CN108085389A - A kind of and the relevant lncRNA of breast cancer and its application - Google Patents
A kind of and the relevant lncRNA of breast cancer and its application Download PDFInfo
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Abstract
The invention discloses a kind of and the relevant lncRNA of breast cancer and its applications, the gene is LOC100507053, present invention firstly discovers that expression of the LOC100507053 in patient with breast cancer is significantly lowered, ROC analyses find that LOC100507053 has higher AUC value, prompting the diagnosis that LOC100507053 is applied to breast cancer has higher accuracy, In vitro cell experiment confirms, LOC100507053 is related to the invasion and attack and migration of breast cancer, prompts LOC100507053 that can be applied to the treatment of breast cancer as molecular target.
Description
Technical field
The invention belongs to biomedicine fields, are related to a kind of and the relevant lncRNA of breast cancer and its application, specifically institute
LncRNA is stated as LOC100507053.
Background technology
Breast cancer is the most common malignant tumour of world wide women, accounts for 22% or so of women whole malignant tumour, is
Endanger one of most important disease of women's health.It is breast cancer that current China, which is increased newly in female malignant case there are about 15%,
Case, breast cancer have become the highest female malignant of China's incidence and less than 45 years old women because malignant tumour is dead
The primary cause of disease died.More severe, data shows all in rising to become in the morbidity and mortality of China's breast cancer
Gesture, seriously endangered numerous women health so that life (Chen W, Zheng R, Baade PD, Zhang S, Zeng H,
Bray F,Jemal A,Yu XQ,He J.Cancer statistics in China,2015.CA Cancer J Clin
2016；66:115-32.).The morbidity of solid tumor patient and it is dead generally by the tumour cell that spreads to body normal function
Destruction causes, tumor cell migration become the research of cancer metastasis potential mechanism a big hot spot (Palmer TD, Ashby WJ,
Lewis JD,Zijlstra A.Adv Drug Deliv Rev 2011；63:568-81).Compared with primary tumor, transfer
Property tumour can not perform the operation removal and to Chemoresistance, 90% is all as caused by the DISTANT METASTASES IN of tumour in cancer mortality case
(Fidler IJ.Nat Rev Cancer 2003；3:453-8.).However, the molecular mechanism of mediation breast cancer cell migration is simultaneously
Not very clearly, the molecular marker that its progress transfer can be predicted is still limited.It was found that the pass in breast cancer progression transfer process
Key molecule and adjusting access are the emphasis in breast cancer research, help that breast cancer early stage diagnosis and treatment is instructed so as to reduce case fatality rate to change
Kind prognosis.
High pass quantity research, which discloses only very small part in mammalian genome, can be transcribed into protein coding gene (An
integrated encyclopedia of DNA elements in the human genome.Nature 2012489:
57-74.), it is most of to be transcribed into non-coding RNA.Long-chain non-coding RNA (long noncoding RNA, 1ncRNA) is
A kind of not coding protein of discovered in recent years, length is more than RNA molecule (the Prensner JR, Chinnaiyan of 200bp
AM.The emergence of 1ncRNAs in cancer biology.Cancer Discov 2011；1:391-407).
Many document report lncRNA play a significant role during tumour progression shifts：As CN201710522240.1,
The differential expression of the lncRNA of CN201710522694.9, CN201710522693.4 report is related to the transfer of liver cancer.
Although early find, early treatment, the strategies such as early operation make Mammary cancer five year survival rate improve to 98% or so,
The transfer and recurrence of breast cancer can not still cure.More and more lncRNA molecules are proved the occurrence and development phase with breast cancer
It closes, the further investigation of lncRNA will provide new thinking for the diagnose and treat of breast cancer.
The content of the invention
In order to make up for the deficiencies of the prior art, it is an object of the invention to provide a kind of relevant with breast cancer occurrence and development
LncRNA so as to provide molecular target for the diagnose and treat of breast cancer, realizes the personalized diagnosis and treatment of patient.
To achieve these goals, the present invention adopts the following technical scheme that：
The first aspect of the present invention provides a kind of reagent, and the reagent can detect the expression of LOC100507053 genes
It is horizontal.
Further, the reagent is selected from：
The probe of specific recognition LOC100507053；Or
The primer of specific amplification LOC100507053.
Further, the institute of primer sequence such as SEQ ID NO.1 of the specific amplification LOC100507053 genes~2
Show.
The second fermentation of the present invention provides a kind of kit, and the kit includes the examination described in first aspect present invention
Agent.
The third aspect of the present invention provides a kind of chip, and the chip includes the reagent described in first aspect present invention.
