CN108192977A - A kind of and relevant molecular marker of gastric cancer occurrence and development - Google Patents

Abstract

The invention discloses a kind of and relevant molecular markers of gastric cancer occurrence and development, the present invention is combined by high throughput sequencing technologies with bioinformatics, the lncRNA that differential expression is presented in stomach organization is screened, and the lncRNA is further demonstrated by molecule experiments and cell experiment and is used as feasibility of the molecular marker applied to the clinical diagnosis of gastric cancer and treatment.

Description

A kind of and relevant molecular marker of gastric cancer occurrence and development

Technical field

The invention belongs to biomedicine fields, are related to a kind of and relevant molecular marker of gastric cancer occurrence and development, specifically Molecular marker is LINC00941.

Background technology

Gastric cancer (GC) is one of most common malignant tumour, while tumour related mortality is located at third position in the whole world (Arnold M,Moore SP,Hassler S,Ellison-Loschmann L,Forman D,Bray F.The burden of stomach cancer in indigenous populations:a systematic review and global assessment.Gut.2014；63:64-71.).The improvement of development, modus operandi although as medicine and medical consultations skill The raising of art, many Patients with Gastric Cancer can be obtained properly with the treatment of specification, and obtained good therapeutic effect, but the early stage hair of gastric cancer Now more difficult, advanced gastric carcinoma recurrence after resection rate and rate of transform height drastically influence therapeutic effect and the life of Patients with Gastric Cancer always Deposit the phase.For other tumours, the early detection ratio of gastric cancer is low, and when making a definite diagnosis, progressive stage is up to more than 70-80%, into Even if 5 years recurrence rates are higher after patient's radical excision of postponing (Vogiatzi P, Vindigni C, Roviello F, Renieri A,Giordano A.Deciphering the underlying genetic and epigenetic events leading to gastric carcinogenesis.J Cell Phvsiol.2007；211:287-295.).Therefore, adding While strong early gastric caacer diagnosis research, illustrate and gastric cancer caused to occur and the specific mechanism of transfer, establish new early carcinoma of stomach or The diagnostic criteria of metastatic gastric carcinoma, and then new intervening measure is explored, the further recurrence and transfer of gastric cancer are prevented, improves gastric cancer The therapeutic effect of preoperative and postoperative, the survival rate for increasing patient are of great significance to the treatment and prevention of disease.

Generation, the development of gastric cancer are related to the unconventionality expression and tumor suppressor gene functionally inactive of oncogene, and gastric cancer occurs and turns Shifting etc. is related to cell functional disorders, and virus-mediated immune microenvironment changes, multiple links such as matrix destruction, angiogenesis, packet Include multifactor induction, complicated pathologic process (Nieman KM, Kenny HA, the Penicka CV, et that polygenes participates in al.Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth.Nat Med.2011；17:1498-1503.).Therefore, it finds effectively related to gastric cancer generation or transfer Gene expression profile and key gene mark, predict gastric cancer be ensure clinical therapeutic efficacy and improve survival rate it is important Step.

In recent years, with the development of biotechnology, some are research shows that the unconventionality expression of 1ncRNA takes part in tumour The formation of tumour and progress, the 1ncRNA of these unconventionality expressions can be as the early diagnosis of malignant tumour or diving for prognosis evaluation At molecular marker (CN201710127268.5/CN201710112636.9/CN201710522694.9), lncRNA can conduct Molecule, signaling molecule are guided, the forms such as bait molecule and molecule of the skeleton participate in tumor-related gene, epigenetics, signal and lead to Road and crosstalk regulation and control (Xing Z, Lin A, Li C, the et al.1ncRNA directs between them cooperative epigenetic regulation downstream of chemokine signals.Cell.2014； 159:1110-1125.), key effect has been played in tumor development.Therefore, it finds relevant with gastric cancer occurrence and development LncRNA has great importance for the early diagnosis and targeted therapy of gastric cancer.

