CN106701986B - Application of the molecular marker in gastric cancer diagnosis and treatment - Google Patents

Abstract

The invention discloses application of the molecular marker in gastric cancer diagnosis and treatment, and specifically the lncRNA is ENSG00000228742, are in specificity overexpression in stomach organization.Experiments have shown that, the expression of ENSG00000228742 is reduced by specificity, the proliferation of stomach cancer cell can be effectively inhibited, prompt ENSG00000228742 can be used as the auxiliary diagnostic index of early gastric caacer diagnosis, and the expression of interference ENSG00000228742 gene can become the new target drone for the treatment of gastric cancer.

Description

Application of the molecular marker in gastric cancer diagnosis and treatment

Technical field

The invention belongs to biomedicine fields, are related to application of the molecular marker in gastric cancer diagnosis and treatment, and specific described point Sub- marker is ENSG00000228742.

Background technique

Gastric cancer is one of most common malignant tumour of alimentary canal, although within the scope of world in recent years the disease incidence of gastric cancer and The death rate is declined, but still occupies the 2nd and the 3rd of malignant tumour respectively, and the Clinics and Practices of gastric cancer are still oncologist The research hotspot paid close attention to jointly.China is the high-incidence country of gastric cancer, and the morbidity and mortality of China's gastric cancer occupy the world for a long time Forefront.Gastric cancer can be divided into early carcinoma of stomach (early gastric cancer, EGC) and advanced gastric carcinoma (advanced Gastric cancer, AGC), wherein early carcinoma of stomach refers to the gastric cancer for being confined to mucous layer and submucosa.Early carcinoma of stomach prognosis is good Good, operability is lower than 5%~10%, and 5 years survival rates are up to 90% or more, and 5 years survival rates of patients with advanced gastric cancer are still insufficient 20%, therefore the early diagnosis of gastric cancer, early treatment play a significant role improvement patient's prognosis, the reduction death rate.

Currently, the control strategy of national tumor disease turns to the prevention of active from passively treatment and early stage knows Not, for gastric cancer, it is preferred that emphasis is the discovery of precancerous lesion and the identification of people at highest risk, while gastroscope is improved to early carcinoma of stomach And the recall rate of precancerous lesion.And the early diagnosis rate of China's gastric cancer and advanced country be there are larger gap, overall gastric cancer early diagnosis rate is low In 10%, most patients with gastric cancer have been in progressive stage when making a definite diagnosis, treatment is costly, and prognosis is bad, are national health thing Industry brings larger burden, needs to be improved.

The difficult point of gastric cancer early diagnosis is that early carcinoma of stomach patient most without specific clinical symptoms and sign, may only show For non-specific epigastric discomfort, and sensibility that the tumor markers such as common CEA, CA19-9 diagnose early carcinoma of stomach and special Property is lower.Therefore new biological targets are found for the canceration monitoring of gastric cancer and are intervened, it is urgently to be resolved to become gastric cancer prevention and treatment The problem of.

LncRNA be a kind of endogenous, fragment length be greater than 200 core former times acid, lack complete special open reading frame and RNA molecule without coding protein ability, it is most to be transcribed by rna plymerase ii and generated through variable sheer.LncRNA and tumour In close relations, discovery has the expression of lncRNA and adjusts different in the kinds of tumors such as liver cancer, breast cancer, leukaemia and colon cancer Often.A variety of lncRNA such as HEIR, HOTAIR, H19 and ANRIL have the function of proto-oncogene or promotion metastases, and The lncRNA such as MEG3 and PTCSC3 then have the function of tumor suppressor gene, above-mentioned it turns out that lncRNA is in tumorigenesis process In all have important function.LncRNA relevant to gastric cancer occurrence and development is found, prevention and treatment and Mechanism Study for gastric cancer all have There is important meaning.

Summary of the invention

In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a kind of molecule marks that can be used for gastric cancer diagnosis and treatment Will object, molecular marker provided by the present invention are a kind of lncRNA, which expresses up-regulation in patients with gastric cancer, can be used as Potential molecular target is used for the diagnosing and treating of gastric cancer.

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

The present invention provides the purposes of ENSG00000228742 gene, it is used to prepare prevention or treats the medicine group of gastric cancer Close object.

Further, described pharmaceutical composition includes the lower adjustment of ENSG00000228742.Wherein, the lower adjustment energy The expression of ENSG00000228742 gene is lowered at the genetic level；The lower adjustment of the ENSG00000228742 is selected from: with ENSG0000018480 gene or its transcript are target sequence and are able to suppress ENSG0000018480 gene expression or gene turn The disturbing molecule of record, comprising: shRNA (children purpura nephritis), siRNA (siRNA), dsRNA, Microrna, antisense nucleic acid, or It can express or be formed the construction of the shRNA, siRNA, Microrna, antisense nucleic acid.

Further, the lower adjustment is selected from the group the siRNA:SEQ ID NO.8 of sequence, SEQ ID NO.9.