The fourth aspect of the present invention provides a kind of pharmaceutical composition, and described pharmaceutical composition includes LOC100507053's
Accelerating agent.
Further, the accelerating agent is the expression vector containing LOC100507053.
Further, described pharmaceutical composition further includes and other medicine classes of the accelerating agent compatibility and pharmaceutically acceptable
Carrier and/or auxiliary material.
The fifth aspect of the present invention provides a kind of method for the potential substance for screening prevention or treatment breast cancer, the side
Method includes：
The system expressed or containing LOC100507053 genes is handled with substance to be screened；With
Detect the expression of LOC100507053 genes in the system；
Wherein, if the substance to be screened can promote expression or the activity of LOC100507053 genes, the screening is shown
Substance is prevention or the potential substance for treating breast cancer.
The sixth aspect of the present invention provides following any one of them application：
A. application of the reagent described in first aspect present invention in the product for preparing Diagnosis of Breast cancer；
B. application of the kit described in second aspect of the present invention in the product for preparing Diagnosis of Breast cancer；
C. application of the chip described in third aspect present invention in the product for preparing Diagnosis of Breast cancer；
D. application of the pharmaceutical composition described in fourth aspect present invention in the product for preparing treatment breast cancer；
E. application of the pharmaceutical composition described in fourth aspect present invention in the product for preparing treatment mammary gland cancerous invasion；
F. application of the pharmaceutical composition described in fourth aspect present invention in the product for preparing treatment Metastasis in Breast Cancer；
Applications of the g.LOC100507053 in the potential substance of screening treatment breast cancer.
Description of the drawings
Fig. 1 is the expression figure in breast cancer tissue using QPCR detection LOC100507053 genes；
Fig. 2 is the expression figure in breast cancer cell line using QPCR detection LOC100507053 genes；
Fig. 3 is the transfected condition figure in breast cancer cell using QPCR detections LOC100507053；
Fig. 4 is the influence figure with CCK-8 methods detection LOC100507053 gene pairs Cells Proliferation of Human Breast Cancer；
Fig. 5 is the influence figure for being migrated and being attacked to breast cancer cell using Transwell cells detection LOC100507053；
Wherein figure A is the influence figure that LOC100507053 migrates breast cancer cell；Figure B is LOC100507053 to mammary gland
The influence figure of cancer cell invasion.
Specific embodiment
The present invention is most wide using current covering database by high throughput method by in-depth study extensively
LncRNA chips detect in breast cancer sample lncRNA in the expression of tumor tissues and cancer beside organism, find wherein to have apparent
The lncRNA of differential expression inquires into its relation between the occurrence and development of breast cancer, so as to the diagnosis and targeting for breast cancer
Better approaches and methods are found in treatment.By screening, present invention firstly discovers that LOC100507053 conspicuousnesses in breast cancer
It lowers.It is demonstrated experimentally that the expression by improving LOC100507053, can effectively inhibit breast cancer cell multiplication and
Invasion and attack.
Biomarker
In the present invention, " biomarker " " gene marker " and " molecular marker " can be substituted for each other, be it
Expression in tissue or cell is the same as any gene for changing compared with the expression of normal or healthy cell or tissue.
The present invention can utilize any method known in the art to measure gene expression.Those skilled in the art should manage
Solution, the means for measuring gene expression are not the importances of the present invention.
LOC100507053 genes
LOC100507053 is to be located at No. 4 long-armed 2nd area 3 of dyeing of people to take, a kind of representative people LOC100507053 bases
LOC100507053 genes (NR_037884.1) in the nucleotide sequence of cause such as current international public nucleic acid database GeneBank
It is shown.The LOC100507053 nucleotide full length sequence or its segment of the present invention can usually use PCR amplification method, recombination method or people
Work synthetic method obtains.LOC100507053 in the present invention includes wild type, saltant type or its segment.
The present invention can utilize any method known in the art to measure gene expression.Those skilled in the art should manage
Solution, the means for measuring gene expression are not the importances of the present invention.The table of biomarker can be detected on transcriptional level
Up to level.
Detection technique
The lncRNA of the present invention is detected using multiple nucleic acids technology known to persons of ordinary skill in the art, these skills
Art includes but not limited to：Nucleic acid sequencing, nucleic acid hybridization and nucleic acid amplification technologies.
The exemplary, non-limitative example of Nucleic acid sequencing techniques includes but not limited to chain terminator (Sanger) sequencing and dye
Expect terminator sequencing.Those of ordinary skill in the art it will be recognized that due to RNA in cell less stable and in an experiment
Be more vulnerable to nuclease attack, thus before sequencing usually by RNA reverse transcriptions into DNA.