Invention content

In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a kind of relevant with gastric cancer occurrence and development LncRNA so as to which the diagnose and treat for gastric cancer provides molecular target, realizes the personalized diagnosis and treatment of patient.

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

The present invention provides detect application of the reagent of LINC00941 levels in the product for preparing diagnosis of gastric cancer.

Further, the product includes detecting in sample by sequencing technologies, nucleic acid hybridization technique, nucleic acid amplification technologies The reagent of the expression of LINC00941 genes.

The present invention provides a kind of product of diagnosis of gastric cancer, product includes chip, preparation, kit or nucleic acid film item, production Product include the reagent of LINC00941 levels in detection sample.

Further, the reagent is selected from：

The probe of specific recognition LINC00941；Or

The primer of specific amplification LINC00941.

Further, the primer sequence of specific amplification LINC00941 genes is as shown in SEQ ID NO.1~2.

The present invention provides a kind of pharmaceutical composition, described pharmaceutical composition includes the inhibition of a effective amount of LINC00941 Agent.The inhibitor is selected from：Using LINC00941 or its transcript as target sequence and can inhibit LINC00941 gene expressions or The disturbing molecule of genetic transcription, including：ShRNA (children purpura nephritis), siRNA (siRNA), dsRNA, Microrna, antisense Nucleic acid or the construction that can express or be formed the shRNA, siRNA, dsRNA, Microrna, antisense nucleic acid.

Further, the inhibitor is siRNA, it is preferred that the sequence of siRNA is as shown in SEQ ID NO.13~14.

In the present invention, described pharmaceutical composition is further included and other medicine classes of the inhibitor compatibility and pharmaceutically may be used The carrier and/or auxiliary material of receiving；The drug of the present invention can also be with the drug combination of other treatment gastric cancer, other therapeutic compound It can be administered simultaneously with main active constituent or even be administered simultaneously in same composition.

Application in the product for treating gastric cancer in preparation the present invention provides the inhibitor of LINC00941.

Further, the inhibitor is siRNA, it is preferred that the institute of the sequence of siRNA such as SEQ ID NO.13~14 Show.

Further, the gastric cancer includes primary gastric cancer, metastatic gastric carcinoma.

A kind of method for the candidate substances for screening treatment gastric cancer of body of the present invention, the method includes：

The system expressed or containing LINC00941 genes is handled with substance to be screened；With

Detect the expression of LINC00941 genes in the system；

Wherein, if the substance to be screened can inhibit LINC00941 genes expression (preferably significantly reduce, Such as low more than 20%, preferably low more than 50%；More preferably low more than 80%), then it is treatment gastric cancer to show the candidate substances Candidate substances.The system is selected from：Cell system, subcellular system, solution system, organizational framework, organ systems or animal body System.

The candidate substances include but is not limited to：It is designed for LINC00941 genes or its upstream or downstream gene Disturbing molecule, nucleic acid inhibitor, micromolecular compound etc..

Description of the drawings

Fig. 1 is the expression figure in stomach organization using QPCR detection LINC00941 genes；

Fig. 2 is the expression figure in gastric carcinoma cell lines using QPCR detection LINC00941 genes；

Fig. 3 is the transfected condition figure in stomach cancer cell using QPCR detections LINC00941；

Fig. 4 is the influence figure of MTS methods detection LINC00941 gene pairs proliferation of human gastric cancer cell；

Fig. 5 is the influence figure for being migrated and being invaded to stomach cancer cell using Transwell cells detection LINC00941；Wherein Figure A is the influence figure that LINC00941 migrates stomach cancer cell；Figure B is the influence figure that LINC00941 invades stomach cancer cell.

Specific embodiment

The present invention is after extensive and in-depth study, most wide using current covering database by high throughput method LncRNA chips detect the expression of lncRNA in gastric cancer sample in tumor tissues and cancer beside organism, by bioinformatic analysis, It was found that wherein having the lncRNA of apparent differential expression, its relationship between the occurrence and development of gastric cancer is inquired into, so as to be gastric cancer Diagnosis and targeted therapy find better approaches and methods.By screening, present invention firstly discovers that LINC00941 in gastric cancer Conspicuousness raises, and further confirms that expression quantity of the LINC00941 in stomach organization is significantly high in large sample qPCR verifications In cancer beside organism.The research that this discovery will further enrich incidence gastric cancer mechanism, is also examined for the early diagnosis of gastric cancer and prognosis Survey provides new tumor markers and therapy target.