The present invention provides a kind of for preventing or treating the lower adjustment of the ENSG00000228742 of gastric cancer, the downward Agent is siRNA.

The present invention provides a kind of for preventing or treating the pharmaceutical composition of gastric cancer, and described pharmaceutical composition contains:

The lower adjustment of ENSG00000228742 recited above；With

Pharmaceutically acceptable carrier.

The present invention provides a kind of purposes of ENSG00000228742 gene, for screening prevention or treating the latent of gastric cancer In substance.

The present invention provides a kind of screening prevention or the methods of the potential substance for the treatment of gastric cancer, which comprises

The system expressed or containing ENSG00000228742 gene is handled with candidate substances；With

Detect the expression of ENSG00000228742 gene in the system；

Wherein, if the candidate substances can reduce the expression or activity (preferably significant drop of ENSG00000228742 gene It is low, such as low 20% or more, preferably low 50% or more, more preferably low 80% or more) then shows that the candidate substances are preventions or control Treat the potential substance of gastric cancer.

Further, the system includes but is not limited to: cell system, subcellular system, solution system, organizer System, organ systems or animal system.

The present invention provides a kind of purposes of ENSG00000228742 gene, are used to prepare the product of diagnosis of gastric cancer.

Further, the product includes chip, preparation or kit.

Further, the chip includes the probe of specific recognition ENSG00000228742；The kit includes special Property amplification ENSG00000228742 primer or specific recognition ENSG00000228742 probe or chip.

Wherein, the chip can be used for detect including ENSG00000228742 gene multiple genes (for example, with The relevant multiple genes of gastric cancer) expression；The kit can be used for detecting including ENSG00000228742 gene Multiple genes (for example, multiple genes relevant to gastric cancer) expression.

The advantages of the present invention:

Present invention firstly discovers that the differential expression of ENSG00000228742 is related to the occurrence and development of gastric cancer, pass through inspection Survey the expression of ENSG00000228742 in subject's sample, it can be determined that whether subject suffers from gastric cancer, to instruct clinical doctor Teacher provides prevention scheme or therapeutic scheme to subject.

Present invention finds a kind of new molecular marker-ENSG00000228742 of gastric cancer, using molecular marker come Prevention or treatment gastric cancer, it is more sensitive, special.

Detailed description of the invention

Fig. 1 is the expression figure using QPCR detection ENSG00000228742 gene in stomach organization；

Fig. 2 is the expression figure using QPCR detection ENSG00000228742 in stomach cancer cell；

Fig. 3 is using QPCR detection ENSG00000228742 gene silencing to the gene expression dose in stomach cancer cell Influence diagram；

Fig. 4 is the influence diagram with MTS method detection ENSG00000228742 gene pairs proliferation of human gastric cancer cell.

Specific embodiment

The present invention obtains lncRNA relevant to gastric cancer after extensive and in-depth study, by lncRNA cDNA microarray, Have found that specificity overexpression is presented in ENSG00000228742 in gastric cancer for the first time.It is demonstrated experimentally that by specifically reducing The expression of ENSG00000228742 can effectively inhibit the proliferation of stomach cancer cell, prompt detection The expression of ENSG00000228742 gene can become one of the auxiliary diagnostic index of early gastric caacer diagnosis, interference ENSG00000228742 gene expression can become the new way of curing gastric cancer.

Molecular marker

Term " molecular marker " is its expression in tissue or cell and normal or healthy cell or tissue Expression compares any gene to change.

It would be recognized by those skilled in the art that practicability of the invention is not limited to marker gene of the invention The gene expression of any specific variants is quantified.As unrestricted example, marker gene can have SEQ ID NO.1 Specified nucleotide sequence.In some embodiments, have and the same or similar cDNA of listed sequence at least 85% Such as above-mentioned listed sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of sequence or at least 99% the same or similar cDNA sequence.

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

In some embodiments, the expression of biomarker is detected on transcriptional level.Hybridize skill using nucleic acid A variety of methods that art carries out specific DNA and RNA measurement are known to the skilled in the art.Certain methods are related to being separated by electrophoresis (for example, the Southern trace for detecting DNA and Northern trace for detecting RNA), but can also be unfavorable With the measurement (for example, passing through Dot blot) for carrying out DNA and RNA in the case where electrophoretic separation.Genomic DNA is (for example, come from People) Southern trace can be used for screening restriction fragment length polymorphism (RFLP), influence polypeptide of the present invention to detect The presence of inherited disorder.It can detecte the RNA of form of ownership.

Pharmaceutical composition

Discovery based on the present inventor, the present invention provides a kind of purposes of ENSG00000228742 gene, are used to prepare The pharmaceutical composition of prevention or treatment gastric cancer.Wherein described pharmaceutical composition includes under a effective amount of ENSG00000228742 It adjusts, and/or its other medicine class and pharmaceutically acceptable carrier and/or auxiliary material with the promotor compatibility.

As used in the present invention, the lower adjustment of ENSG00000228742 includes but is not limited to inhibitor, blocking agent, nucleic acid Mortifier etc., as long as the expression of ENSG00000228742 gene can be lowered at the genetic level.

