CN106636444A

CN106636444A - Application of FAM78A gene

Info

Publication number: CN106636444A
Application number: CN201710112638.8A
Authority: CN
Inventors: 杨承刚; 宋宏涛
Original assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2017-02-28
Filing date: 2017-02-28
Publication date: 2017-05-10
Anticipated expiration: 2037-02-28
Also published as: CN106636444B

Abstract

The invention discloses application of a FAM78A gene to preparation of a product used for diagnosis of lung adenocarcinoma and/or a pharmaceutical composition used for treatment of lung adenocarcinoma. The invention discloses a method for screening potential substances used for treating lung adenocarcinoma. If candidate substances added into a system expressing or containing the FAM78A gene or protein coded by the FAM78A gene can improve the expression and/or activity of FAM78A, the substances are potential substances for treating lung adenocarcinoma. The FAM78A gene related to generation and development of lung adenocarcinoma can be used as a biological marker for lung adenocarcinoma, thereby assisting a doctor in formulation of individualized treatment schemes.

Description

The purposes of FAM78A genes

Technical field

The invention belongs to biomedicine field, is related to the purposes of FAM78A genes, gene FAM78A is specifically related in lung Purposes in gland cancer early diagnosis and therapy.

Background technology

With the deterioration of environment for human survival, the incidence of disease of whole world cancer is raised year by year, and malignant tumour becomes generation One of fatal rate highest disease in boundary.And lung cancer has become modal malignant tumour, its incidence of disease and fatal rate shelter have First of malignant tumour.Lung cancer can be divided into non-small cell lung cancer (non small cell lung cancer, NSCLC) and little thin Born of the same parents' lung cancer (small cell lung cancer, SCLC), wherein NSCLC accounts for the 80%-85% of lung cancer sum, in NSCLC Adenocarcinoma of lung accounting raise year by year.In China, the incidence of disease and the death rate of lung cancer have leapt to the first position, and replacing liver cancer to become causes The dead principal disease of tumor patient.Show according to Third National disease coroner's inquest situation, increased due to smoking population within nearly 30 years Plus and the factor such as increasing environmental pollution, China's lung cancer mortality rises 465%, while the incidence of disease is also gradually rising, to complete The health and lives of the social people cause great threat.

Tumour is a kind of disease of molecular level, and the research for carrying out tumour from genes protein level is final approach.In recent years Genomics, proteomics and CAD in integral level based on high flux molecule scanning means New opportunity is provided for the research of tumour etc. the integration and the application etc. of " Technology Chain " of being mutually related of technology, and is existed Great successes are achieved in the research of breast cancer, lung cancer, cancer of the stomach, colon cancer, oophoroma, melanoma etc..Wherein gene table It is most typical representative up to spectrum chip, the difference of tumor tissues and normal structure can be detected and screened by chip gene expression profile Allogene and oncogene, the change of tumor suppressor gene and variation relation, therefrom find the cause of disease, and to early diagnose and effecting a radical cure line is provided Rope and foundation.

At present the conventional method for the treatment of NSCLC has operative treatment, platinum bigeminy chemotherapy, radiotherapy and targeted therapy etc., but this The purpose of healing can not be reached for a little conventional treatments centering late tumor patients, therefore, survive after most of patients treatment Phase is still very short, and survival rate is less than 15% within 5 years.Because most NSCLC patients early stage is without specific clinical performance, when making a definite diagnosis into There is transfer and local Invasion in late period, most of patients.Therefore, the morbidity regulatory mechanism of NSCLC is inquired into, early intervention tumour is thin The growth of born of the same parents, invasion and attack and transfer ability, are favorably improved the survival rate of patient NSCLC, reduce its death rate, are the gene of lung cancer Diagnosis and targeted therapy provide more structurally sound foundation.

The content of the invention

In order to make up the deficiencies in the prior art, it is an object of the invention to a kind of biomarker of adenocarcinoma of lung is in adenocarcinoma of lung Application in early diagnosis and therapy.

To achieve these goals, the present invention is adopted the following technical scheme that：

The invention provides a kind of purposes of FAM78A genes, for preparing the product of early diagnosis adenocarcinoma of lung.

Further, the product includes：The product of adenocarcinoma of lung is diagnosed with RT-PCR, real-time quantitative PCR, in situ hybridization or chip Product.

Further, the RT-PCR diagnoses the product of adenocarcinoma of lung at least includes a pair of specific amplification FAM78A genes Primer；The real-time quantitative diagnoses the product of adenocarcinoma of lung at least includes the primer of a pair of specific amplification FAM78A genes；Institute State the probe included with the product of in situ hybridization diagnosis adenocarcinoma of lung with the nucleic acid array hybridizing of FAM78A genes；The chip is examined The product of disconnected adenocarcinoma of lung includes the probe with the nucleic acid array hybridizing of FAM78A genes or is combined with FAM78A protein-specifics Antibody or part..

The invention provides a kind of product of diagnosis adenocarcinoma of lung, the product can be by the expression of FAM78A in detection sample Level is diagnosing adenocarcinoma of lung.

Further, described product includes chip, preparation or kit.

Further, the chip can be used to detect the albumen including the multiple genes including FAM78A genes or its coding The expression of (for example, the multiple genes related to adenocarcinoma of lung)；The kit can be used for detection includes that FAM78A genes exist The expression of the albumen (for example, the multiple genes related to adenocarcinoma of lung) of interior multiple genes or its coding.

The invention provides a kind of purposes of FAM78A genes, for screening prevention or the medicine for the treatment of adenocarcinoma of lung.

The invention provides a kind of screen the method prevented or treat adenocarcinoma of lung medicine, methods described includes：

System with candidate substances process expression or containing FAM78A genes or the albumen of its coding；With

Detect the expression of the albumen of FAM78A genes or its coding or activity in the system；

Wherein, the candidate substances can increase the expression of FAM78A, then show that the candidate substances are to prevent or treat lung gland The potential material of cancer.