LINC00941 genes

LINC00941 is to be located at 1 areas 1 of the dyeing galianconism of people No. 12 to take, the LINC00941 in the present invention include wild type, Saltant type or its segment.In an embodiment of the present invention, a kind of nucleotide sequence such as mesh of representative people LINC00941 genes In the preceding public nucleic acid database GeneBank in the world shown in LINC00941 genes (NR_040245.1).The present invention's LINC00941 nucleotide full length sequences or its segment can usually be obtained with PCR amplification method, recombination method or artificial synthesized method .

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 are it will be recognized that since RNA less stable and is being tested in cell In be more vulnerable to nuclease attack, therefore before sequencing usually by RNA reverse transcriptions into DNA.

In general, PCR uses denaturation, primer pair and the annealing of opposite strand and multiple cycles of primer extend, with index side Formula increases the copy number of target nucleic acid sequence；RT-PCR then by reverse transcriptase (RT) for preparing complementary DNA (cDNA) from mRNA, Then by cDNA by PCR amplification to generate multiple copies of DNA；TMA is in the temperature of substantial constant, ionic strength and pH Under the conditions of autocatalytically synthesize multiple copies of target nucleic acid sequence, multiple RNA copies of wherein target sequence are autocatalytically given birth to Into other copy, TMA is optionally included using blocking, part, terminate part and other modified parts, to improve TMA processes Sensitivity and accuracy；LCR uses the two groups of complementary DNA oligonucleotides hybridized with the adjacent area of target nucleic acid.DNA few nucleosides Acid is covalently attached in thermal denaturation, hybridization and the multiple cycles of the repetition of connection by DNA ligase, to generate detectable double-strand Connect oligonucleotide product；SDA uses multiple cycles of following steps：Primer sequence pair and the opposite strand of target sequence move back Fire carries out primer extend to generate (hemiphosphorothioated) of half thiophosphorylation of double-strand under there are dNTP α S Primer extension product, the nicking for the endonuclease mediation that semi-modified restriction enzyme enzyme recognition site carries out and from cutting The polymerase-mediated object extension drawn that mouth 3' ends carry out is put with replacing existing chain and generating for next round primer annealing, nicking and chain The chain changed expands so as to cause the geometry of product.

Nucleic acid hybridization technique in the present invention include but not limited in situ hybridization (ISH), microarray and Southern or Northern traces.In situ hybridization (ISH) be it is a kind of use label complementary DNA or RNA chains as probe with position tissue one Part or slice (original position) or if organize it is sufficiently small if for entirely organize (full organization embedding ISH) in specific DNA or The hybridization of RNA sequences.DNA ISH can be used for determining the structure of chromosome.RNA ISH for measure and position tissue slice or MRNA and other transcripts (for example, ncRNA) in full organization embedding.Usually sample cell and tissue are handled with original position Fixed target transcript, and increase the entrance of probe.Probe hybridizes at high temperature with target sequence, then washes off extra probe. Respectively using autoradiograph, fluorescence microscopy or immunohistochemistry, to using the alkali of radiation, fluorescence or antigenic mark in tissue The probe of disjunction mark note is positioned and is quantified.ISH can also be used two or more to pass through radioactivity or other on-radiation marks The probe of substance markers is remembered, to detect two or more transcripts simultaneously.

Southern and Northern traces are respectively used to detection specific DNA or RNA sequence.Make to extract from sample DNA or RNA fracture, it is separated by electrophoresis on matrix gel, be then transferred on molecular filter.Make filter combine DNA or RNA with and the label probe of sequence of interest complementation hybridize.Detection is attached to the hybridization probe of filter.A kind of change of the program Change form is reverse northern trace, wherein the substrate nucleic acid fixed to film is the set of the DNA fragmentation of separation, and probe is From tissue extraction and the RNA that is marked.