As a kind of preferred embodiment of the invention, the lower adjustment of the ENSG00000228742 is a kind of The siRNA of ENSG00000228742 specificity." siRNA " refers to a kind of short-movie section double stranded rna molecule, Can be using the mRNA of homologous complementary sequence as 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 a positive-sense strand and one anti- Adopted chain, this two chains only form double-strand under conditions of hybridization.One double-stranded RNA compound can be by the positive-sense strand that is separated from each other It is prepared with antisense strand.Therefore, for example, complementary positive-sense strand and antisense strand are chemical synthesis, and can pass through annealing thereafter Hybridization, generates the double-stranded RNA compound of synthesis.

When screening effective siRNA sequence, the present inventor is by largely comparing analysis, to find out optimal effective 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 verifying, as a result detects 2 kinds of preferable disturbing molecules of interference effect, they are respectively provided with SEQ ID NO.8, SEQ IDNO.9 Shown in sequence, further cellular level test, as a result prove for cell experiment inhibit efficiency it is very high.

Nucleic acid inhibitor of the invention such as siRNA can be with chemical synthesis, can also be by a recombinant nucleic acid structure Expression cassette is prepared after being transcribed into single stranded RNA.The nucleic acid inhibitors such as siRNA, can be by using transfection reagent quilt appropriate It is transported into the cell, or multiple technologies known in the art also can be used and be transported into the cell.

As a kind of optional way of the invention, the lower adjustment of the ENSG00000228742 is also possible to a kind of " small Hairpin RNA ", is the non-coding small RNA molecular for being capable of forming hairpin structure, children purpura nephritis can by RNA interference channel come The expression of suppressor.As above-mentioned, shRNA can be expressed by the double-stranded DNA template of the transcript of coding needs.Coding needs The double-stranded DNA template of transcript be inserted into a carrier, such as plasmid or viral vectors, be then connected in vitro or in vivo One promoter is expressed.ShRNA under the action of DICER enzyme, can be cut into siRNA molecule in eukaryocyte, Hence into RNAi approach." shRNA expression vector " refers to plasmid of some this fields conventionally used for constructing shRNA structure, leads to In the presence of " intervening sequence " and positioned at the multiple cloning sites on " intervening sequence " both sides or for replacing sequence on the normal plasmid, thus people Can by shRNA (or the like) corresponding DNA sequence dna is inserted into multiple cloning sites or replacement by way of forward and reverse Thereon for replace sequence, the DNA sequence dna transcription after RNA can form shRNA (Short Hairpin) structure.Described " shRNA expression vector " can be bought by commercially available approach completely obtain at present, such as some viral vectors.

Expression vector can be various carriers known in the art, and such as commercially available carrier including plasmid, are bitten clay Thallus, virus etc..Electroporation, calcium phosphate method, liposome method, DEAE can be used in importing of the expression vector into host cell Glucan method, microinjection, virus infection, the known method such as combination of liposome transfection and cell-membrane permeable peptide.

Those skilled in the art can use the well known method expression vector required in the building present invention.These methods Including recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc..The expression vector preferably include one or Multiple selected markers, to provide the phenotypic character for selecting the host cell of conversion, as kalamycin, celebrating are big mould Element, hygromycin, amicillin resistance.

In the present invention, term " host cell " includes prokaryotic cell and eukaryocyte.The example of common prokaryotic host cell Attached bag includes Escherichia coli, hay bacillus etc..Common eukaryotic host cell includes that yeast cells, insect cell and mammal are thin Born of the same parents.Preferably, the host cell is eukaryocyte, such as Chinese hamster ovary celI, COS cell.

Term " effective quantity " refers to can generate function or active and can be connect by people and/or animal to 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 medicament carriers some in this way: themselves not being necessary active constituent, and does not have excessive toxicity after applying.Properly Carrier be well known to those of ordinary skill in the art.Pharmaceutically acceptable carrier can contain liquid in the composition, such as Water, salt water, buffer.In addition, there is likely to be complementary substance in these carriers, as filler, lubricant, glidant, Wetting agent or emulsifier, pH buffer substance etc..Lipofectamine can also be contained in the carrier.

In the present invention, can using a variety of methods well known in the art by the promotor or its open gene or Its pharmaceutical composition delivers medicine to mammal.Including but not limited to: subcutaneous injection, intramuscular injection, for percutaneous administration of, administer locally to, Implantation, sustained release are given；Preferably, the administration mode is that non-bowel is given.

Preferably, the means that gene therapy can be used carry out.For example, can be directly by the lower adjustment of ENSG00000228742 Subject is delivered medicine to by the methods of such as injecting；It is lowered alternatively, ENSG00000228742 can will be carried by certain approach The ceneme (such as expression vector or virus etc. or siRNA or shRNA) of agent is delivered on target spot, and concrete condition need to regard institute Depending on the type for the lower adjustment stated, these are well-known to those skilled in the art.

