CN108707672A - Applications of the DUXAP8 in diagnosis of hepatoma and treatment - Google Patents

Abstract

The invention discloses applications of the DUXAP8 in diagnosis of hepatoma and treatment, present invention firstly discovers that DUXAP8 up-regulated expressions in patients with hepatocellular carcinoma, and DUXAP8 is demonstrated by large sample and can be used as the auxiliary diagnosis that detection target is applied to hepatocellular carcinoma.The present invention is found that DUXAP8 is related to the proliferation of liver cancer cells, migration and invasion simultaneously, and DUXAP8 is prompted to can be used as drug targets applied to hepatocellular carcinoma and the treatment of hepatocellular carcinoma invasion and transfer.

Description

Applications of the DUXAP8 in diagnosis of hepatoma and treatment

Technical field

The invention belongs to biomedicine fields, are related to applications of the DUXAP8 in diagnosis of hepatoma and treatment.

Background technology

In recent years, hepatocellular carcinoma has become one of most common malignant tumour in China, and the death rate occupies malignant tumour Two, seriously threaten Chinese national economy development and population health (Jemal A, Bray F, Center MM, Ferlay J, Ward E,Forman D.Global statistics.CA:a cancer journal for clinicians.2011,61 (2):69-90).Surgical resection, liver transfer operation, chemicotherapy, interventional treatment, molecular targeted therapy etc. are the main of current liver cancer Therapeutic modality, but since onset of liver cancer is hidden, non-evident sympton, most of patients has just lost surgical radical treatment chance, art when medical The factors such as recurrence and transfer drastically influence the therapeutic effect and life cycle (Ling TC, Kang JI, Bush of hepatocarcinoma patient afterwards DA,Slater JD,Yang GY.Proton therapy for hepatocellular carcinoma.Chinese journal of cancer research.2012,24(4):361-7.).Therefore, it is explored on molecular level and illustrates liver cell liver The precise mechanism of generation, the development, transfer of cancer seeks new drug target, and screening effective early diagnosis molecular marked compound is Among in the hot spot studied at present, and the weight of breakthrough liver cancer treatment bottleneck.

With the completion of the Human Genome Project (human genome project, HGP), new-generation sequencing technology also obtains To rapid development.With the development of high throughput sequencing technologies, a large amount of non-coding RNAs are annotated.Non-coding RNA, which is one kind, to be compiled The RNA of code protein, their main function are the growth and development of the transcript and expression and then influence biology that adjust encoding gene, The differentiation of cell, proliferation, apoptosis and various biologicals behavior stress be waited.The research of non-coding RNA is concentrated mainly at present On microRNA and long-chain non-coding RNA (long noncoding RNA, 1ncRNA).1ncRNA is that known transcript length is big In the non-coding RNA of 200 nucleotide, by with DNA, the interaction of RNA and protein participates in generation and the hair of disease Exhibition.It is in differential expression that previously some researches show that lncRNA in liver cancer tissue, and changing its expression can inhibit liver cancer thin The proliferation (CN201710404209.8, CN201710404203.0) of born of the same parents.

It is still in infancy at present about research of the lncRNA molecules in liver cancer regulatory mechanism.Therefore, by analysis, LncRNA is studied in the occurrence and development mechanism of liver cancer, is played an important role to diagnosis, the prevention and treatment of liver cancer, and be liver cancer Gene target treatment provide may.

Invention content

In order to make up for the deficiencies of the prior art, an object of the present invention provides a kind of biomarker, is used for liver cell The early diagnosis of cancer or targeted therapy.

The second object of the present invention provides a kind of method of screening treatment hepatocellular carcinoma drug candidate, waits sieving by detecting Whether select substance can change the expression of marker to judge whether it is the drug candidate for treating hepatocellular carcinoma.

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

The present invention provides application of the reagent in the product for preparing diagnosing hepatocellular carcinoma of detection DUXAP8 expressions, Wherein, DUXAP8 expressions in patients with hepatocellular carcinoma raise.