The nucleic acid of non-amplification or amplification can be detected by any conventional means in the present invention.

Chip, preparation, nucleic acid film item, kit

The present invention provides the product of the expression of LINC00941 genes in detection, the product includes (but unlimited In) microarray biochip, preparation, nucleic acid film item or kit.Its chips includes：Solid phase carrier；And it is orderly fixed on described solid Oligonucleotide probe on phase carrier, the oligonucleotide probe specifically correspond to LINC00941 shown in part or Full sequence.

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 preciseness of hybridization conditions, probe energy and with the probe Target polynucleotide combines.Probe can make direct or indirect label, and range 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 connect 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 have usually relative to the specific base sequence 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 pass through PNA (Polyamide nucleic in part of it or whole nucleotides Acid, peptide nucleic acid), LNA (registered trademark, locked nucleic acid, Bridged Nucleic Acid, Cross-linked core Acid), ENA (registered trademark, 2 '-O, 4 '-C-Ethylene-bridged nucleic acids), GNA (Glycerol Nucleic acid, glycerine nucleic acid), the artificial replacement nucleic acid such as TNA (Threose nucleic acid, threose nucleic acid) obtains Polynucleotides.

In the present invention, nucleic acid film item includes substrate and the oligonucleotide probe being fixed in the substrate；The substrate Can be any substrate suitable for immobilized oligonucleotide probe, for example, nylon membrane, nitrocellulose filter, polypropylene screen, sheet glass, Silica gel chip, micro magnetic bead etc..

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 connect with molecular barcode and The probe being fixed on pearl.

The present invention provides a kind of kit, the kit can be used for the expression of detection LINC00941.The kit Specific primer pair including being used to expand LINC00941；Standard DNA template；PCR reaction solution.In a preferred embodiment party In case, the specific primer is to including sense primer and downstream primer, and sequence is as shown in SEQ ID NO.1~2.

Embodiment more preferably, the kit are fluorescent quantificationally PCR detecting kit, and the primer is suitable for The detection of SYBR Green, TaqMan probe, molecular beacon, double cross probe, combined probe.

In a further preferred embodiment, the PCR reaction solution in the kit is fluorescence quantitative PCR reaction solution, And one step include fluorescent dye.

In a further preferred embodiment, the fluorescence quantitative PCR reaction solution includes dNTP, Mg²⁺, Taq enzyme and Buffer buffer solutions, the fluorescent dye are SYBR Green II, and Taq enzyme is thermal starting enzyme.

Inhibitor 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 inhibitor of LINC00941.

The inhibitor of the LINC00941 refers to any substance for reducing LINC00941 gene levels, these substances The present invention is used equally for, the substance useful as the expression for downward LINC00941 genes, so as to be used to treat gastric cancer. For example, inhibitor of the invention can be using LINC00941 genes as target sequence and can inhibit LINC00941 genes Disturbing molecule, including：ShRNA (children purpura nephritis), siRNA (siRNA), dsRNA, Microrna, antisense nucleic acid or energy Express or formed the construction of the shRNA, siRNA, dsRNA, Microrna, antisense nucleic acid.

As a kind of preferred embodiment of the present invention, the inhibitor of the LINC00941 is a kind of LINC00941 specificity SiRNA molecule.As used herein, " siRNA " refers to a kind of short-movie section double stranded rna molecule, can be with same The mRNA of source complementary series is the target specific mRNA of degradation, this process is exactly RNA interference (RNA interference) Process.SiRNA can be prepared into the form of double-strandednucleic acid, it contains, and there are one positive-sense strand and an antisense strand, this two chains Double-strand is only formed under conditions of hybridization.One double-stranded RNA compound can be made by the positive-sense strand that is separated from each other and antisense strand It is standby.Therefore, for example, complementary positive-sense strand and antisense strand are chemical synthesis, and can generate synthesis by anneal thereafter Double-stranded RNA compound.