The effective quantity of the lower adjustment of ENSG00000228742 of the present invention can be with the mode and disease to be treated of administration Severity etc. of disease and change.Preferred a effective amount of selection can by those of ordinary skill in the art depending on various factors Lai It determines (such as passing through clinical test).The factor includes but is not limited to: the lower adjustment of the ENSG00000228742 Pharmacokinetic parameter such as bioavailability, metabolism, half-life period etc.；Patient institute the severity of disease to be treated, patient Weight, immune state, the approach of administration of patient etc..For example, can be given once daily several times by an urgent demand for the treatment of situation Separated dosage, or dosage is reduced pari passu.

Drug screening

The present invention provides purposes of the ENSG00000228742 in screening human gastric cancer diagnosis and treatment drug.That is: candidate substances are used The system of processing expression ENSG00000228742；With the expression or activity for detecting ENSG00000228742 in the system；If The candidate substances can lower the expression or activity of ENSG00000228742, then show that the candidate substances are to inhibit diving for gastric cancer In substance.The system of the expression ENSG00000228742 for example can be cell (or cell culture) system, described Cell can be the cell of endogenous expression ENSG00000228742；Or it can be the thin of recombinant expression ENSG00000228742 Born of the same parents.The system of the expression ENSG00000228742 can also be subcellular system, solution system, organizational framework, organ body System or animal system (such as animal model, the preferably animal model of non-human mammal, such as mouse, rabbit, sheep, monkey)) etc..

The expression of ENSG00000228742 gene in the system of test group is detected, and compared with the control group, wherein described Control group is the expression for not adding the candidate substances or the system containing ENSG00000228742 gene；If in test group The expression of ENSG00000228742 gene is statistically lower than control group, indicates that the substance is prevention or treatment gastric cancer Potential substance.

Chip

The lncRNA chip of the invention includes: solid phase carrier；And it is orderly fixed on the solid phase carrier Oligonucleotide probe, the oligonucleotide probe some or all of specifically correspond to shown in ENSG00000228742 Sequence.

Specifically, can lncRNA according to the present invention, design suitable probe, be fixed on solid phase carrier, shape At " oligonucleotide arrays "." oligonucleotide arrays " refer to (addressable i.e. with distinctive with addressable point The position that address is characterized) array, each addressable point is containing a coupled characteristic oligonucleotides.According to It needs, oligonucleotide arrays can be divided into multiple sub- battle arrays.

Term " probe " refers to can be with the molecule in conjunction with the particular sequence of another molecule or subsequence or other parts.Unless another It points out, term " probe " is often referred to match and another polynucleotides (often referred to as " target polynucleotide ") by complementary base In conjunction with polynucleotide probes.Lack sufficient sequence complementarity according to the stringency of hybridization conditions, probe energy and with the probe Target polynucleotide combines.Probe can make direct or indirect 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.

Term " complementary " or " complementarity " are for referring to through the relevant polynucleotides of basepairing rule (that is, nucleosides The sequence of acid).For example, sequence " 5'-A-G-T-3' " is complementary with sequence " 3'-T-C-A-5' ".Complementarity can be " part ", The base of only few of them nucleic acid is matched according to basepairing rule.Alternatively, can also exist between nucleic acid " complete " or It is " total " complementary.Complementarity between nucleic acid chains between nucleic acid chains hybridization efficiency and intensity there is significant impact. This amplified reaction and rely on nucleic acid between combination detection method in be even more important.

Term " stringency ", which is used to refer to, carries out the locating condition of nucleic acid hybridization: temperature, ionic strength and other compounds The presence of (such as organic solvent).Under " low stringency condition ", nucleic acid sequence of interest will with its exact complementary sequence, have The individually sequence, closely related sequence (for example, sequence with 90% or more high homology) of base mispairing and only portion Divide sequence (for example, sequence with 50-90% homology) hybridization of homology.It is of interest under " medium stringent conditions " Nucleic acid sequence by only with its exact complementary sequence, the sequence with single base mispairing and closely related sequence (for example, 90% or more high homology) hybridization.Under " high stringency condition ", nucleic acid sequence general of interest and its exact complementary sequence (condition depending on such as temperature) there is the sequence of single base mispairing to hybridize.In other words, under the conditions of high stringency, Temperature can be increased to exclude to hybridize with the sequence with single base mispairing.

It is embedding that oligonucleotide probe in the present invention for ENSG00000228742 gene can be DNA, RNA, DNA-RNA Fit, PNA or other derivatives.There is no limit as long as complete specific hybrid and purpose nucleotides sequence for the length of the probe Column specific binding, any length are ok.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Together Sample, the length of the probe can be grown to 60,80,100,150,300 base-pairs or longer or even whole genes.Due to difference Probe length have different influences to hybridization efficiency, signal specificity, the length of the probe is typically at least 14 bases Right, longest is usually no more than 30 base-pairs, and the length complementary with purpose nucleotide sequence is best with 15-25 base-pair.Institute Probe self-complementary sequences are stated most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.