Further, in test group, candidate substances are added to the albumen expressed or containing FAM78A genes or its coding System in；And/or detection test group system in FAM78A genes or its coding albumen expression or activity, and with compare Group compares, wherein described control group is without the expression of the candidate substances or or containing FAM78A genes or its volume The system of the albumen of code；If the expression of the albumen of FAM78A genes or its coding or activity are higher than control group in test group, i.e., Show that the candidate substances are the potential materials for preventing or treating adenocarcinoma of lung.

The invention provides a kind of purposes of FAM78A genes, for preparing prevention or treating the drug regimen of adenocarcinoma of lung Thing.

Further, described pharmaceutical composition includes FAM78A genes, albumen or its accelerator.

Further, the FAM78A albumen include total length FAM78A albumen, ripe FAM78A albumen, FAM78A with Domain or the FAM78A active fragments containing the domain that DNA is combined；The accelerator is produced including FAM78A gene expressions Thing, promoted type miRNA, promoted type transcription regulatory factor or promoted type targeting micromolecular compound.

Further, described pharmaceutical composition also includes pharmaceutically acceptable carrier and/or auxiliary material.

The advantages of the present invention：

Present invention firstly discovers that FAM78A is related to the generation of adenocarcinoma of lung development, can as adenocarcinoma of lung diagnosis base Because of mark, for distinguishing people's early stage adenocarcinoma of lung.

Human lung adenocarcinoma gene marker FAM78A provided by the present invention, can be applied to face as potential drug target spot The treatment of adenocarcinoma of lung on bed.

Description of the drawings

Fig. 1 is to detect expression figure of the FAM78A genes in pulmonary adenocarcinoma using QPCR；

Fig. 2 is to detect transfected condition figures of the FAM78A in lung adenocarcinoma cell using QPCR；

Fig. 3 is the impact figure that FAM78A gene pairs lung adenocarcinoma cell propagation is detected with CCK-8 methods；

Fig. 4 is with the impact figure of flow cytomery FAM78A gene pairs Apoptosis of Lung Adenocarcinoma Cell；

Fig. 5 is to detect the impact figure that FAM78A is migrated to lung adenocarcinoma cell and attacked using Transwell cells；Wherein scheme A is the impact figure that FAM78A is migrated to lung adenocarcinoma cell；Figure B is the impact figure that FAM78A is attacked to lung adenocarcinoma cell.

Specific embodiment

The present invention, by high throughput method, using current the most wide base of database is covered through extensively in-depth study Because of chip, detect that gene is in the expression of tumor tissues and cancer beside organism in adenocarcinoma of lung sample, find wherein have substantially expression poor Different genetic fragment, inquires into itself relation and the generation of adenocarcinoma of lung between, so as to for the early detection and targeted therapy of adenocarcinoma of lung Find more preferable approaches and methods.By screening, present invention firstly discovers that FAM78A conspicuousnesses are lowered in adenocarcinoma of lung.Experiment card It is bright, by the expression for increasing FAM78A, can effectively suppress the growth and invasion and attack of lung adenocarcinoma cell, so as to reach suppression The effect of adenocarcinoma of lung.

FAM78A genes

FAM78A genes are taken positioned at 3rd area of No. 9 chromosome long arms of people 4, a kind of representational people FAM78A genes Nucleotide sequence and amino acid sequence are as shown in SEQ ID NO.1 and SEQ ID NO.2.FAM78A in the present invention includes wild Type, saltant type or its fragment.

People's FAM78A nucleotides full length sequence or its fragment of the present invention can generally use PCR TRAPs, recombination method or people The method of work synthesis is obtained.For PCR TRAPs, can be according to published relevant nucleotide sequence, especially ORFs Sequence designing primer, and with commercially available cDNA storehouses or the cDNA storehouses as prepared by conventional method well known by persons skilled in the art As template, expand and obtain relevant sequence.When sequence is longer, it is often necessary to carry out twice or multiple PCR is expanded, then again will The fragment for amplifying for each time is stitched together by proper order.

Once obtain relevant sequence, it is possible to obtain relevant sequence in large quantity with recombination method.This typically will It is cloned into carrier, then proceeds to cell, then isolated relevant sequence in the host cell by conventional method from after propagation. Additionally, relevant sequence can also be synthesized with artificial synthesized method, when especially fragment length is shorter.Generally, by first synthesizing Multiple small fragments, being then attached again can obtain the very long fragment of sequence.

It is optimized for obtaining the gene of the present invention using the method for round pcr DNA amplification/RNA.For the primer of PCR Can be properly selected according to the sequence information of invention disclosed herein, and available conventional method synthesis.Conventional method can be used The DNA/RNA fragments of amplification are such as separated and purified by gel electrophoresis.

It would be recognized by those skilled in the art that the practicality of the present invention is not limited to the marker gene to the present invention The gene expression of any specific variants is carried out quantitatively.If when nucleic acid or its fragment most preferably compare with other nucleic acid (or its complementary strand) Pair when (have appropriate nucleotides inserted or disappearance), nucleotide base at least about 60%, usually at least about 70%, More generally at least about 80%, it is preferably at least about 90% and more preferably at least about there is core in 95-98% nucleotide bases Nucleotide sequence homogeny, then the two sequences are " substantially homologous " (or basic simlarities).