The present invention simultaneously can expand nucleic acid (for example, ncRNA) before detection or with detection.Nucleic acid amplification technologies
Exemplary, non-limitative example include but not limited to：PCR (PCR), reverse transcriptase polymerase chain reaction (RT-
PCR), the amplification (TMA) of transcriptive intermediate, ligase chain reaction (LCR), strand displacement amplification (SDA) and based on nucleotide sequence
It expands (NASBA).Those of ordinary skill in the art will be it will be recognized that some amplification techniques (for example, PCR) needs will before amplification
RNA reverse transcriptions are into DNA (for example, RT-PCR), and other amplification techniques then direct cloning RNA (for example, TMA and NASBA).
The PCR of commonly referred to as PCR uses annealing and the primer extend of denaturation, primer pair and opposite strand
Multiple cycling, exponentially increase target nucleic acid sequence copy number；The amplification of the transcriptive intermediate of TMA is (in substantial constant
Temperature, multiple copies of target nucleic acid sequence are autocatalytically synthesized under conditions of ionic strength and pH, wherein target sequence is more
A RNA copies autocatalytically generate other copy；The ligase chain reaction of LCR uses miscellaneous with the adjacent area of target nucleic acid
The two groups of complementary DNA oligonucleotides handed over；Other amplification methods are included for example：The expansion based on nucleotide sequence of commonly referred to as NASBA
Increase；Use rna replicon enzyme (the commonly referred to as Q β replicase) amplification of amplification probe molecule in itself；Amplification method based on transcription；
And the sequence amplification of self―sustaining.
The nucleic acid of non-amplification or amplification can be detected by any conventional means in the present invention.
Chip, kit
The present invention provides the product of the expression of LOC100507053 genes in detection, the product is included (but not
It is limited to) preparation, chip or kit.Its chips includes：Solid phase carrier；And the widow on the solid phase carrier is fixed in order
Nucleotide probe, the oligonucleotide probe specifically correspond to the part or all of sequence shown in LOC100507053.
The solid phase carrier includes inorganic carrier and organic carrier, the inorganic carrier include but not limited to have silicon carrier,
Glass carrier, ceramic monolith etc.；The organic carrier includes polypropylene film, nylon membrane etc..
Term " probe " refers to the molecule that can be combined with the particular sequence or subsequence or other parts of another molecule.It is unless another
It points out, term " probe " is often referred to match by complementary base and another polynucleotides (often referred to as " target polynucleotide ")
With reference to polynucleotide probes.Lack sufficient sequence complementarity according to the stringency of hybridization conditions, probe energy and with the probe
Target polynucleotide combines.Probe can make direct or indirect mark, and scope includes primer.Crossing system, including, but it is unlimited
In：Solution phase, solid phase, mixed phase or in situ hybridization measuring method.
Exemplary probe in the present invention includes PCR primer and gene specific DNA oligonucleotide probe, such as fixed
In micro probe array, the quantitative nucleic acid enzyme protection on microarray substrate examine probe, the probe that is connected with molecular barcode and
The probe being fixed on pearl.
These probes have the base sequence with the specific base sequence complementary of target gene.Here, so-called " complementation ",
As long as hybridization, can not be complete complementary.These polynucleotides, which are commonly angled relative to the specific base sequence, to be had
More than 80%, preferably more than 90%, more preferable more than 95%, particularly preferred 100% homology.These probes can be DNA,
Can also be RNA, furthermore it is possible to be artificial by PNA, LNA, ENA, GNA, TNA etc. in part of it or whole nucleotides
The polynucleotides that replacement nucleic acid obtains.
The present invention provides a kind of kit, the kit can be used for the expression of detection LOC100507053.Preferably,
In the preparation or kit also containing be useful for labeled RNA sample label and with the corresponding bottom of the label
Object.In addition, may also include to extract the various reagents needed for RNA, PCR, hybridization, colour developing etc. in the kit, including
But it is not limited to：Extract, amplification liquid, hybridization solution, enzyme, comparison liquid, developing solution, washing lotion etc..In addition, it is also wrapped in the kit
Include operation instructions and/or chip image analysis software.
Accelerating agent and pharmaceutical composition
Based on the discovery of the present invention, the present invention provides a kind of pharmaceutical composition, described pharmaceutical composition includes
The accelerating agent of LOC100507053.