The various common used materials in genetic chip field, such as, but not limited to nylon can be used in heretofore described solid phase carrier Film, slide or silicon wafer, unmodified slide, plastic sheet through active group (such as aldehyde radical, amino) modification etc..

The conventionally fabricated side of biochip known in the art can be used in the preparation of the ENSG00000228742 chip Method.For example, 5 ' ends of probe are gone here and there containing amido modified poly- dT if solid phase carrier is using modification slide or silicon wafer, it can Oligonucleotide probe is configured to solution, then point sample instrument is used on modification slide or silicon wafer, to be arranged in its point scheduled Sequence or array, are then fixed by standing overnight, so that it may obtain lncRNA chip of the invention.

Kit

The present invention provides a kind of kit, the kit can be used for detecting the expression of ENSG00000228742.

Preferably, in the preparation or kit also containing for labeled RNA sample marker, and with the mark Remember the corresponding substrate of object.In addition, needed for may also include in the kit for extracting RNA, PCR, hybridization, colour developing etc. Various reagents, including but not limited to: extract, amplification liquid, hybridization solution, enzyme, comparison liquid, developing solution, washing lotion etc..In addition, described Kit in further include operation instructions and/or chip image analysis software.

Drug in the present invention can also be with individual composition or the dosage form different from main active constituent Individually give other therapeutic compounds.The Fractional of main component can be administered simultaneously with other therapeutic compounds, and Other dosage can be administered alone.It over the course for the treatment of, can be according to the severity of symptom, the frequency of recurrence and therapeutic scheme Physiologic response, adjust the dosage of pharmaceutical composition of the present invention.

Drug of the invention can also with the drug combination of other treatment gastric cancer, other therapeutic compound can with it is main Active constituent is administered simultaneously, or even is administered simultaneously in same composition.

Term " sample " is with the use of its broadest sense.In a kind of meaning, it is intended that including what is obtained from any source Sample or culture, and biology and environmental samples.Biological sample is available from animal (including people) and covers liquid, solid, group It knits and gas.Biological sample includes blood product, blood plasma, serum etc..It is applicable in however, such sample should not be construed as limitation In sample type of the invention.

The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.

Embodiment 1 screens gene marker relevant to gastric cancer

1, sample collection

8 gastric cancer cancer beside organisms are collected respectively and gastric cancer tissue sample all cases do not receive chemotherapy before surgery and put It treats, all patients informed consent, and obtains the agreement for passing through the committee, organizational ethics.

2, the preparation of RNA sample (utilizes E.Z.N.A.Kit is operated)

1) clinical samples are taken out from -80 DEG C of ultra low temperature freezers, is placed in and thaws on ice.About 20mg tissue is weighed to be placed in In 2mL centrifuge tube, it is placed in addition 1mL Trizol on ice.Using hand-held, tissue is sufficiently homogenized to without solid by equal pulp grinder automatically Body is stored at room temperature 10min.

2) 13000rpm, 4 DEG C of centrifugation 10min.

3) with pipettor, carefully Aspirate supernatant is transferred in the l.5mL centrifuge tube of clean RNase-Free.

4) methylene chloride of 0.2mL is added, shakes 15s with vortex mixer, is stored at room temperature 10min.

5) 3000rpm, 4 DEG C of centrifugation 10min.

6) water phase carefully is drawn with pipettor and be transferred in the centrifuge tube of 1.5mL RNase-free.

7) isopropanol with the medium volume of step 6) is added, with vortex mixer concussion 4s to precipitate total serum IgE, is stored at room temperature 30min。

8) 13000rpm is centrifuged 10min at 4 DEG C, abandons supernatant.75% alcohol 1mL is added, mixes again.13000rpm It is centrifuged 10min at 4 DEG C, abandons supernatant.

9) it is placed in and dries 10min at room temperature, 12 μ L RNase-Free water dissolution RNA is added and is placed in spare on ice.

3, total serum IgE is quantitative and purity analysis

Use OD value of the Bio-Red ultraviolet specrophotometer measurement total serum IgE at 280nm and 260nm.The amount of RNA By 1OD_260nm- 40 μ g RNA are calculated, and work as OD₂₆₀/OD₂₈₀Value think at 1.8~2.0 total serum IgE purity be it is reliable, can Experiment for next step.

4, lncRNA expression chip is analyzed

Stomach organization and cancer beside organism are detected using the Arraystar Human 1ncRNA Array of Arraystar company The difference of middle lncRNA express spectra.Arraystar 1ncRNA chip designs the oligonucleotides that probe is 60nt length, these length Oligonucleotide probe available highly sensitive and specificity gedanken experiment result under the stringent hybridization conditions of height.For Every sequence all devises a plurality of probe, increases the reliability of signal.

5, data are analyzed

Chip results are analyzed using Agilent GeneSpring software, screening expression quantity has significant difference The lncRNA of (standard differs 2 times or more, and p < 0.05 with the expression quantity by cancer in cancer for the lncRNA).

6, result

The results show that expression quantity of the ENSG00000228742 in stomach organization is significantly higher than the expression in cancer beside organism Amount.