Or, when nucleic acid or its fragment with another nucleic acid (or its complementary strand), a chain or its complementary series in selectivity When hybridizing under hybridization conditions, then exist therebetween substantially homologous or (homogeny).When hybridization has more than specific general loss When selective, there is cross selection.Typically, when one section of sequence at least about 14 nucleotides is present at least about 55% homogeny, preferably at least about 65%, more preferably at least about 75% and most preferably at least about 90% homogeny when, send out Raw selective cross.As described herein, the length of cognate pair ratio can be longer sequence section, lead in certain embodiments Often it is at least about 20 nucleotides, more frequently at least about 24 nucleotides, typically at least about 28 nucleotides, more Typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides.

Therefore, polynucleotides of the invention and SEQ ID NO.1 preferably have at least 75%, more preferably at least 85%, more Preferably at least 90% homology.It is highly preferred that there is at least 95%, more preferably at least 98% homology.

" FAM78A albumen " includes any function equivalent of FAM78A albumen and FAM78A albumen.The functional equivalent Thing includes FAM78A albumen conservative variation protein or its active fragment, or its reactive derivative or its mutant.Mutant Including allelic variant, natural mutation, induced mutants, its amino acid sequence by disappearance, replacement, increase and/or insertion The mutant that morphs, can be with the albumen coded by the DNA of the DNA hybridization of people FAM78A under high or low stringent condition Matter.

Preferably, FAM78A albumen is the protein with following amino acid sequences：

(1) protein being made up of the amino acid sequence in sequence table shown in SEQ ID NO.2；

(2) by the amino acid sequence shown in SEQ ID NO.2 is through the replacement of one or several amino acid residues and/or lacks Lose and/or add and have with the amino acid sequence shown in SEQ ID NO.2 the ammonia by shown in SEQ ID NO.2 of identical function Protein derived from base acid sequence.The number of the amino acid for replace, lacking or adding is usually 1-50, preferably 1-30 It is individual, more preferably 1-20, most preferably 1-10.

(3) there is at least 80% homology (being also called sequence iden) with the amino acid sequence shown in SEQ ID NO.2, Preferably, with the homology of the amino acid sequence at least about 90% to 95% shown in SEQ ID NO.2, be often 96%, 97%, 98%th, the polypeptide that the amino acid sequence of 99% homology is constituted.

In specific embodiments of the present invention, the FAM78A albumen is with the amino acid shown in SEQ ID NO.2 The protein of sequence.

Generally, the modification of one or more amino acid does not interfere with the function of protein in a protein.This area skill Art personnel can approve the amino acid for changing single amino acids or little percentage or the indivedual additions to amino acid sequence, disappearance, insert It is conservative modification to enter, replace, and the change of wherein protein produces the protein with identity function.Intimate amino is provided The Conservative substitution tables of acid are well known in the art.

The modification of amino acid sequence can be derived from after spontaneous mutation or heredity and modify, it is also possible to which artificial induction's natural gene is produced It is raw.Example by the protein for adding an amino acid or more amino acid modification is the fusion egg of PCSK1N albumen In vain.For the peptide or protein with PCSK1N protein fusions is not limited, as long as the fusion protein of gained retains PCSK1N eggs White BA.

The present invention can determine gene expression using any method known in the art.Those skilled in the art should manage Solution, the means for determining gene expression are not the importances of the present invention.The table of biomarker can be detected on transcriptional level Up to level.

Chip, kit

The described genetic chip of the present invention includes：Solid phase carrier；And the widow being fixed in order on the solid phase carrier Nucleotide probe, described oligonucleotide probe is specifically corresponding to the part or all of sequence shown in FAM78A.

Specifically, suitable probe can be designed according to gene of the present invention, is fixed on solid phase carrier, be formed " oligonucleotide arrays ".Described " oligonucleotide arrays " are referred to addressable point (i.e. with distinctive, addressablely The position that location is characterized) array, each addressable point is containing a coupled characteristic oligonucleotides.According to need Will, oligonucleotide arrays can be divided into multiple sub- battle arrays.

Term " probe " refers to the molecule that can be combined with the particular sequence of another molecule or subsequence or other parts.Unless another Point out, term " probe " is often referred to can be by complementary base pairing 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 is combined.Probe can make direct or indirect mark, and its scope includes primer.Crossing system, including, but do not limit In：Solution, solid phase, mixed phase or in situ hybridization determination method.

In the present invention for FAM78A genes oligonucleotide probe can be DNA, RNA, DNA-RNA chimera, PNA or Other derivatives.The length of the probe is not limited, as long as completing specific hybrid and purpose nucleotide sequence specificity knot Close, any length can.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 gene.Due to different probe lengths pair Hybridization efficiency, signal specificity have different impacts, and the length of the probe is typically at least 14 base-pairs, most it is long it is general not More than 30 base-pairs, complementary length is optimal with 15-25 base-pair with purpose nucleotide sequence.The probe self-complementary Sequence is most preferably less than 4 base-pairs, in order to avoid affect hybridization efficiency.

Heretofore described solid phase carrier can adopt the various common used materials in genetic chip field, for example, include but is not limited to Plastic products, microparticle, membrane carrier etc..The plastic products can be resisted by non-covalent or physical absorption mechanism and antibody or albumen Original combines, and the most frequently used plastic products are small test tube, globule and micro-reaction plate made by polystyrene；The microparticle is The microballoon aggregated into by high polymer monomer or particle, its diameter mostly is micron, due to carrying the functional group that can be combined with protein, Easily chemical coupling is formed with antibody (antigen), binding capacity is big；The membrane carrier includes nitrocellulose filter, glass fibre element film And the miillpore filter such as nylon membrane.

Preparing for described FAM78A chips can be using the common manufacturing method of biochip known in the art.For example, If solid phase carrier uses modification slide or silicon chip, 5 ' ends of probe are gone here and there containing amido modified poly- dT, can be by few nucleosides Acid probe is configured to solution, then using point sample instrument by its point modification slide or silicon chip on, be arranged in predetermined sequence or battle array Row, are then fixed by standing overnight, so that it may obtain the genetic chip of the present invention.