The accelerating agent of the LOC100507053 refers to any improve LOC100507053 genes or expression product is steady
Qualitative, up-regulation LOC100507053 expression, the effective acting time for increasing lncRNA LOC100507053 or promotion
The substance of the transcription of LOC100507053 genes, these substances are used equally for the present invention, as raising LOC100507053
Gene expresses useful substance, so as to be used to prevent or treat adenocarcinoma of lung.
As a kind of preferred embodiment of the present invention, the accelerating agent of the LOC100507053 is that one kind contains
The expression vector of LOC100507053.The expression vector usually also contains promoter, replication orgin and/or marker gene
Deng.
Method well-known to those having ordinary skill in the art can be used to build the required expression vector of the present invention.These methods include
Recombinant DNA technology in vi, DNA synthetic technologys, In vivo recombination technology etc..The expression vector preferably includes one or more
Selected marker selects the phenotypic character of the host cell of conversion, such as kalamycin, gentamicin, tide to provide
Mycin, amicillin resistance.
In the present invention, be various carriers known in the art, such as commercially available carrier, including plasmid, clay, bacteriophage,
Virus etc..Importing of the expression vector into host cell can use electroporation, calcium phosphate method, liposome method, DEAE Portugals to gather
The known methods such as sugared method, microinjection, viral infection, the combination of liposome transfection and cell-membrane permeable peptide.
Term " host cell " includes prokaryotic cell and eukaryocyte.The example of common prokaryotic host cell includes large intestine
Bacillus, hay bacillus etc..Common eukaryotic host cell includes yeast cells, insect cell and mammalian cell.It is preferred that
The host cell is eukaryocyte, such as Chinese hamster ovary celI, COS cells.
Pharmaceutical composition
Pharmaceutical composition in the present invention include LOC100507053 accelerating agent, and/or with the accelerating agent compatibility
Other medicine classes and pharmaceutically acceptable carrier and/or auxiliary material.
Pharmaceutically acceptable carrier can be that one kind can also be a variety of, and the carrier includes but not limited to diluent such as
Lactose, sodium chloride, glucose, urea, starch, water etc.；Adhesive such as starch, pregelatinized starch, dextrin, maltodextrin, sugarcane
Sugar, Arabic gum, gelatin, methylcellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyethylene
Than pyrrolidone, alginic acid and alginate, xanthans, hydroxypropyl cellulose and hydroxypropyl methyl cellulose etc.；Surfactant
Such as polyoxyethylene sorbitan aliphatic ester, lauryl sodium sulfate, glyceryl monostearate, hexadecanol；Humectant
Such as glycerine, starch；Absorption carrier such as starch, lactose, bentonite, silica gel, kaolin and soap clay etc.；Lubricant such as stearic acid
Zinc, glycerin monostearate, polyethylene glycol, talcum powder, calcium stearate and magnesium, polyethylene glycol, boric acid powder, hydrogenated vegetable oil,
Sodium stearyl fumarate, polyoxyl 40 stearate, single bay sucrose acid ester, sldium lauryl sulfate, magnesium laurylsulfate, 12
Alkylsurfuric acid magnesium etc.；Filler such as mannitol (granular or powdery), xylitol, sorbierite, maltose, erythrose, microcrystalline cellulose
Element, polymerization sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, carbon
Sour calcium and sodium acid carbonate etc.；Disintegrant such as cross-linked ethylene pyrrolidones, sodium carboxymethyl starch, low-substituted hydroxypropyl ylmethyl, crosslinking
Sodium carboxymethylcellulose, soybean polyoses etc..
Pharmaceutical composition in the present invention can also include stabilizer, fungicide, buffer, isotonic agent, chelating agent, pH controls
The additives such as preparation and surfactant.
As used herein, described " effective quantity " refer to that people and/or animal can be generated function or activity and can by people and/
Or the amount that animal is received." pharmaceutically acceptable carrier " refers to the carrier for Therapeutic Administration, including various figurations
Agent and diluent.The term refers to some such medicament carriers：Themselves it is not necessary active ingredient, and does not have after applying
Undue toxicity.Suitable carrier is well known to those of ordinary skill in the art.Pharmaceutically acceptable load in the composition
Body can contain liquid, such as water, brine, buffer solution.In addition, there is likely to be complementary substance in these carriers, as filler,
Lubricant, glidant, wetting agent or emulsifier, pH buffer substance etc..Cell can also be contained in the carrier, and (host is thin
Born of the same parents) transfection reagent.