When screening effective siRNA sequence, the present inventor is best effective so as to find out by largely comparing analysis Segment.The present inventor's design has synthesized a variety of siRNA sequences, and by they respectively by transfection reagent transfect gastric carcinoma cell lines into Row verification, selects the best siRNA of interference effect, further tests, is as a result proved for the siRNA in energy in cellular level It is effective to inhibit the expression of LINC00941 genes and the proliferation of stomach cancer cell in cell.

As a kind of optional mode of the present invention, the inhibitor of the SIGLEC10 can also be a kind of " children purpura nephritis (Small hairpin RNA, shRNA) ", is the non-coding small RNA molecular that can form hairpin structure, children purpura nephritis energy Enough by RNA interference channels come the expression of suppressor.As above-mentioned, shRNA can be expressed by double-stranded DNA template.Double-stranded DNA Template is inserted into a carrier, such as plasmid or viral vectors, is then connected to a promoter carry out table in vitro or in vivo It reaches.ShRNA under the action of DICER enzymes, can be cut into siRNA molecule in eukaryocyte, hence into RNAi approach. " shRNA expression vectors " refers to plasmid of some this fields conventionally used for building shRNA structures, exist on the usual plasmid " Every sequence " and positioned at " intervening sequence " both sides multiple cloning sites or for replace sequence, so as to people can by shRNA (or Analog) corresponding DNA sequence dna be inserted by way of forward and reverse multiple cloning sites or replace thereon for replacing sequence, RNA after DNA sequence dna transcription can form shRNA (Short Hairpin) structure." the shRNA expression vectors " is current It can be bought and obtained, such as some viral vectors by commercially available approach completely.

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, to provide the phenotypic character for selecting the host cell converted, such as kalamycin, gentamicin, tide Mycin, amicillin resistance.

In the present invention, expression vector is 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, The known methods such as DEAE dextran method, microinjection, viral infection, the combination of liposome transfection and cell-membrane permeable peptide.

Term " host cell " is including 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.Preferably, The host cell is eukaryocyte, such as Chinese hamster ovary celI, COS cells.

Pharmaceutical composition

Pharmaceutical composition in the present invention includes the inhibitor of a effective amount of LINC00941, and/or matches with the inhibitor 5 other medicine classes and pharmaceutically acceptable carrier and/or auxiliary material.

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：Active constituent themselves is not necessary, and is not had after applying Excessive 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 used with a variety of methods well known in the art by the inhibitor 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.

Preferably, the means that gene therapy can be used carry out.For example, directly the inhibitor of LINC00941 can be passed through all The methods of such as injecting delivers medicine to subject；Alternatively, the expression list of the inhibitor of LINC00941 can will be carried by certain approach Position (such as expression vector or virus etc. or siRNA or shRNA) is delivered on target spot, and be allowed to the LINC00941 of expression activity Inhibitor, concrete condition need to be depending on the types of the inhibitor, these are well-known to those skilled in the art.

The pharmaceutical composition of the present invention can further include one or more anticancer agents.In specific embodiments, Pharmaceutical composition includes at least one compound for inhibiting LINC00941 gene expressions and at least one chemotherapeutics.For this hair Bright chemotherapeutics, including but not limited to：Micro-pipe activator, alkylating agent, nti-neoplastic antimetabolite, platinum-like compounds, DNA- alkanisations Agent, antitumor antibiotics agent, antimetabolite, microtubule stabilizing agent, tubulin destabilizing agent, hormone antagonist, topology are different Structure enzyme inhibitor, kinases inhibitor, HMG-COA inhibitor, CDK inhibitor, cyclin inhibitors, Guang day albumen Virus, the bacterium of enzyme inhibitor, metal protease inhibitors, antisense nucleic acid, triple helix DNA, aptamer and molecular modification With exotoxin reagent.