The invention provides a kind of kit, the kit can be used to detect the expression of FAM78A.Preferably, it is described Also contain the label for labeled RNA sample, and the substrate corresponding with the label in preparation or kit.This Outward, the various reagents needed for for extracting RNA, PCR, hybridization, colour developing etc. are may also include in described kit, including but do not limit In：Extract, amplification liquid, hybridization solution, enzyme, comparison liquid, nitrite ion, washing lotion etc..Additionally, also including using in described kit Specification and/or chip image analysis software.

Accelerator and pharmaceutical composition

Based on the discovery of the present invention, the invention provides a kind of pharmaceutical composition, described pharmaceutical composition includes FAM78A Expression product or FAM78A accelerator.The accelerator can promote the expression of the albumen of FAM78A genes or its coding And/or activity, so as to suppress adenocarcinoma of lung.The FAM78A accelerator include FAM78A gene expression products, promoted type miRNA, Promoted type transcription regulatory factor or promoted type targeting micromolecular compound.

Generally, these accelerator can be formulated in nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, Wherein pH ordinarily be about 5-8, and preferably pH is about 6-8, although pH value can be with the property for being formulated material and disease to be treated Disease and be varied from.The pharmaceutical composition for preparing can be administered by conventional route, including (but being not limited to)： In knurl, intramuscular, intraperitoneal, intravenous, subcutaneous, intracutaneous or local be administered.

Used as a kind of preferred embodiment of the present invention, the accelerator of described FAM78A is that a kind of expression containing FAM78A is carried Body.Described expression vector is generally also containing promoter, replication orgin and/or marker gene etc..

Method well-known to those having ordinary skill in the art can be used to build the expression vector needed for the present invention.These methods include Recombinant DNA technology in vi, DNA synthetic technologys, In vivo recombination technology etc..Described expression vector preferably includes one or more Selected marker, to provide the phenotypic character for selecting the host cell of conversion, such as kalamycin, gentamicin, tide 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..Expression vector to the importing in host cell can be gathered using electroporation, calcium phosphate method, liposome method, DEAE Portugals The known methods such as the combination of sugared method, microinjection, virus infection, liposome transfection and cell-membrane permeable peptide.

In the present invention, " host cell " born of the same parents can be prokaryotic, such as bacterial cell；Or low eukaryotic, such as Yeast cells；Or higher eucaryotic cells, such as mammalian cell.Representative example has：Escherichia coli, the bacterium of streptomyces Cell；Fungal cell's such as yeast；Plant cell；The insect cell of fruit bat S2 or Sf9；The animal of CHO, COS or 293 cells is thin Born of the same parents etc..

Can be carried out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.When host is original When core biology is such as Escherichia coli, can absorb the competent cell of DNA can harvest after exponential phase of growth, use CaCl₂Method process, institute With the step of it is generally well-known in the art.Another kind of method is to use MgCl₂.If desired, conversion also can be with the side of electroporation Method is carried out.When host is eucaryote, following DNA transfection methods are can select：Calcium phosphate precipitation, conventional mechanical methods are such as Microinjection, electroporation, liposome packaging etc..

The FAM78A albumen of the pharmaceutical composition in the present invention including safe and effective amount or its accelerator, and/or with it is described Other medicine classes of accelerator compatibility and pharmaceutically acceptable carrier and/or auxiliary material.

Term " effective dose " is referred to can producing function or activity to people and/or animal and can connect by people and/or animal The amount received." pharmaceutically acceptable carrier " refers to the carrier for Therapeutic Administration, including various excipient and diluent.The art Language refers to such some medicament carriers：Themselves it is not necessary active component, and without undue toxicity after applying.Properly Carrier be well known to those of ordinary skill in the art.In the composition pharmaceutically acceptable carrier can contain liquid, such as Water, salt solution, buffer solution.In addition, there is likely to be complementary material in these carriers, such as filler, lubricant, glidant, Wetting agent or emulsifying agent, pH buffer substance etc..Lipofectamine can also be contained in described carrier.

In the present invention, can using various methods well known in the art by described accelerator 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, Implantation, sustained release give；Preferably, the administering mode is that non-bowel gives.

Preferably, the means of gene therapy can be adopted to carry out.Such as, can be directly by the accelerator of FAM78A by such as noting The method such as penetrate and deliver medicine to experimenter；Or, the ceneme (ratio that can be adjusted the promotion for carrying FAM78A by certain approach Such as expression vector or virus) it is delivered on target spot, concrete condition need to be depending on the type of described accelerator, and these are these Known to art personnel.

The effective dose of the accelerator of FAM78A of the present invention can be with the serious of the pattern and disease to be treated being administered Degree etc. and change.The selection of preferred effective dose can determine (example by those of ordinary skill in the art according to various factors Such as pass through clinical testing).Described factor is included but is not limited to：The pharmacokinetic parameter example of the accelerator of described FAM78A Such as bioavailability, metabolism, half-life；The order of severity of patient's disease to be treated, the body weight of patient, the immunity of patient Situation, approach of administration etc..For example, by an urgent demand for the treatment of situation, dosage separate several times can daily be given, or by agent Amount is reduced pari passu.

The pharmaceutical composition of the present invention can also can be with the drug combination of other treatment adenocarcinoma of lung, other therapeutic compound It is administered simultaneously with main active component, or even is administered simultaneously in same composition.

The pharmaceutical composition of the present invention can be with single composition or the dosage shape different from main active component 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.Over the course for the treatment of, can be according to the order of 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 invention provides a kind of screening prevention or the method for treating adenocarcinoma of lung medicine, i.e.,：

In experimental group, testing compound is added in cell culture system, and determine the expression of FAM78A；Right According to group, testing compound is added without in same cultivating system, and determines the expression of FAM78A；Wherein, such as fruit The expression of FAM78A in group is tested more than control group, then illustrates the accelerator that the candidate compound is FAM78A.