The present invention may be employed with a variety of methods well known in the art by the accelerating agent or its encoding gene or its
Pharmaceutical composition delivers medicine to mammal.Including but not limited to：Hypodermic injection, intramuscular injection, for percutaneous administration of, administer locally to, plant
Enter, be sustained and give；Preferably, the administering mode is that non-bowel is given.
The pharmaceutical composition of the present invention can further include one or more anticancer agents.In specific embodiments,
Pharmaceutical composition includes the compound of at least one inhibition LOC100507053 gene expressions and at least one chemotherapeutics.For this
The chemotherapeutics of the method for invention, includes but not limited to, DNA- alkylating agents, antitumor antibiotics agent, antimetabolite, and tubulin is steady
Determine agent, tubulin destabilizing agent, hormone antagonist, topoisomerase enzyme inhibitor, kinases inhibitor, HMG-COA inhibition
Agent, CDK inhibitor, cyclin inhibitors, caspase inhibitors, metal protease inhibitors, antisensenucleic acids, three
Virus, bacterium and the exotoxin reagent of chain helical dna, aptamer and molecular modification.
The pharmaceutical composition of the present invention can also can be with the drug combination of other treatment breast cancer, other therapeutic compound
It is administered simultaneously with main active ingredient or even is administered simultaneously in same composition.
The pharmaceutical composition of the present invention can also be with individual composition or the dosage shape different from main active ingredient
Formula individually gives other therapeutic compounds.The Fractional of main component can be administered simultaneously with other therapeutic compounds,
And other dosage can be administered alone.It over the course for the treatment of, can be according to the severity of symptom, the frequency of recurrence and treatment side
The physiologic response of case adjusts the dosage of pharmaceutical composition of the present invention.
Drug screening
The present invention provides it is a kind of screen prevention or treatment breast cancer medicines method, i.e.,：
In experimental group, testing compound is added in into cultivating system, and measures the expression of LOC100507053；
In control group, testing compound is added without into same cultivating system, and measures the expression of LOC100507053；Its
In, if the expression of LOC100507053 is more than control group in experimental group, illustrate that the substance to be screened is
The potential substance of LOC100507053.
In the present invention, the method further includes：The potential substance obtained to previous step further tests its inhibition
The effect of breast cancer if test compound has breast cancer significant inhibition, illustrates the compound for prevention or treatment
The potential substance of breast cancer.
The cultivating system includes but is not limited to cell system, subcellular fraction system, solution system, organizational framework, organ
System or animal system (animal model of such as animal model, preferably non-human mammal, such as mouse, rabbit, sheep, monkey) etc..
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.The experimental method of actual conditions is not specified 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 condition proposed by manufacturer.
Embodiment 1 is screened and the relevant gene marker of breast cancer
1st, sample collection
The cancer beside organism of 8 patient with breast cancers and the breast cancer tissue's sample that pathology is clarified a diagnosis are collected, writes sample name exactly
Situations such as title, organization type, number, Date of Sampling, sample process process, patient sign informed consent form, above-mentioned all samples
Acquirement pass through the agreement of the committee of organizational ethics.
2nd, the preparation (being operated using the tissue RNA extracts kits of QIAGEN) of RNA sample
RNA sample is extracted using the tissue RNA extracts kits of QIAGEN, concrete operations refer to specification.
3rd, reverse transcription and mark
With Low RNA Input Linear Amplification Kit by mRNA reverse transcriptions into cDNA, while use Cy3
Labelling experiment group and control group respectively.
4th, hybridize
Genetic chip uses Kang Cheng biology-Human lncRNA Array, is carried out by the step of chip operation instructions miscellaneous
It hands over.
5th, data analysis
Chip results are analyzed using Agilent GeneSpring softwares, screening expression quantity has significant difference
(standard differs 2 times or more, and p in cancer for the lncRNA with the expression quantity by cancer<0.05) lncRNA.
6th, result
The results show that differential expression is presented in LOC100507053 in patient with breast cancer, compared with cancer beside organism,
Expression in cancerous tissue is significantly lowered.
The differential expression of embodiment 2QPCR sequence verification LOC100507053 genes
1st, large sample QPCR verifications are carried out to LOC100507053 gene differential expressions.It is received according to the sample in embodiment 1
Mode set selects breast cancer cancer beside organism and each 50 of breast cancer tissue.
2nd, RNA is extracted
RNA sample is extracted using the tissue RNA extracts kits of QIAGEN, concrete operations refer to specification.
3、QPCR
1) reaction system：
1 μ l of RNA templates, 1 μ l of random primer, distilled water add to 12 μ l, mixing, slow-speed of revolution centrifugation, 65 DEG C of 5min, Ran Houfang
In cooled on ice.