Pharmaceutical acceptable carrier may include but be not limited to：Virus, micro-capsule, liposome, nano particle or polymer and its arbitrary group It closes.Relevant delivering supporting agent may include but be not limited to：Liposome, biocompatible polymer are (including natural polymer and synthesis Polymer), lipoprotein, polypeptide, polysaccharide, lipopolysaccharides, artificial viral envelope, inorganic (including metal) particle and bacterium or disease Malicious (such as baculoviral, adenovirus and retrovirus), bacteriophage, sticking grain or plasmid vector.

Drug screening

The present invention provides it is a kind of screen treatment gastric cancer medicament method, i.e.,：

In experimental group, untested compound is added in into cultivating system, and measures the expression of LINC00941；Right According in group, untested compound is added without into same cultivating system, and measures the expression of LINC00941；Wherein, if The expression of LINC00941 is less than control group in experimental group, then illustrates the potential object that the substance to be screened is LINC00941 Matter.

In the present invention, the method further includes：The potential substance obtained to previous step further tests its inhibition The effect of gastric cancer, if test compound has gastric cancer significant inhibition, it is the potential for the treatment of gastric cancer to illustrate the compound Substance.

The cultivating system includes but is not limited to cell system, subcellular 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..

When regarding the compound detached by the screening technique of the present invention as medicament administration in people or other mammals, Including but not limited to mouse, rat, cavy, rabbit, cat, dog, sheep, pig, ox, monkey, baboon, chimpanzee when, the compound of separation It can directly apply or various dosage forms can be configured to using known process for preparing medicine.For example, as needed, it is described Drug can be used as sugar coated tablet, capsule, elixir and microcapsules to be administered orally；Or it is subjected to water or any other drug Liquid dosage into sterile solution or suspension, the non-oral application in the form of injection.It for example, can be by compound with general Unit dosage forms (unit dose) and pharmaceutically acceptable carrier or medium needed for the medicament administration mode of receiving are blended in one Rise, the carrier or medium include but not limited to sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, Stabilizer, flavoring agent, excipient (excipient), medium (vehicle), preservative, adhesive etc..According to these preparations The content of middle active ingredient can obtain suitable dosage within the specified range.

Statistical analysis

In a specific embodiment of the present invention, experiment all completed according to being at least repeated 3 times, result data be all with The mode of mean+SD represents, using SPSS18.0 statistical softwares come for statistical analysis, difference between the two It is different to be examined using t, it is believed that work as P<There is statistical significance when 0.05.

It being further illustrated the present invention with reference to specific embodiment, the embodiment of the present invention is only used for explaining the present invention, It is not intended to limit protection scope of the present invention.

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Embodiment 1 is screened and the relevant gene marker of gastric cancer

1st, sample collection

8 gastric cancer cancer beside organisms and gastric cancer tissue sample are collected respectively, and with normal gastric tissue 3, all cases are in hand It is preoperative not receive chemotherapy and radiation, all patients informed consent, and obtain and pass through the same of the committee of organizational ethics Meaning.

2nd, the preparation of RNA sample

The extraction of tissue RNA is carried out using the tissue RNA extracts kits of QIAGEN, the specific steps of by specification carry out Operation.

3rd, total serum IgE is quantitative and purity analysis

OD value of the total serum IgE at 280nm and 260nm is measured using Bio-Red ultraviolet specrophotometers, works as OD₂₆₀/ OD₂₈₀Value think that the purity of total serum IgE is reliable at 1.8~2.0, for the experiment of next step.

4th, lncRNA expression chips are analyzed

Stomach organization and cancer beside organism are detected using the Arraystar Human 1ncRNA Array of Arraystar companies The difference of middle lncRNA express spectras.

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 LINC00941 in patients with gastric cancer, with cancer beside organism and normal gastric mucosa It compares, significantly about 10.5 times of the up-regulation of the expression in cancerous tissue.

The differential expression of 2 QPCR sequence verification LINC00941 genes of embodiment

1st, large sample QPCR verifications are carried out to LINC00941 gene differential expressions.According to the sample collection side in embodiment 1 Formula selects gastric cancer cancer beside organism and stomach organization each 50 and normal stomach tissue 10.