In the present invention, described method also includes step：The candidate compound that previous step is obtained further is tested It suppresses the effect of liver cancer, if test compound has significant inhibition to adenocarcinoma of lung, illustrates that the compound is FAM78A Accelerator.

In the present invention, term " sample " is used with its broadest sense.In a kind of implication, it is intended that include from it is any come Sample or culture that source obtains, and biological and environmental samples.Biological specimen available from animal (including people) and cover liquid, Solid, tissue and gas.Biological specimen includes blood product, blood plasma, serum etc..However, such sample should not be construed as Limit the sample type for being applied to the present invention.

With reference to the accompanying drawings and examples the present invention is further detailed explanation.Following examples are merely to illustrate this Invention rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in embodiment, generally according to conventional strip Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) described in condition, or according to the condition proposed by manufacturer.

Embodiment 1 screens the gene marker related to adenocarcinoma of lung

1st, sample collection

Respectively collect 8 adenocarcinoma of lung cancer beside organisms and pulmonary adenocarcinoma sample.Adenocarcinoma of lung tumor tissues specimen sampling position is Vital tumor areas, positioned at tumor mass China and foreign countries 1/3 and normal structure junction, exclude tumor center substantially necrosis, calcification portion Divide and tumour periphery normal lung tissue；Ai Pang normal lung tissues sample takes from the position of more than borderline tumor 5cm, visually observes Without significant change.The acquirement of above-mentioned all samples is by the agreement of the committee of organizational ethics.

2nd, the preparation of RNA sample (utilizes E.Z.N.A.MiRNA kit are operated)

Liquid nitrogen is imported in mortar, the tissue for taking above-mentioned acquisition is put into mortar, is shredded in liquid nitrogen and grind into powder, Put into liquid nitrogen after shredding and be ground to powder, in then continuing at glass homogenizer；Tissue homogenate adds in glass homogenizer Enter Trizol reagents, in tissue abrasion on ice.Tissue homogenate after homogenate is proceeded in the EP pipes without RNase, is stood under room temperature 5min.Extract according to the specification in kit and separate RNA.It is specific as follows：

1) separation of RNA：

0.2m1 chloroforms are added in EP pipes, EP lids are covered tightly, 15s is acutely vibrated manually so as to fully mix.Under room temperature Hatching 5min.Then 15min is centrifuged with 14000g at 4 DEG C.Sample is divided into three layers after centrifugation, and RNA is present in upper strata aqueous phase.

2) RNA precipitate

Separate water is mutually taken into 450 μ l to move in the new EP pipes without RNase, according to 1:1 ratio adds 450 μ l different Propyl alcohol, hatches at room temperature 10min after mixing of turning upside down, 4 DEG C of 14000g are centrifuged 10min.

3) RNA wash-outs

Carefully remove supernatant after centrifugation, add the ethanol of 1ml 75% (destroy the enzyme treatment, matching while using and in precooling on ice) punching RNA is washed, subsequent 4 DEG C of 7500g are centrifuged 5min.

4) RNA re-dissolveds

The supernatant after washing is carefully removed, EP pipe lids are opened in superclean bench, RNA samples are placed at room temperature 5-10min, dries.Add and process water 20-50 μ l, water-bath 10min in 55-60 DEG C of water bath without RNase.

5) quality analysis of RNA sample

Spectrophotometer is detected：

NanoDrop1000 spectrophotometers detect RNA sample, the sample requirement of RNA-seq sequencings：OD260/OD280 is 1.8-2.2。

Agarose gel electrophoresis is detected：

The RNA of said extracted is entered into row agarose gel electrophoresis, Agilent Technologies 2100Bioanalyzer detects RNA sample quality, and observation 28S rRNA and 18S rRNA master tapes are obvious, complete without degraded, RNA Sex index is qualified, concentration reaches requirement, can be used for the gene expression profile and screening experiment of chip.

3rd, reverse transcription and mark

With Low RNA Input Linear Amplification Kit by mRNA reverse transcriptions into cDNA, while using Cy3 Difference labelling experiment group and control group.

4th, hybridize

Genetic chip adopts people's full-length genome chip of expression spectrum of Aglient companies.The step of by chip operation instructions Carry out.

5th, data analysis

Chip results are analyzed using Agilent GeneSpring softwares, screening expression has significant difference (standard is that the gene differs more than 2 times, and p with the expression by cancer in cancer<0.05) gene.

6th, result

As a result show, expressions of the FAM78A in pulmonary adenocarcinoma is substantially less than the expression in cancer beside organism.

The differential expression of the QPCR sequence verification FAM78A genes of embodiment 2

1st, large sample QPCR checkings are carried out to FAM78A gene differential expressions.According to the sample collection mode in embodiment 1 Select adenocarcinoma of lung cancer beside organism and pulmonary adenocarcinoma each 50.

2nd, RNA extraction steps are as described in Example 1.

3rd, reverse transcription

1) reaction system：

The μ l of RNA templates 1, the μ l of random primer 1, distilled water adds to 12 μ l, mixes, slow-speed of revolution centrifugation, 65 DEG C of 5min, Ran Houfang In cooled on ice.

Continuation adds following ingredients in 12 μ l reactant liquors：

The μ l of 5 × reaction buffer 4, the μ l of RNase inhibitor (20U/ μ l) 1, the μ l of 10mM dNTP mixed liquors 2, AMV reverse transcriptions The μ l of enzyme (200U/ μ l) 1；Fully mix and carry 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：

According to the coded sequence design QPCR amplimers of FAM78A genes and GAPDH genes in Genebank, stepped by rich Moral biotech firm synthesizes.Concrete primer sequence is as follows：

FAM78A genes：

Forward primer is 5 '-TCAGCATGAATGACAACT-3 ' (SEQ ID NO.3)；

Reverse primer is 5 '-CGGTAGATATTGGTGAGC-3 ' (SEQ ID NO.4).