Continuation adds in following ingredients in 12 μ l reaction solutions：
5 × reaction buffer, 4 μ l, 1 μ l, 10mM dNTP mixed liquors of RNase inhibitor (20U/ μ l), 2 μ l, AMV reverse transcriptions
1 μ l of enzyme (200U/ μ l)；Abundant mixing simultaneously carries out centrifugal treating；
2) reverse transcription reaction condition
25 DEG C of 5min, 42 DEG C of 60min, 70 DEG C of 5min.
3) polymerase chain reaction
Design of primers：
QPCR amplimers are designed according to the coded sequence of LOC100507053 genes and GAPDH genes in Genebank,
It is synthesized by Bo Maide biotech firms.Specific primer sequence is as follows：
LOC100507053 genes：
Forward primer is 5 '-TTAGAGAAGGACAAGAATAG-3 ' (SEQ ID NO.1)；
Reverse primer is 5 '-CAATATGAACAGACAACAG-3 ' (SEQ ID NO.2).
GAPDH genes：
Forward primer is 5 '-AATCCCATCACCATCTTCCAG-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-GAGCCCCAGCCTTCTCCAT-3 ' (SEQ ID NO.4).
Prepare PCR reaction systems：
2 × qPCR mixed liquors, 12.5 μ l, 2.0 μ l of gene primer, reverse transcription product 2.5 μ l, ddH2O 8.0μl。
PCR reaction conditions：95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 60s) × 40 Xun Huans, 60 DEG C of 5min extensions.75
DEG C to 95 DEG C, heat up 1 DEG C per 20s, draw solubility curve.Using SYBR Green as fluorescent marker, in Light Cycler
PCR reactions are carried out on fluorescence quantitative PCR instrument, purpose band are determined by melt curve analysis analysis and electrophoresis, Δ Δ CT methods carry out phase
To quantitative.
4th, ROC curve is analyzed
Using the Receiver Operating Characteristics of the pROC bags analysis LOC100507053 in R language, the accurate confidence of binomial is calculated
ROC curve is drawn in space.
5th, statistical method
Experiment is all completed according to being repeated 3 times, and result data is represented in a manner of mean+SD,
Statistical analysis is carried out using SPSS18.0 statistical softwares, the paired comparisons of cancer and cancer beside organism are examined using t, it is believed that work as P<
There is statistical significance when 0.05.
6th, result
The results are shown in Figure 1 by QPCR, and compared with breast cancer cancer beside organism, LOC100507053 is in expression in breast
It lowers, difference has statistical significance (P<0.05) it is, consistent with chip testing result；ROC curve the results show that
The AUC value of LOC100507053 is up to 0.955, has higher specificity and sensibility, and LOC100507053 is prompted to be applied to
The diagnosis of breast cancer has higher accuracy.
Expressions of the embodiment 3LOC100507053 in breast cancer cell line
1st, cell culture
Cultivate human breast carcinoma cell lines MCF-7, SK-BR-3, MDA-MB-231 and normal breast epithelium cell system MCF-
L0A, wherein, MDA-MB-231 is incubated in the L15 culture mediums of 10% hyclone, and SKBR3 is incubated at containing 10% hyclone
RPMI-1640 culture mediums in, MCF-7 and normal breast epithelial MCF-10A are incubated at containing 10% hyclone
In DMEM culture mediums.1%P/S is added in culture solution.In 37 DEG C, 5%CO2, relative humidity be 90% incubator in cultivate.2-
It changes within 3 days liquid 1 time, is passed on using the 0.25% trypsase conventional digestion containing EDTA.
2nd, the extraction of RNA and concentration mensuration
1) by cell to be collected with PBS cleaning is entered, appropriate RNAiso Plus are added in, 2min cracking is incubated on ice, scrapes
Bottom of bottle cell, with micro sample adding appliance pipettor is blown and beaten, is stored at room temperature repeatedly, takes supernatant spare after centrifugation.
2) Total RNAs extraction：The chloroform of proper volume is added to and is received in cell or tissue supernatant, is fully mixed to solution
It is creamy white；It is centrifuged after being stored at room temperature.Aspirate supernatant is managed to new EP, adds in the isopropanol of 1/2 volume, is turned upside down fully mixed
It is even, it is centrifuged after being stored at room temperature, adds in isometric pre-cooled ethanol (75%), turn upside down mixing, and supernatant is abandoned in 4 DEG C of centrifugations, and room temperature is done
Dry precipitation adds in the deionized water solution without RNAase in right amount.