Further, the reagent is selected from：

The probe of specific recognition DUXAP8；Or

The primer of specific amplification DUXAP8.

Further, the primer sequence of the specific amplification DUXAP8 is as shown in NO.3~4 SEQ ID.

The present invention provides a kind of product of diagnosing hepatocellular carcinoma, the product includes DUXAP8 expression water in detection sample Flat reagent.As long as product of the present invention can detect the expression of DUXAP8, and be not limited to common detection Product such as chip, nucleic acid film item, preparation or kit etc.." sample " includes cell, tissue, internal organs, body fluid (blood, leaching Bar liquid etc.), digestive juice, expectoration, alveole bronchus cleaning solution, urine, excrement etc..Preferably, the sample is tissue, blood.

Further, the reagent includes the probe of specific recognition DUXAP8 or the primer of specific amplification DUXAP8.

In the specific implementation mode of the present invention, the reagent includes the primer of specific amplification DUXAP8, described special Property amplification DUXAP8 primer sequence as shown in NO.3~4 SEQ ID.

The present invention provides application of the DUXAP8 genes in hepatocellular carcinoma drug candidate is treated in screening.

The present invention provides it is a kind of screening treatment hepatocellular carcinoma drug candidate method, the method includes：

The system expressed or containing DUXAP8 genes is handled with substance to be screened；With

Detect the expression of DUXAP8 genes in the system；

Wherein, if the expression that the substance to be screened can reduce DUXAP8 genes (preferably significantly reduces, such as low 20% More than, preferably low 50% or more；More preferably low 80% or more) then shows that the substance to be screened is the time for treating hepatocellular carcinoma Select drug.

The system is selected from：Cell system, subcellular system, solution system, organizational framework, organ systems or animal body System.

The drug candidate includes but is not limited to：The interference designed for DUXAP8 genes or its upstream or downstream gene Molecule, nucleic acid inhibitor, micromolecular compound etc..

The present invention provides a kind of composition, the composition includes：The inhibitor of DUXAP8 functional expressions；And pharmacy Upper acceptable carrier.Wherein, the inhibitor of the DUXAP8 functional expressions is selected from：Using DUXAP8 or its transcript as target sequence Row and the disturbing molecule that DUXAP8 gene expressions or genetic transcription can be inhibited, including：ShRNA (children purpura nephritis), small interference RNA (siRNA), dsRNA, Microrna, antisense nucleic acid, or can express or be formed the shRNA, siRNA, dsRNA, micro- The construction of tiny RNA, antisense nucleic acid.

Pharmaceutically acceptable carrier includes but is not limited to buffer, emulsifier, suspending agent, stabilizer, preservative, life Manage salt, excipient, filler, coagulating agent and blender, interfacial agent, diffusant, antifoaming agent.

Preferably, the inhibitor is siRNA.

More preferably, the sequence of the siRNA is as shown in NO.7~8 SEQ ID.

The present invention provides applications of the DUXAP8 in the pharmaceutical composition for preparing treatment hepatocellular carcinoma.

The present invention provides applications of the DUXAP8 in the pharmaceutical composition for preparing treatment hepatocellular carcinoma invasion.

The present invention provides applications of the DUXAP8 in the pharmaceutical composition for preparing treatment hepatocellular carcinoma transfer.

The present invention provides the compositions for the inhibitor for including DUXAP8 functional expressions to prepare treatment hepatocellular carcinoma Drug in application.

The present invention provides the compositions for the inhibitor for including DUXAP8 functional expressions to prepare treatment hepatocellular carcinoma Application in the drug of invasion.

The present invention provides the compositions for the inhibitor for including DUXAP8 functional expressions to prepare treatment hepatocellular carcinoma Application in the drug of transfer.