GAPDH genes：

Forward primer is 5 '-AATCCCATCACCATCTTCCAG-3 ' (SEQ ID NO.5)；

Reverse primer is 5 '-GAGCCCCAGCCTTCTCCAT-3 ' (SEQ ID NO.6).

Prepare PCR reaction systems：

The μ l of 2 × qPCR mixed liquors 12.5, the μ l of gene primer 2.0, 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 circulations, 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 The enterprising performing PCR reaction of quantitative real time PCR Instrument, by melt curve analysis analysis and electrophoresis purpose band is determined, Δ Δ CT methods carry out phase To quantitative.

5th, statistical method

Experiment is all completed according to being repeated 3 times, result data be all in the way of mean+SD representing, Statistical analysis is carried out using SPSS18.0 statistical softwares, cancer is checked with the paired comparisons of cancer beside organism using t, it is believed that work as P< There is statistical significance when 0.05.

6th, result

As a result as shown in figure 1, compared with adenocarcinoma of lung cancer beside organism, FAM78A expresses downward, difference in pulmonary adenocarcinoma With statistical significance (P<0.05) it is, consistent with chip detection result.

The FAM78A overexpression of embodiment 3

1st, cell culture

Human A459 lung cancer cell line, with the RPMI1640 culture mediums containing 10% hyclone and 1%P/S 37 DEG C, 5% CO₂, cultivate in the incubator that relative humidity is 90%.Liquid 1 time is changed within 2-3 days, using the 0.25% trypsase routine containing EDTA Had digestive transfer culture.

Cell in blake bottle is digested with pancreatin and is seeded in 6 orifice plates, it is ensured that cell number is 2-8 × 10⁵Individual/ Hole, adds cell culture medium.Overnight, second day observation of cell density, cell density can be transfected for more than 70%.

2nd, the structure of gene overexpression carrier

Special pcr amplification primer thing, primer sequence are synthesized according to the cDNA sequence (as shown in SEQ ID NO.1) of FAM78A It is as follows：

Forward primer：5’-CCGAAGCTTGCCACCATGCCTGGTTTTTTCTGTGA-3’(SEQ ID NO.7)

Reverse primer：5’-CGGCTCGAGCCGGTGCTTGGGCGGGATCACCACC-3’(SEQ ID NO.8)

From cDNA library (clontech companies, article No. into Human fetal spleen：638831) the GDPD2 genes of amplification total length in Coded sequence, above-mentioned cDNA sequence is inserted into Jing restriction enzymes Jing after restriction enzyme HindIII and XhoI double digestion In the eukaryotic expression vector pcDNA3.1 of HindIII and XhoI double digestions, connect the recombinant vector pcDNA3.1-1 for obtaining For subsequent experimental.

3rd, transfect

Lung adenocarcinoma cell is divided into 3 groups, respectively control group (A549), blank control group (transfection pcDNA3.1-NC), real Test group (transfection pcDNA3.1-1).The transfection of carrier, concrete transfection method finger to specifications are carried out using liposome 2000 Showing is carried 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 FAM78A genes

The extraction of 4.1 cell total rnas

1) cell culture fluid in 6 orifice plates is outwelled, is rinsed twice with PBS, each hole adds 1ml Trizol reagents, room temperature Place 5min.

2) 0.2m1 chloroforms are added, acutely shakes 15s, 4 DEG C, 12000g is centrifuged 15min.

3) water is mutually transferred in new pipe, adds 4.5m1 isopropanols, room temperature to place 10min；4 DEG C, 10000g centrifugation 10min.

4) liquid is outwelled, with 75% ethanol of l ml EP tube walls is washed.4 DEG C, 7500g is centrifuged 5min.

5) 75% ethanol after cleaning is outwelled, room temperature hangs 5-10min.

6) DEPC water of 25 μ 1 without RNase, -70 DEG C of preservations are added.

4.2 reverse transcription steps are with embodiment 2.

4.3QPCR amplification steps are with embodiment 2.

5th, statistical method

Experiment is all completed according to being repeated 3 times, result data be all in the way of mean+SD representing, Statistical analysis is carried out using SPSS18.0 statistical softwares, the difference between FAM78A gene overexpressions group and control group is adopted T is checked, it is believed that work as P<There is statistical significance when 0.05.

6th, result

As a result as Fig. 2 shows, compare with non-transfection group with transfection empty plasmid group, overexpression in transfection FAM78A groups FAM78A, difference has statistical significance (P<0.05).

The impact of the FAM78A gene pairs lung adenocarcinoma cell of embodiment 4 propagation

Affected using CCK-8 experiment detection FAM78A gene pairs lung adenocarcinoma cells multiplication capacities.

1st, cell culture and transfection procedure are with embodiment 3.

2nd, cell was taken out in second day, basis of microscopic observation cell growth status, 1ml/ holes add the pancreatin containing EDTA, enter Row cell dissociation, removes pancreatin after the completion of waiting to digest, adding cell culture medium to mix makes cell suspend, and then carries out cytometer Number.

3rd, concentration of cell suspension is diluted into 1.5 × 10⁴Individual/ml, is inoculated with afterwards toward 96 orifice plates, is added per hole thin The μ 1 of born of the same parents' suspension 200, cell is controlled at 3000 or so, is inoculated with 8 multiple holes.PcDNA3.1-1 experimental groups and pcDNA3.1- are set NC control groups.4 piece of 96 orifice plate is spread altogether is respectively used to 24h, 48h, 72h, 96h4 detection time point.