3) agarose gel electrophoresis analyzes RNA purity, and RNA concentration is measured under spectrophotometer.
3rd, QPCR specific steps are the same as embodiment 2
4th, statistical analysis
Experiment is all completed according to being repeated 3 times, and result data is represented in a manner of mean+SD,
Statistical analysis is carried out using SPSS18.0 statistical softwares, the paired comparisons of cancer and cancer beside organism are examined using t, it is believed that work as P<
There is statistical significance when 0.05.
5th, result
The results are shown in Figure 2, and expression of the LOC100507053 in breast cancer cell is substantially less than normal cell system, difference
With statistical significance (P<0.05).
The overexpression of embodiment 4LOC100507053 genes
1st, cell culture
Human breast carcinoma cell lines MCF-7, with the culture medium DMEM containing 10%FBS and 1%P/S in 37 DEG C, 5%CO2, it is opposite
Humidity is to be cultivated in 90% incubator.It changes within 2-3 days liquid 1 time, is passed on using the 0.25% trypsase conventional digestion containing EDTA.
Cell in blake bottle with pancreatin is digested and is seeded in 6 orifice plates, guarantee cell number is 2-8 × 105A/
Hole adds in cell culture medium.Overnight, second day observation cell density, cell density can be transfected for more than 70%.
2nd, the structure of gene overexpression carrier
Special PCR amplification primer is synthesized according to the cDNA sequence of LOC100507053, in 5 ' end primers and 3 ' end primers
Two restriction enzyme sites of HindIII and XhoI are added respectively.It is obtained with the extraction of patients with lung adenocarcinoma blood and reverse transcription
CDNA as amplification template, above-mentioned cDNA sequence be inserted into after restriction enzyme HindIII and XhoI double digestion through
In the eukaryotic expression vector pcDNA3.1 of HindIII and XhoI double digestions, the recombinant vector pcDNA3.1-1 of acquisition is connected
For subsequent experimental.
3rd, transfect
Experiment is divided into three groups：Control group (MCF-7), negative control group (pcDNA3.1-NC) and experimental group
(pcDNA3.1-1), the transfection of carrier is carried out using liposome 3000, the instruction of specific transfection method to specifications carries out.
The transfection concentrations of pcDNA3.1 empty carriers and pcDNA3.1-1 are 0.5 μ g/ml.
4th, QPCR detects the transcriptional level of LOC100507053 genes
1) the extraction specific steps of cell total rna are the same as embodiment 3
2) QPCR expands specific steps with embodiment 2
5th, statistical method
Experiment is all completed according to being repeated 3 times, and result data is represented in a manner of mean+SD,
Statistical analysis, the difference between LOC100507053 Gene Experiments group and control group are carried out using SPSS18.0 statistical softwares
It is examined using t, it is believed that work as P<There is statistical significance when 0.05.
6th, result
As a result such as Fig. 3 shows that compared with the control group, the expression of the LOC100507053 in experimental group dramatically increases,
Difference has statistical significance (P<0.05).
The influence of embodiment 5LOC100507053 gene pairs Cells Proliferation of Human Breast Cancer
Detection LOC100507053 gene pairs Cells Proliferation of Human Breast Cancer capacities are tested using CCK-8.
1st, with embodiment 3,6h changes liquid after transfection, places cell incubator and stays overnight for cell culture and transfection procedure.
2nd, cell to be taken out in second day, micro- Microscopic observation cell growth status, 1ml/ holes add in the pancreatin containing EDTA, into
Row cell dissociation waits removal pancreatin after the completion of digesting, and adding in cell culture medium mixing makes cell suspend, and then carries out cytometer
Number.
3rd, concentration of cell suspension is diluted to 15000/ml, is inoculated with afterwards into 96 orifice plates, cell is added in per hole
200 μ l of suspension, cell are controlled at 3000 or so, are inoculated with 8 multiple holes.PcDNA3.1-1 experimental groups and pcDNA3.1-NC are set
Control group.Altogether spread 4 piece of 96 orifice plate be respectively used to for 24 hours, 48h, 72h, 96h4 detection time points..
4th, after for 24 hours, first piece of 96 orifice plate is taken out, the CCK-8 detection liquid of 10 μ l is added in every hole, 96 orifice plates are continued to put
Enter and 4h or so is incubated in cell incubator, absorbance of each hole at 450nm wavelength is detected with microplate reader and record data.
5th, the operation in 4 is respectively repeated steps after 48h, 72h, 96h, finally counts the absorbance at each time point,
Make growth curve chart.