Description of the drawings

Fig. 1 is the expression figure in patients with hepatocellular carcinoma using QPCR detections DUXAP8；

Fig. 2 is the expression figure in hepatocellular carcinoma cells using QPCR detections DUXAP8；

Fig. 3 is expression influence diagrams of the detection transfection siRNA to DUXAP8 in hepatocellular carcinoma cells；

Fig. 4 is the influence diagram to hepatoma cell proliferation using CCK8 detection DUXAP8；

Fig. 5 is the influence diagram for detecting the migration of DUXAP8 gene pairs hepatocellular carcinoma cells；

Fig. 6 is the influence diagram for detecting the invasion of DUXAP8 gene pairs hepatocellular carcinoma cells.

Specific embodiment

The present invention after extensive and in-depth study, by high-flux sequence method, detects by Tissues of Hepatocellular Carcinoma and cancer The expression of lncRNA in tissue finds the wherein lncRNA segments with apparent differential expression, inquires into itself and hepatocellular carcinoma Generation between relationship, to for hepatocellular carcinoma early detection and targeted therapy find better approaches and methods.Pass through Screening, present invention firstly discovers that DUXAP8 conspicuousnesses raise in hepatocellular carcinoma.It is demonstrated experimentally that siRNA interferes silence DUXAP8, The proliferation that hepatocellular carcinoma cells can be effectively inhibited provides new way for the personalized treatment of hepatocellular carcinoma.

DUXAP8 genes

DUXAP8 genes are located at No. 22 1 areas 1 of chromosome long arm and take, and the DUXAP8 in the present invention includes wild type, mutation Type or its segment.In an embodiment of the present invention, a kind of nucleotide sequence of representative people DUXAP8 genes is such as international at present In public nucleic acid database GeneBank shown in DUXAP8 genes (NR_122113.1).The DUXAP8 nucleotide overall lengths of the present invention Sequence or its segment can usually use PCR amplification method, recombination method or artificial synthesized method to obtain.

It would be recognized by those skilled in the art that the practicability of the present invention is not limited to appointing to the target gene of the present invention The gene expression of what specific variants is quantified.If when nucleic acid or its segment and other nucleic acid (or its complementary strand) optimal comparison When (have nucleotides inserted appropriate or missing), at least about 60% nucleotide base, usually at least about 70%, more Usually at least about 80%, it is preferably at least about 90% and there are nucleosides more preferably at least about in 95-98% nucleotide bases The acid sequence phase same sex, then the two sequences are " substantially homologous " (or substantially similar).

Alternatively, when nucleic acid or its segment and another nucleic acid (or its complementary strand), a chain or its complementary series are in selectivity When hybridizing under hybridization conditions, then there is substantially homologous or (the phase same sex) therebetween.When hybridization has more than specific general loss When selective, there are cross selections.Typically, when one section of sequence at least about 14 nucleotide exists at least about When the 55% phase same sex, preferably at least about 65%, more preferably at least about 75% and the most preferably at least about 90% phase same sex, hair Raw selective cross.As described herein, cognate pair than length can be longer sequence section, lead in certain embodiments Often it is at least about 20 nucleotide, more frequently at least about 24 nucleotide, typically at least about 28 nucleotide, more Typically at least about 32 nucleotide, and preferably at least about 36 or more nucleotide.

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 skilled in the art it will be recognized that due to RNA in cell less stable and in an experiment It is more vulnerable to nuclease attack, therefore usually by RNA reverse transcriptions at DNA before sequencing.