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 It cools down on ice.Add in 5 × reaction buffer, 4 μ l, 1 μ l, 10mM dNTP mixed liquors of RNase inhibitor (20U/ μ l) 2 μ l, AMV 1 μ l of reverse transcriptase (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 LINC00941 genes and GAPDH genes in Genebank, by winning Mai De biotech firms synthesize.Specific primer sequence is as follows：

LINC00941 genes：

Forward primer is 5 '-ATAGGCATACTGACAATA-3 ' (SEQ ID NO.1)；

Reverse primer is 5 '-GCTCATTACATCATCAAT-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 cycles, 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 packets analysis LINC00941 in R language, it is empty to calculate the accurate confidence of binomial Between, draw ROC curve.

5th, result

The results are shown in Figure 1 by QPCR, and compared with gastric cancer cancer beside organism, LINC00941 is raised in expression in gastric carcinoma, poor It is different that there is statistical significance (P<0.05)；Positive rate=up-regulated expression number of cases/always detects number of cases × 100%=47/50= 94%, LINC00941 is prompted to can be applied to the diagnosis of gastric cancer, and ROC curve analysis is prompted it is found that AUC is up to 0.8923 The diagnosis that LINC00941 is applied to gastric cancer has higher accuracy.

Expressions of 3 LINC00941 of embodiment in gastric carcinoma cell lines

1st, cell culture

People immortalizes gastric epithelial cell system GES-1, human gastric cancer cell line HGC-27, MGC-803, AGS (are purchased from wide Your bio tech ltd of state Rider) the RPMI1640 cultures containing 10% fetal calf serum and 1%P/S based on 37 DEG C, 5% CO₂, relative humidity be 90% incubator in cultivate.Change within 2-3 days liquid 1 time, it is conventional using 0.25% trypsase containing EDTA Had digestive transfer culture takes the cell in exponential phase for testing.

2nd, the extraction of RNA and concentration mensuration

Cell total rna is extracted using the cell RNA extracts kit of QIAGEN, concrete operations are with reference to specification.

3rd, QPCR specific steps are the same as embodiment 2

4th, result

The results are shown in Figure 2, and compared with gastric epithelial cell, LINC00941 genes are in stomach cancer cell HGC-27, MGC- 803rd, significantly up-regulation is expressed in AGS, difference has statistical significance (P<0.05), selection HGC-27 cells carry out subsequent reality It tests, effects of the research LINC00941 to stomach cancer cell.

The silence of 4 LINC00941 genes of embodiment

1st, cell culture step is the same as embodiment 3

2nd, the design of siRNA

For the sequence design siRNA of LINC00941 genes, the sequence such as SEQ ID NO.5 of negative control siRNA-NC Shown in~6；The sequence of experimental group siRNA1~4 is as shown in SEQ ID NO.7~14.

3rd, it transfects

HGC-27 cells are pressed 2 × 10⁵/ hole is inoculated into six porocyte culture plates, in 37 DEG C, 5%CO₂It is thin in incubator Born of the same parents cultivate for 24 hours；In without DMEM culture mediums dual anti-, containing 10%FBS, transfect and (be purchased from according to lipofectamine 3000 Invitrogen companies) specification transfection.

Experiment be divided into blank control group (HGC-27), negative control group (siRNA-NC) and experimental group (siRNA1, SiRNA2, siRNA3, siRNA4), the sequence of wherein negative control group siRNA and LINC00941 genes is a concentration of without homology 20nM/ holes, while transfected respectively.

4th, QPCR detects the transcriptional level of LINC00941 genes

The extraction of 4.1 cell total rnas

The total serum IgE in cell is extracted using QIAGEN cell RNAs extracts kit, specific steps refer to specification.

4.2 reverse transcription steps are the same as embodiment 2.

4.3QPCR amplification steps are the same as embodiment 2.