4th, after 24h, first piece of 96 orifice plate is taken out, the CCK-8 that 10 μ 1 are added in every hole detects liquid, and 96 orifice plates are continued to put Enter and be incubated in cell culture incubator 4h or so, with ELIASA absorbance and record data of each hole at 450nm wavelength is detected.

5th, the operation in 4 is respectively repeated steps after 48h, 72h, 96h, the absorbance of each time point is finally counted, Make growth curve chart.

6th, statistical analysis

Experiment is all completed according to being repeated 3 times, and using SPSS18.0 statistical softwares statistical analysis is carried out, both it Between difference using t check, it is believed that work as P<There is statistical significance when 0.05.

7th, result

As a result as shown in figure 3, compared with the control, after transfection pcDNA3.1-1, the propagation of cell is substantially subject to experimental group Suppress, difference has a statistical significance (P<0.05) illustrate that FAM78A has the effect for suppressing cell propagation.

The impact of the FAM78A gene pairs Apoptosis of Lung Adenocarcinoma Cell of embodiment 5

Using the apoptotic impact of flow cytomery FAM78A gene pairs.

1st, cell culture step is with embodiment 3.

2nd, cell transfecting step is with embodiment 3.

3rd, step

1) by 10 × sample-loading buffers of 3m1 27m1 distilled water dilutings.

2) collection of cellular samples and with the PBS of precooling.

3) 1 × sample-loading buffers of lml, 300g centrifugation 10min is added to suction out buffer solution in cell.

4) 1 × sample-loading buffers of lml are added again, cell concentration in cell suspension is adjusted into 1 × 10⁶Individual/ml.

5) cell suspension is taken out into 100 μ 1, in adding EP pipes.

6) the Annexin V FITC of 5 μ l are added in EP pipes, mixes the liquid in EP pipes, at room temperature lucifuge incubation 10min。

7) 5 μ 1PI dye liquors are added in EP pipes, at room temperature lucifuge 5min.

8) PBS solution of 500 μ l is added in EP pipes, is gently mixed, flow cytometer is detected in 1h.

3rd, statistical method

Experiment is all completed according to being repeated 3 times, result data be all in the way of mean+SD representing, Statistical analysis is carried out using SPSS13.0 statistical softwares, the t inspections of difference employing between the two, it is believed that work as P<When 0.05 With statistical significance.

4th, result：

As a result as shown in figure 4, experimental group is compared with control group, apoptosis rate has significant change (P<0.05), should As a result illustrate, the low expression of FAM78A reduces the apoptosis rate of cell.

The cell migration of embodiment 6 and Matrigel

1st, prepared by Transwell cells

Matrigel is ice bath melted under aseptic condition, and 20 times of dilutions are carried out 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 Upper strata separates out liquid.The serum-free medium containing BSA of 50 μ l is added in per hole carries out hydration process, 37 DEG C of placements to basilar memebrane 30min。

2nd, cell suspension is configured

Cell removes serum starvation and processes 12-24h, and digestion process is carried out to cell, terminates being centrifuged after digestion, in removal Layer nutrient solution.Sedimentation cell is cleaned with PBS, adds the serum free medium containing BSA to carry out it resuspended.Adjustment is thin The density of born of the same parents is to 5 × l0⁵Individual/ml.

3rd, cell inoculation

The μ 1 (migration experiment is 100 μ 1, and Matrigel is 200 μ 1) of obtained cell suspension 200 is added to Transwell cells In.Room adds 500 1640 culture mediums of the μ 1 containing FBS under 24 orifice plates.Cell is put into cell culture incubator and cultivates 24h.

4th, dye

Cell is dyeed after culture terminates using DAPI.Little ventricular cell is first rinsed 2 times with PBS, DAPI working solutions are put into Middle room temperature dyes 5-20min.Rinsed 2 times with PBS, be put into fluorescence microscopy Microscopic observation and count.

5th, result

As a result as shown in figure 5, after lung adenocarcinoma cell transfected plasmids, compared with control group, the migration of experimental group and invade Attack ability to be decreased obviously, as a result illustrate that FAM78A can suppress the migration and invasion and attack of lung adenocarcinoma cell.

The explanation of above-described embodiment is only intended to understand the method for the present invention and its core concept.It should be pointed out that for this For the those of ordinary skill in field, under the premise without departing from the principles of the invention, some improvement can also be carried out to the present invention And modification, these are improved and modification also will be fallen into the protection domain of the claims in the present invention.