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 is used for reverse transcriptase (RT) to prepare complementary DNA (cDNA) from mRNA, Then cDNA is generated to multiple copies of DNA by PCR amplification；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 At 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 that the endonuclease that semi-modified restriction enzyme enzyme recognition site carries out mediates, and from cutting Mouth 3'The polymerase-mediated object drawn that end carries out, which extends, to be set 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 sequence.DNA ISH can be used for determining the structure of chromosome.RNA ISH are for measuring with position tissue slice or entirely MRNA in organization embedding and other transcripts (for example, ncRNA).Usually sample cell and tissue are handled in situ solid Targeting transcript, and increase the entrance of probe.Probe hybridizes with target sequence at high temperature, then washes off extra probe.Point Not Shi Yong autoradiograph, fluorescence microscopy or immunohistochemistry, in tissue with radiation, fluorescence or antigenic mark base The probe of label is positioned and is quantified.ISH can also be used two or more to pass through radioactivity or other nonradioactive labelings The probe of substance markers, 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, nucleic acid film item, kit

Chip includes in the present invention：Solid phase carrier；And the oligonucleotides being orderly fixed on the solid phase carrier is visited Needle, the oligonucleotide probe is specifically corresponding to sequence some or all of shown in DUXAP8.

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..

" probe " refers to the molecule that can be combined with the particular sequence of another molecule or subsequence or other parts.Unless otherwise finger Go out, term " probe " is often referred to match by complementary base and (often referred to as " target polynucleotide ") be combined with another polynucleotides Polynucleotide probes.According to the preciseness of hybridization conditions, probe energy and the target for lacking sufficient sequence complementarity with the probe are more Nucleotide combines.Probe can make direct or indirect label, and range includes primer.Crossing system includes, but are not limited to：It is molten Liquid 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 80% or more, preferably 90% or more, more preferable 95% or more, 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 that is 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..

The present invention provides a kind of kit, the kit can be used for detecting the expression of DUXAP8.The kit packet Include the specific primer pair for expanding DUXAP8；Standard DNA template；PCR reaction solution.In a preferred embodiment, The specific primer is to including sense primer and downstream primer, and sequence is as shown in NO.3~4 SEQID.

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.

Gene detecting kit or genetic chip can be used for detecting multiple bases including DUXAP8 genes in the present invention Because of the expression of (for example, with the relevant multiple genes of hepatocellular carcinoma), multiple markers of hepatocellular carcinoma are carried out at the same time inspection It surveys, is greatly improved the accuracy rate of diagnosis of hepatoma.

Inhibitor and pharmaceutical composition

Discovery based on inventor, the present invention provides a kind of inhibitor of DUXAP8, and the property of the inhibitor is to this It is not important for invention, as long as it inhibits the functional expression of DUXAP8 genes, these inhibitor to be used as lowering Substance useful DUXAP8 can be used for preventing or treat hepatocellular carcinoma.

As a kind of preferred embodiment of the present invention, the inhibitor of the DUXAP8 is a kind of small interference of DUXAP8 specificity RNA molecule.As used herein, " siRNA " refers to a kind of short-movie section double stranded rna molecule, can be with homologous complementary The mRNA of sequence is the target specific mRNA of degradation, this process is exactly RNA interference (RNA interference) processes.It is small RNA interfering can be prepared into the form of double-strandednucleic acid, it contains there are one positive-sense strand and an antisense strand, this two chains are only hybridizing Under conditions of form double-strand.One double-stranded RNA compound can be prepared by the positive-sense strand that is separated from each other and antisense strand.Therefore, For example, complementary positive-sense strand and antisense strand are chemical synthesis, and can generate the double-strand of synthesis by anneal thereafter RNA compounds.

When screening effective siRNA sequence, the present inventor is best effective to find out by largely comparing analysis Segment.The present inventor's design has synthesized a variety of siRNA sequences, and they are passed through transfection reagent transfected hepatocytes cancer cell respectively System is verified, and the best siRNA of interference effect is selected, and is further tested, is as a result proved for the siRNA in cellular level In the proliferation for the expression and hepatocellular carcinoma cells that can effectively inhibit DUXAP8 genes in cell.

The nucleic acid inhibitor such as siRNA of the present invention 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 mode of the present invention, the inhibitor of the DUXAP8 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, 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 be obtained by commercially available approach completely, such as some viral vectors.