The differential expression of embodiment 2QPCR sequence verification ENSG00000228742 gene

1, large sample QPCR verifying is carried out to ENSG00000228742 gene differential expression.According to the sample in embodiment 1 Collection mode selects gastric cancer cancer beside organism and stomach organization each 50.

2, RNA extraction step is as described in Example 1.

3, reverse transcription

1) reaction system:

1 μ l of RNA template, 1 μ l of random primer, distilled water add to 12 μ l, mix, then slow-speed of revolution centrifugation, 65 DEG C of 5min are put It cools down on ice.

Following ingredients are added in 12 μ l reaction solutions in continuation:

5 × reaction buffer, 4 μ l, 1 μ l, 10mM dNTP mixed liquor of RNase inhibitor (20U/ μ l), 2 μ l, AMV reverse transcription 1 μ l of enzyme (200U/ μ l)；It mixes well and carries out centrifugal treating；

2) reverse transcription reaction condition

25 DEG C of 5min, 42 DEG C of 60min, 70 DEG C of 5min, 4 DEG C of heat preservation 60min.

3) polymerase chain reaction

Design of primers:

According to the sequence design QPCR amplimer of ENSG00000228742 gene in Genebank and GAPDH gene, by The synthesis of Shanghai bioengineering Co., Ltd.Specific primer sequence is as follows:

ENSG00000228742 gene:

Forward primer is 5 '-CTCTTCAGCACAGAATCAG-3 ' (SEQ ID NO.2)；

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

GAPDH gene:

Forward primer is 5 '-AATCCCATCACCATCTTCCAG-3 ' (SEQ ID NO.4)；

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

Prepare PCR reaction system:

2 × qPCR mixed liquor, 12.5 μ l, 2.0 μ l of gene primer, reverse transcription product 2.5 μ l, ddH₂O 8.0μl。

PCR reaction condition: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 60s) × 40 circulations, 60 DEG C of 5min extensions. Using SYBR Green as fluorescent marker, PCR reaction is carried out on Light Cycler fluorescence quantitative PCR instrument, by melting Tracing analysis and electrophoresis determine that purpose band, Δ Δ CT method carry out relative quantification.

5, statistical method

Experiment is completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD, Using SPSS18.0 statistical software come for statistical analysis, the paired comparisons of cancer and cancer beside organism are examined using t, it is believed that when P < There is statistical significance when 0.05.

6, result

As a result as shown in Figure 1, compared with gastric cancer cancer beside organism, ENSG00000228742 is raised in expression in gastric carcinoma, Difference has statistical significance (P < 0.05), consistent with chip test result.

Expression of the embodiment 3ENSG00000228742 in stomach cancer cell

1, cell culture

People immortalizes gastric epithelial cell system GES-1, human gastric cancer cell line HGC-27, MGC-803, AGS (are purchased from wide State Rider that Biotechnology Co., Ltd) it cultivates in the RPMI1640 containing 10% fetal calf serum and 1%P/S based on 37 DEG C, 5% CO₂, relative humidity be 90% incubator in cultivate.It changes within 2-3 days liquid 1 time, trypsase of the use 0.25% containing EDTA is conventional Had digestive transfer culture takes the cell in logarithmic growth phase for testing.

2, the extraction of RNA

Complete medium is discarded, trypsin digestion cell is used after cleaning 2 times with PBS, cell suspension is made, 2mL centrifuge tube is added In, 1200rpm is centrifuged 8min, and addition 1mL Trizol is blown and beaten 15 times or more repeatedly with pipettor after abandoning supernatant, keeps cell abundant Cracking.Remaining operating procedure is the same as embodiment 1.

3, reverse transcription

Specific steps are the same as embodiment 2.

4, statistical method

Experiment is completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD, Using SPSS18.0 statistical software come for statistical analysis, difference between the two is examined using t, it is believed that is had as P < 0.05 It is statistically significant.

5, result

As a result as shown in Fig. 2, compared with gastric epithelial cell, ENSG00000228742 gene is in stomach cancer cell HGC- 27, it expresses in MGC-803, AGS and raises, difference has statistical significance (P < 0.05), consistent with RNA-sep result.

The silencing of embodiment 4ENSG00000228742 gene

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

2, the design of siRNA

Negative control siRNA sequence (siRNA-NC):

Positive-sense strand is 5 '-UUCUCCGAACGUGUCACGU-3 ' (SEQ ID NO.6)

Antisense strand is 5 '-ACGUGACACGUUCGGAGAA-3 ' (SEQ ID NO.7)

SiRNA-1:

Positive-sense strand is 5 '-AAAUGGUUUAUAGUACAAGAU-3 ' (SEQ ID NO.8)

Antisense strand is 5 '-CUUGUACUAUAAACCAUUUGU-3 ' (SEQ ID NO.9)

SiRNA-2:

Positive-sense strand is 5 '-UUUAGACUCCCAUUUGGAGAG-3 ' (SEQ ID NO.10)

Antisense strand is 5 '-CUCCAAAUGGGAGUCUAAAGG-3 ' (SEQ ID NO.11)

Cell is pressed 4 × 10⁴/ hole is inoculated into six porocyte culture plates, in 37 DEG C, 5%CO₂Cell culture in incubator For 24 hours, in DMEM culture medium without double antibody, containing 10%FBS, transfection (is purchased from according to lipofectamine 2000 Invitrogen company) specification transfection.