SEQUENCE LISTING

<110>Beijing Yang Shen biology information technologies Co., Ltd

<120>The purposes of FAM78A genes

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 852

<212> DNA

<213>People source

<400> 1

atgcctggtt ttttctgtga ctgctggcct tccctggaga tcagagcgct cctgtatgcc 60

atgggctgta ttcagagcat cggaggcaaa gccagagtct tccgggaagg gatcacggtg 120

attgatgtga aagcctccat cgaccccgtc cccactagca tcgatgagtc ctccagcgtg 180

gtgctccgct accggacacc ccacttccgg gcctcggccc aggtggtcat gccgcccatc 240

cccaagaagg agacttgggt agttggctgg atccaggcgt gcagccacat ggagttctac 300

aaccagtacg gcgagcaggg catgtccagc tgggagctcc ccgacctcca ggagggcaag 360

atccaagcca tcagcgactc ggatggggtg aactacccct ggtacggcaa caccacagag 420

acctgcacca tcgtgggccc caccaagagg gactccaagt tcatcatcag catgaatgac 480

aacttttacc ccagcgtcac atgggccgtg cccgtcagcg agagcaacgt ggccaagctc 540

accaatatct accgggacca gagcttcacc acctggctgg tggccaccaa cacctccacc 600

aacgacatga tcatcctgca gacgctgcac tggcgcatgc agctcagcat cgaggtgaac 660

cccaaccggc ccctgggcca gcgcgcccgg ctgcgggagc ccatcgccca ggaccagccc 720

aaaatcctga gcaagaatga gcccatcccg cccagcgccc tggtcaagcc caatgccaac 780

gatgcccagg tcctcatgtg gcggcccaag tacgggcagc cgctggtggt gatcccgccc 840

aagcaccggt ga 852

<210> 2

<211> 283

<212> PRT

<213>People source

<400> 2

Met Pro Gly Phe Phe Cys Asp Cys Trp Pro Ser Leu Glu Ile Arg Ala

1 5 10 15

Leu Leu Tyr Ala Met Gly Cys Ile Gln Ser Ile Gly Gly Lys Ala Arg

20 25 30

Val Phe Arg Glu Gly Ile Thr Val Ile Asp Val Lys Ala Ser Ile Asp

35 40 45

Pro Val Pro Thr Ser Ile Asp Glu Ser Ser Ser Val Val Leu Arg Tyr

50 55 60

Arg Thr Pro His Phe Arg Ala Ser Ala Gln Val Val Met Pro Pro Ile

65 70 75 80

Pro Lys Lys Glu Thr Trp Val Val Gly Trp Ile Gln Ala Cys Ser His

85 90 95

Met Glu Phe Tyr Asn Gln Tyr Gly Glu Gln Gly Met Ser Ser Trp Glu

100 105 110

Leu Pro Asp Leu Gln Glu Gly Lys Ile Gln Ala Ile Ser Asp Ser Asp

115 120 125

Gly Val Asn Tyr Pro Trp Tyr Gly Asn Thr Thr Glu Thr Cys Thr Ile

130 135 140

Val Gly Pro Thr Lys Arg Asp Ser Lys Phe Ile Ile Ser Met Asn Asp

145 150 155 160

Asn Phe Tyr Pro Ser Val Thr Trp Ala Val Pro Val Ser Glu Ser Asn

165 170 175

Val Ala Lys Leu Thr Asn Ile Tyr Arg Asp Gln Ser Phe Thr Thr Trp

180 185 190

Leu Val Ala Thr Asn Thr Ser Thr Asn Asp Met Ile Ile Leu Gln Thr

195 200 205

Leu His Trp Arg Met Gln Leu Ser Ile Glu Val Asn Pro Asn Arg Pro

210 215 220

Leu Gly Gln Arg Ala Arg Leu Arg Glu Pro Ile Ala Gln Asp Gln Pro

225 230 235 240

Lys Ile Leu Ser Lys Asn Glu Pro Ile Pro Pro Ser Ala Leu Val Lys

245 250 255

Pro Asn Ala Asn Asp Ala Gln Val Leu Met Trp Arg Pro Lys Tyr Gly

260 265 270

Gln Pro Leu Val Val Ile Pro Pro Lys His Arg

275 280

<210> 3

<211> 18

<212> DNA

<213>Artificial sequence

<400> 3

tcagcatgaa tgacaact 18

<210> 4

<211> 18

<212> DNA

<213>Artificial sequence

<400> 4

cggtagatat tggtgagc 18

<210> 5

<211> 21

<212> DNA

<213>Artificial sequence

<400> 5

aatcccatca ccatcttcca g 21

<210> 6

<211> 19

<212> DNA

<213>Artificial sequence

<400> 6

gagccccagc cttctccat 19

<210> 7

<211> 35

<212> DNA

<213>Artificial sequence

<400> 7

ccgaagcttg ccaccatgcc tggttttttc tgtga 35

<210> 8

<211> 34

<212> DNA

<213>Artificial sequence

<400> 8

cggctcgagc cggtgcttgg gcgggatcac cacc 34

Claims

1. a kind of purposes of FAM78A genes, it is characterised in that for preparing the product of early diagnosis adenocarcinoma of lung.

2. purposes according to claim 1, it is characterised in that the product includes：With RT-PCR, real-time quantitative PCR, original Position hybridization or chip diagnose the product of adenocarcinoma of lung.

3. application according to claim 2, it is characterised in that the product for diagnosing adenocarcinoma of lung with RT-PCR at least includes The primer of a pair of specific amplification FAM78A genes；The product that the real-time quantitative diagnoses adenocarcinoma of lung is special at least including a pair Property amplification FAM78A genes primer；The product for diagnosing adenocarcinoma of lung with situ hybridization includes the nucleic acid sequence with FAM78A genes The probe of row hybridization；The product for diagnosing adenocarcinoma of lung with chip include probe with the nucleic acid array hybridizing of FAM78A genes or The antibody combined with FAM78A protein-specifics or part.

4. it is a kind of diagnosis adenocarcinoma of lung product, it is characterised in that the product can pass through detection sample in FAM78A genes table Adenocarcinoma of lung is diagnosed up to level.

5. product according to claim 4, it is characterised in that described product includes chip, preparation or kit.

6. a kind of purposes of FAM78A genes, it is characterised in that for screening prevention or treat the potential material of adenocarcinoma of lung.

7. a kind of method that screening prevents or treats adenocarcinoma of lung medicine, it is characterised in that methods described includes：

Wherein, the candidate substances can increase expression or the activity of FAM78A, then show that the candidate substances are to prevent or treat lung The potential material of gland cancer.

8. a kind of purposes of FAM78A genes, it is characterised in that for preparing prevention or treating the pharmaceutical composition of adenocarcinoma of lung.

9. purposes according to claim 8, it is characterised in that described pharmaceutical composition include FAM78A genes, albumen or Its accelerator.

10. purposes according to claim 9, it is characterised in that described pharmaceutical composition also includes pharmaceutically acceptable Carrier and/or auxiliary material.