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 of the host cell for selecting conversion, 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 " includes prokaryotic cell and eukaryocyte.The example of common prokaryotic host cell includes large intestine Bacillus, hay bacillus etc..Common eukaryotic host cell includes yeast cells, insect cell and mammalian cell.Preferably, The host cell is eukaryocyte, such as Chinese hamster ovary celI, COS cells.

The present invention also provides a kind of pharmaceutical compositions, it contains the inhibitor of a effective amount of DUXAP8 functional expressions, And pharmaceutically acceptable carrier.The composition can be used for inhibiting hepatocellular carcinoma.The inhibition of any DUXAP8 above-mentioned Agent is used equally for the preparation of pharmaceutical composition.

As used herein, described " effective quantity " refer to people and/or animal can be generated function or it is active 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：It themselves is not necessary active constituent, 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, can be directly by the inhibitor of DUXAP8 by such as noting It the methods of penetrates and to deliver medicine to subject；Alternatively, can will be carried by certain approach the inhibitor of DUXAP8 ceneme (such as Expression vector or virus etc. or siRNA or shRNA) it is delivered on target spot, and it is allowed to the DUXAP8 inhibitor of expression activity, have Body situation need to be depending on the type 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 DUXAP8 gene expressions and at least one chemotherapeutics.For the present invention's Chemotherapeutics, including but not limited to：Micro-pipe activator, alkylating agent, nti-neoplastic antimetabolite, platinum-like compounds, DNA- alkylating agents resist Anti-neoplastic antibiotic agent, antimetabolite, microtubule stabilizing agent, tubulin destabilizing agent, hormone antagonist, topoisomerase suppression Preparation, kinases inhibitor, HMG-COA inhibitor, CDK inhibitor, cyclin inhibitors, Caspase inhibit Virus, bacterium and the outer poison of agent, metal protease inhibitors, antisense nucleic acid, triple helix DNA, aptamer and molecular modification Plain reagent.

The pharmaceutical composition of the present invention can also be with the drug combination of other treatment hepatocellular carcinoma, and other therapeutic compound can To be administered simultaneously with main active constituent, or even it is administered simultaneously in same composition.

The pharmaceutical composition of the present invention can also be with individual composition or the dosage shape different from main active constituent Formula individually gives other therapeutic compounds.The Fractional of main component can be administered simultaneously with other therapeutic compounds, And other dosage can be administered alone.It over the course for the treatment of, can be according to the severity of symptom, the frequency of recurrence and treatment side The physiologic response of case adjusts the dosage of pharmaceutical composition of the present invention.

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 indicates, 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 hepatocellular carcinoma

1, sample collection

Respectively collect the cancerous tissue of the patients with hepatocellular carcinoma of 50 HBV infections and corresponding cancer beside organism's sample, Cong Zhongsui Machine chooses 8 progress high-flux sequences, and the acquirement of the equal informed consent of patient, above-mentioned all samples passes through the committee of organizational ethics Agreement.

2, 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.

3, the quality analysis of RNA sample

By the RNA of said extracted into row agarose gel electrophoresis, using Nanodrop2000 to the concentration of carried RNA and pure Degree is detected, and agarose gel electrophoresis detects RNA integralities, and Agilent2100 measures RIN values.Single requirement for construction data base RNA is total 5ug is measured, concentration >=200ng/ μ L, OD260/280 is between 1.8~2.2.

4, rRNA is removed

The rRNA in total serum IgE is removed using Ribo-Zero kits.

5, construction cDNA library

The structure of cDNA library, concrete operations are carried out using Illumina TruseqTM RNA sample Prep Kit By specification carries out.

6, upper machine sequencing

CDNA library is sequenced using Illumina X-Ten microarray datasets, concrete operations by specification carries out.

