cell；
Anti-human VSIG10L monoclonal antibody (1:50) is added in experimental group 2:NCI-H520 cell.
By NCI-H520 cell in 37 DEG C, 5%CO2After incubator incubation acts on 24 hours, is digested and is counted using pancreatin,
Take 105A cell is placed in 1.5mL EP pipe, 200 μ L serum free mediums is added, cell is resuspended, the cell transwell is added
In, the DMEM culture medium of 10% fetal calf serum is added in bottom chamber, is put into 37 DEG C, 5%CO2Incubator culture is for 24 hours.It takes
The cell transwell wipes the cell of the inside with cotton swab, and the inside remaining cell is gently washed off with PBS.It is micro- after fixed dyeing
8 random fields are taken to be counted under mirror.
3, Matrigel
Cell is divided into two processing groups,
Experimental group 1 (control group): unrelated monoclonal antibody (1:50) is added in NCI-H520 cell；
Anti-human VSIG10L monoclonal antibody (1:50) is added in experimental group 2:NCI-H520 cell.
By NCI-H520 cell in 37 DEG C, 5%CO2After incubator incubation acts on 24 hours, is digested and is counted using pancreatin,
Take 105A cell is placed in 1.5mL EP pipe, 200 μ L serum free mediums is added, cell is resuspended, and is added by paving matrigel
In the cell transwell, the DMEM culture medium of 10%FBS is added in bottom chamber, is put into 37 DEG C, 5%CO2Incubator culture is for 24 hours.It takes
The cell transwell wipes the cell of the inside with cotton swab, and the inside remaining cell is gently washed off with PBS.It is micro- after fixed dyeing
8 random fields are taken to be counted under mirror.
4, result
Migration experiment: compared with the control group, the groups of cells of anti-human VSIG10L monoclonal antibody passes through the cell transwell basilar memebrane
Leukopenia about 64%.
Matrigel: compared with the control group, the groups of cells of anti-human VSIG10L monoclonal antibody, which passes through, had spread matrigel
The Leukopenia of the cell transwell basilar memebrane 68%.
It is above-mentioned the experimental results showed that, inhibit VSIG10L protein function can significantly inhibit NCI-H520 cell migration, invasion
Ability, while showing that VSIG10L albumen is conducive to the migration and invasion of NCI-H520 cell.
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 VSIG10L gene expression in the tool of preparation diagnosis lung squamous cancer transfer.
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 VSIG10L gene expression are to diagnose the production that lung squamous cancer shifts
Product；The product with RT-PCR diagnosis lung squamous cancer transfer includes at least the primer of a pair of of specific amplified VSIG10L gene；It is described
The primer of a pair of of specific amplified VSIG10L gene is included at least with the product of real-time quantitative PCR diagnosis lung squamous cancer transfer；The use
The product of immune detection diagnosis lung squamous cancer transfer includes: the antibody in conjunction with VSIG10L protein-specific；It is described to use in situ hybridization
The product of diagnosis lung squamous cancer transfer includes: the probe with the nucleic acid array hybridizing of VSIG10L gene；It is described to use chip Diagnosis of pulmonary squama
The product of metastasis of cancer includes: protein chip and genetic chip；Wherein, protein chip includes in conjunction with VSIG10L protein-specific
Antibody, genetic chip includes the probe with the nucleic acid array hybridizing of VSIG10L gene.
3. application according to claim 2, which is characterized in that the production with real-time quantitative PCR diagnosis lung squamous cancer transfer
The primer for a pair of of specific amplified VSIG10L gene that product include at least is as shown in SEQ ID NO.3 and SEQ ID NO.4.
4. application according to claim 1, which is characterized in that the tool includes the examination for detecting VSIG10L gene expression
Agent；The reagent includes the primer and/or probe, the antibody for detecting VSIG10L albumen for detecting VSIG10L gene mRNA.
5. application according to claim 4, which is characterized in that the primer of the detection VSIG10L gene mRNA includes SEQ
Primer pair shown in ID NO.3 and SEQ ID NO.4.
Application of the inhibitor of 6.VSIG10L gene and/or its expression product in the drug of preparation treatment lung squamous cancer transfer.
7. application according to claim 6, which is characterized in that the inhibitor includes inhibiting VSIG10L gene expression
Reagent, and/or the reagent for inhibiting VSIG10L gene expression product.
8. application according to claim 7, which is characterized in that the reagent packet for inhibiting VSIG10L gene expression product
Include the antibody for inhibiting siRNA the, and/or VSIG10L albumen for VSIG10L gene.
9. application according to claim 8, which is characterized in that the siRNA sequence such as SEQ for VSIG10L gene
Shown in ID NO.7 and SEQ ID NO.8.
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Non-Patent Citations (3)
| Title |
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| Differential gene expression between African American and European American colorectal cancer patients;Jovov B et al;《PLoS One》;20120119;1-2 |
| Expression and functions of long noncoding RNAs during human T helper cell differentiation;Spurlock CF 3rd;《Nat Commun》;20150423;1-27 |
| SOX15 Governs Transcription in Human Stratified Epithelia and a Subset of Esophageal Adenocarcinomas;Rita Sulahian et al.;《Cell Mol Gastroenterol Hepatol》;20150805;598–609 |
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