5th, result

As a result such as Fig. 3 is shown, compared with HGC-27 and transfection zero load siRNA-NC groups, experimental group (siRNA1~4) can drop The effect of the level of low LINC00941, wherein siRNA4 is the most notable, therefore siRNA4 is selected to carry out subsequent experimental.

The influence of 5 LINC00941 gene pairs proliferation of human gastric cancer cell of embodiment

Detection LINC00941 gene pairs proliferation of human gastric cancer cell capacities are tested using MTS.

1st, cell culture and transfection procedure are the same as embodiment 4.

2nd, cell is resuspended after pancreatin digests, counts for the cell of each group processing, and adjustment cell concentration is l × 10⁵/ ml is pressed The density in 100 μ l/ holes is inoculated in 96 orifice plates, i.e., is 1 × 10 per hole cell number⁴It is a.

3rd, it after cell reaches corresponding detection time point (0d, for 24 hours, 48h, 72h, 96h), is added in by 10 μ L/ holes Mono- Solution Cell Proliferation detection (MTS) reagents of CelITiter96AQ, micro oscillator vibrate 1~2min；It is placed in 5%CO₂、37 DEG C cell incubator in be incubated 4h.

4th, microplate reader read plate measures its absorbance (A) value in 490nm wavelength.

5th, result

The results are shown in Figure 4, and compared with the control, the vitro growth rates of experimental group (siRNA4) are significantly lower than control group The speed of growth of cell, difference have statistical significance (P<0.05), the above results show to reduce the expression inhibiting of LINC00941 The proliferation of stomach cancer cell prompts LINC00941 that can be applied to the treatment of gastric cancer as molecular target.

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, are gently sucked 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 Nature enemy 12-24h, pancreatin digestion centrifugation, is resuspended using serum free medium, adjusts cell density For 5 × l0⁵A/ml.

3rd, cell inoculation

200 μ l of cell suspension is taken to be added in Transwell cells, room adds in 500 DMEMs of the μ l containing FBS under 24 orifice plates Culture medium.Cell is put into cell incubator and is cultivated for 24 hours.

4th, it dyes

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 decreased significantly, and as a result say The reduction of bright LINC00941 levels can inhibit the migration and invasion of gastric cancer, and LINC00941 is prompted target can be used as to be applied to stomach Metastasis of cancer and the treatment of invasion.

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.

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Claims (10)

1. detect application of the reagent of LINC00941 levels in the product for preparing diagnosis of gastric cancer.

2. application according to claim 1, which is characterized in that the product includes hybridizing skill by sequencing technologies, nucleic acid The reagent of the expression of LINC00941 genes in art, nucleic acid amplification technologies detection sample.

3. a kind of product of diagnosis of gastric cancer, product includes chip, preparation, kit or nucleic acid film item, which is characterized in that product packet Include the reagent of LINC00941 levels in detection sample.

4. product according to claim 3, which is characterized in that the reagent includes：

The probe of specific recognition LINC00941；Or

The primer of specific amplification LINC00941.

5. product according to claim 4, which is characterized in that the primer sequence of specific amplification LINC00941 such as SEQ Shown in ID NO.1~2.

6. a kind of pharmaceutical composition, which is characterized in that described pharmaceutical composition includes the inhibitor of a effective amount of LINC00941.

7. pharmaceutical composition according to claim 6, which is characterized in that the inhibitor is siRNA, it is preferred that siRNA Sequence as shown in SEQ ID NO.7~8.

8. application of the pharmaceutical composition in the product for preparing treatment gastric cancer described in claim 6 or 7.

9. application according to claim 8, which is characterized in that the gastric cancer includes primary gastric cancer, metastatic gastric carcinoma.

A kind of 10. method for the candidate substances for screening treatment gastric cancer, which is characterized in that the method includes：

The system expressed or containing LINC00941 genes is handled with substance to be screened；With

Detect the expression of LINC00941 genes in the system；

Wherein, if the substance to be screened can inhibit the level of LINC00941 genes, show that the substance to be screened is to control Treat the candidate substances of gastric cancer.