Experiment is divided into three groups: control group (HGC-27), negative control group (siRNA-NC) and experimental group (siRNA1, SiRNA2), wherein for the sequence of negative control group siRNA and ENSG00000228742 gene without homology, concentration is the hole 20nM/, It is transfected respectively simultaneously.

3, QPCR detects the transcriptional level of ENSG00000228742 gene

The extraction step of 3.1 cell total rnas is the same as embodiment 3.

3.2 reverse transcription steps are the same as embodiment 2.

3.3 QPCR amplification steps are the same as embodiment 2.

4, statistical method

Experiment is completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD, Using SPSS18.0 statistical software come for statistical analysis, between ENSG00000228742 Gene Experiments group and control group Difference is examined using t, it is believed that has statistical significance as P < 0.05.

5, result

As a result such as Fig. 3 is shown, compared with non-transfection group, transfection siRNA-NC group, experimental group can be significantly reduced The expression of ENSG00000228742 gene, difference have statistical significance (P < 0.05).

The influence of embodiment 5ENSG00000228742 gene pairs proliferation of human gastric cancer cell

Detection ENSG00000228742 gene pairs proliferation of human gastric cancer cell capacity is tested using MTS.

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

2, 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 holes μ L/ is inoculated in 96 orifice plates, i.e., every hole cell number is 1 × 10⁴It is a.

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

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

5, statistical analysis

Experiment is completed according to being repeated 3 times, using SPSS18.0 statistical software come for statistical analysis, the two it Between difference using t examine, it is believed that as P < 0.05 have statistical significance.

6, result

As a result as shown in figure 4, compared with the control, for experimental group after transfecting siRNA-1, the proliferation of cell obviously receives suppression System, difference have statistical significance (P < 0.05), illustrate that ENSG00000228742 has the function of promoting cell Proliferation.

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

SEQUENCE LISTING

<110>Taishan Hospital

<120>application of the molecular marker in gastric cancer diagnosis and treatment