6th, statistical analysis
Experiment is all completed according to being repeated 3 times, and statistical analysis is carried out using SPSS18.0 statistical softwares, the two it
Between difference using t examine, it is believed that work as P<There is statistical significance when 0.05.
7th, result
The results are shown in Figure 4, and compared with the control, for experimental group after siRNA-1 is transfected, the multiplication of cell substantially receives suppression
System, difference have statistical significance (P<0.05) illustrate that LOC100507053 plays an important role of to promote cell Proliferation.
6 cell migration of embodiment and Matrigel
1st, prepared by Transwell cells
Matrigel is ice bath melted under aseptic condition, carries out 20 times of dilutions with PBS, is layered on the volume in 50 μ l/ holes
On the polycarbonate membrane of Transwell cells.37 DEG C of placement 4h, take out after Matrigel glue aggregates into gel, gently suction out
Liquid is precipitated in upper strata.The serum-free medium containing BSA that 50 μ l are added in per hole carries out basilar memebrane hydration process, 37 DEG C of placements
30min。
2nd, cell suspension is configured
Cell removes serum starvation processing 12-24h, carries out digestion process to cell, is centrifuged after terminating digestion, in removal
Layer culture solution.Sedimentation cell is cleaned with PBS, the serum free medium containing BSA is added in and it is resuspended.Adjustment is thin
The density of born of the same parents is to 5 × l05A/ml.
3rd, cell inoculation
200 μ l of cell suspension (migration experiment is 100 μ l, and Matrigel is 200 μ l) is taken to be added to Transwell cells
In, room adds in DMEM culture mediums of the 500 μ l containing FBS under 24 orifice plates.Cell is put into cell incubator and is cultivated for 24 hours.
4th, dye
Cell is dyed after culture using DAPI.Small ventricular cell first with PBS is rinsed 2 times, is put into DAPI working solutions
Middle room temperature dyes 5-20min.It is rinsed 2 times with PBS, is put into fluorescence microscopy Microscopic observation and counts.
5th, result
The results are shown in Figure 5, and compared with the control group, the migration of experimental group and invasive ability are decreased obviously, and are as a result said
The increase of bright LOC100507053 expressions can inhibit the migration and invasion and attack of breast cancer.
The explanation of above-described embodiment is only intended to understand the method and its core concept of the present 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 be also fallen into the protection domain of the claims in the present invention.
Sequence table
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Claims (10)
1. a kind of reagent, which is characterized in that the reagent can detect the expression of LOC100507053 genes.
2. reagent according to claim 1, which is characterized in that including：
The probe of specific recognition LOC100507053；Or
The primer of specific amplification LOC100507053.
3. reagent according to claim 2, which is characterized in that the primer sequence of specific amplification LOC100507053 is such as
Shown in SEQ ID NO.1~2.
4. a kind of kit, which is characterized in that the kit includes claim 1-3 any one of them reagents.
5. a kind of chip, which is characterized in that the chip includes claim 1-3 any one of them reagents.
6. a kind of pharmaceutical composition, which is characterized in that described pharmaceutical composition includes the accelerating agent of LOC100507053.
7. pharmaceutical composition according to claim 6, which is characterized in that the accelerating agent is to contain LOC105371720's
Expression vector.
8. the pharmaceutical composition according to claim 6 or 7, which is characterized in that described pharmaceutical composition further include with it is described
Other medicine classes and pharmaceutically acceptable carrier and/or auxiliary material of accelerating agent compatibility.
A kind of 9. method for the potential substance for screening treatment breast cancer, which is characterized in that the described method includes：
The system expressed or containing LOC100507053 genes is handled with candidate substances；With
Detect the expression of LOC100507053 genes in the system；
Wherein, if the candidate substances can promote expression or the activity of LOC100507053 genes, show that the candidate substances are
Prevention or the potential substance for the treatment of breast cancer.
10. following any one of them application：
A. application of claim 1-3 any one of them reagent in the product for preparing Diagnosis of Breast cancer；
B. application of the kit described in claim 4 in the product for preparing Diagnosis of Breast cancer；
C. application of the chip described in claim 5 in the product for preparing Diagnosis of Breast cancer；
D. application of claim 6-8 any one of them pharmaceutical composition in the product for preparing treatment breast cancer；
E. application of claim 6-8 any one of them pharmaceutical composition in the product for preparing treatment mammary gland cancerous invasion；
F. application of claim 6-8 any one of them pharmaceutical composition in the product for preparing treatment Metastasis in Breast Cancer；
Applications of the g.LOC100507053 in the potential substance of screening treatment breast cancer.
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