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 3049

<212> DNA

<213>source of people

<400> 1

gaaatttggg tgcagacagg cacacaggga gaacaccatg tcaacacgaa ggcagaagtt 60

ggagtgatga gtctacaagc caaggattgc cagcaattca tcagaggcca ggacagcagc 120

ctggggcagc cccctacatg gtaccaactc catcaacacc ttgatctcag acttccaacc 180

tccagaactg tccagccctc catgaatgag gaacttcctg gggccactag tgcatgctgc 240

agctcacaca gtcaactcca tccacacctc cacaaacagc tgccctttgg ccactactca 300

ggcgtaagga cacacccagc agggtatgcc ctctggaggt cagaccaggg aagaagccct 360

tgcacagtca ggagccagct cagcacctgt gatgctgaat tctatgtggc aacttgactg 420

gacgcaggtg ctcggattaa acctgatttc tgggtatgtc tgtgagggta tttccagagg 480

agattagaat ttgaatccgt ggattggcaa agttggtgtc tgccccagtg tgggtggtca 540

ccatctactc tcctgagggc ctgaagagaa caaaaggcaa aggaaggagg aattcgccct 600

taacttgctt gccgcctgcc tgcttgagca gagacatttc atcacatctt ctctggccct 660

cgactgagat tcacaccatt ggcttccctg gttctcaggc attttgactt ggactgaact 720

acaccattgg ctttcctggg ctccagcttg cacatagcag attgtgggat ttctcagcct 780

ctattattac aggcgccagt tcttcaaaat caacctctct ctctctgtct ctctctctct 840

ccctagatca cagtctggaa gtctgctcac agtctcaagg ttggcatatc ccctagagcc 900

cccagactcc tcatcccatg gagaccaaca tggccagggg agggccagag cagggccttc 960

taaatcacag accccagggc aggagcccct ccggtctgga tctaaggatg gtattttcaa 1020

caattcctaa atttttatcc taggctctcc aaatgggagt ctaaaggtct caaaacatgg 1080

tccagaagaa gtatgcaaaa atgtaaattc ctaggcccca acccagacct atgaatcaga 1140

tgatctgtat gggggttcaa ggaatctata ttattaaaca atcttcctaa atgacttgta 1200

ttcatactaa agcttgagaa ttgctatcaa acagctagaa gttggagtga agggtcctgc 1260

ctttcccatg aaattcatca actctatgcc ctcaaaagct gggctcttgg atgagctgct 1320

ctccattcta gagacattac cagctaggta gcctgctggt cctcaggaag ttaagcccag 1380

actcttcagc acagaatcag ataaacactg attacattgt actctacagt tctgagaaca 1440

ccattactta attcccggca agttgcaatg taattgagta ggttgtagtt ttttgggttt 1500

ggggtttttt tgttttttaa atatatcagg cgtagttagc cagcttccta gaaacaccat 1560

gatctcttgg tgaaaattcc agaaatatgc atcacttcct aattaaagta gggaattttt 1620

aaaaattatt aaaccacatg ctttctaatt cctattgccc tttcttttca cccaccattc 1680

agcagtcagc agacactttc tgggtacaac agggtaggac catccaggca gaacaaaagc 1740

cattgatcat gaactacaga ggagagtttg gtaaatggac accagaaacc atggcctaca 1800

ggggcctggt tttctcccat ctaaatacta actaggccca acgctagttg ggcttagctt 1860

cctagatcag atgaaattgg gcacatttag ggttgggggt atggctctct attgtaagta 1920

gcaagagttt gagagaagaa aaaagggatt ttcttttttt tttcttttaa attttagagc 1980

attaacttta aacatcaaga tccaaacccc aaggattcag agaatcttgt actataaacc 2040

atttgtatta gtcagggttc tccagagaaa cagaacccat aggaggtata gagatagaga 2100

aatatggaat tggctcccat gattatggag gctgagaagt ccctcaatct gctgtctgtg 2160

agctggagac ctaggaaagc ttagctggtg gtacaaacca gcccaagtgc aaaggcccaa 2220

gaactaagag caccattgtc agagagcagg aaaagacaga tgtcccagct caaaaagaga 2280

gagcaaattc accttccttc caccatttta ttccgtcctg ttccattcct caacaggttg 2340

gatgatgagg gagatcttta tccatctcaa gttttttgtg caaatatgga atacaaatga 2400

aaaatactta aagcacatca tcattgattt ccaacttaga ctagtttaac ttcccttttt 2460

cttctatcct gtcagttcct tgagggatcc tctctccttg ttcatctctg catctctagt 2520

aattagcaca gtgccaggca catggtaata tggctaaaac agtggattat tggtctccaa 2580

cataattaag cccatccaaa agaaaaacca acctcataaa tctgctttgc aaagttcata 2640

aacaatggat atgctatgtc tcaaaggtgc tggccaaaaa aatttaagaa cttgggaatt 2700

atgtgttatt ttgtttgttt ttttcaccaa gcctactttt ctgtcacaag agcccatatc 2760

aacaacacac ccatgtatgt ccttcttttt atctactttt ctccatgaag tttgtcacca 2820

tcaactgtta aaactgtaaa gatttgatca gctaaataag tggtctttga actaattcct 2880

ctcgtacttc ctaaaggaac tttgaaaaac tatgtatccc cttgcacatt ttcaagcaga 2940

cacctaagat ttttcatcat aaatttgaat aacttcaaaa tatgtactta ttagcatatt 3000

gtaaatattg atttttaaat aaaatatatc acactttgta tccaatgaa 3049

<210> 2

<211> 19

<212> DNA

<213>artificial sequence

<400> 2

ctcttcagca cagaatcag 19

<210> 3

<211> 23

<212> DNA

<213>artificial sequence

<400> 3

actacaacct actcaattac att 23

<210> 4

<211> 21

<212> DNA

<213>artificial sequence

<400> 4

aatcccatca ccatcttcca g 21

<210> 5

<211> 19

<212> DNA

<213>artificial sequence

<400> 5

gagccccagc cttctccat 19

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<211> 19

<212> RNA

<213>artificial sequence

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uucuccgaac gugucacgu 19

<210> 7

<211> 19

<212> RNA

<213>artificial sequence

<400> 7

acgugacacg uucggagaa 19

<210> 8

<211> 21

<212> RNA

<213>artificial sequence

<400> 8

aaaugguuua uaguacaaga u 21

<210> 9

<211> 21

<212> RNA

<213>artificial sequence

<400> 9

cuuguacuau aaaccauuug u 21

<210> 10

<211> 21

<212> RNA

<213>artificial sequence

<400> 10

uuuagacucc cauuuggaga g 21

<210> 11

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<212> RNA

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cuccaaaugg gagucuaaag g 21

Claims (3)

1.ENSG00000228742 lower adjustment purposes, which is characterized in that be used to prepare treatment gastric cancer pharmaceutical composition, The lower adjustment is siRNA, wherein positive-sense strand is SEQ ID NO.8, antisense strand is SEQ ID NO.9.

2. a kind of purposes of ENSG00000228742 gene, which is characterized in that screen the latent for the treatment of gastric cancer for non-treatment purpose In substance.

3. a kind of method of the potential substance of non-treatment purpose screening treatment gastric cancer, which is characterized in that the described method includes:

The system expressed or containing ENSG00000228742 gene is handled with candidate substances；With

Detect the expression of ENSG00000228742 gene in the system；

Wherein, if the candidate substances can reduce the expression or activity of ENSG00000228742 gene, show the candidate substances It is the potential substance for treating gastric cancer.