7, high-throughput transcript profile sequencing data analysis

Bioinformatic analysis is carried out using the DESeq2 in R-3.3.3 kits to sequencing result, passes through structure dds squares Battle array, standardization, carry out variance analysis, the screening criteria of differential expression lncRNA：FDR<0.05, abs (log₂FC)>2。

8, result

RNA-seq is prompted the results show that expression quantity of the DUXAP8 genes in Tissues of Hepatocellular Carcinoma is significantly higher than control group DUXAP8 may be applied to the diagnosis of hepatocellular carcinoma as detection target.

The differential expression of 2 QPCR sequence verification DUXAP8 genes of embodiment

1, the 50 patient's cancerous tissue samples and cancer beside organism's sample collected using front are to DUXAP8 gene differential expressions Carry out large sample QPCR verifications.

2, RNA extraction steps are the same as embodiment 1.

3, reverse transcription：

1) by the total serum IgE template of 1 μ g and 2 10 × buffer solutions of μ l, 2 μ l dATP (10mM), 0.5 μ l polyA polymerases, 0.5 μ l ribalgilases (RNase) inhibitor and deoxyribonuclease water (RNase free water) mixing, volume are finally 20 μ l, 37 DEG C of incubation 1h.

2) 1 μ l 0.5 μ g/ μ l Oligo (dT) specific RT primer, 70 DEG C of incubation 5min are added in reaction tube.

3) it sets immediately and is incubated at least 2min on ice, interrupt the secondary structure of RNA and primer.

4) by above-mentioned 20 μ l reaction mixtures and 45 × buffer solutions of μ l, 1 μ l dNTP (10mM), 0.5 μ l M-MLV are reversed Record enzyme, 0.5 μ l ribalgilases (RNase) inhibitor, 10 μ l polyA reaction mixtures and 4 μ l deoxyribonuclease water (RNase free water) is mixed, 42 DEG C of incubation 1h.

4, QPCR amplifications are examined

1) design of primers：

The primer sequence of house-keeping gene GAPDH is：

Forward primer：5'-CCGGGAAACTGTGGCGTGATGG-3'(SEQ ID NO.1)

Reverse primer：5'-AGGTGGAGGAGTGGGTGTCGCTGTT-3'(SEQ ID NO.2)

The primer sequence of DUXAP8 genes is：

Forward primer：5'-CCTCATCAATACCTTCACTCA-3'(SEQ ID NO.3)

Reverse primer：5'-CTGGATTCTGGACTCTTCTG-3'(SEQ ID NO.4)

2) reaction system

SYBR Green PCRs system 12.5 μ l, forward and reverse primer (5 μM) each 1 μ l, 2.0 μ of template cDNA L, ddH₂O 8.5μl.Operations are carried out on ice.3 parallel pipes are arranged in each sample, and all amplified reactions repeat three It is secondary above to ensure the reliability of result.

3) reaction condition

95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 60s) × 45 cycles.Using SYBR Green as fluorescent marker, On Light Cycler fluorescence quantitative PCR instruments carry out PCR reactions, using GAPDH as reference gene, by melt curve analysis analysis with Electrophoresis determines that purpose band, Δ Δ CT methods carry out relative quantification.

5, result

The results are shown in Figure 1, and compared with cancer beside organism, DUXAP8 genes expression in Tissues of Hepatocellular Carcinoma raises, Difference has statistical significance (P<0.05)；Wherein the patient of up-regulated expression is 48, low expression 1, normal expression 1, sun Property recall rate=48/50 × 100%=96%；Prompt DUXAP8 can be used as the diagnosis that Testing index is applied to hepatocellular carcinoma.

Differential expression of the 3 DUXAP8 genes of embodiment in hepatocellular carcinoma cells system

1, cell culture

Human hepatocellular carcinoma Cell Line HepG2, Huh7 and normal liver cell system HL-7702, with contain 10% fetal calf serum and The culture medium DMEM of 1%P/S is in 37 DEG C, 5%CO₂, relative humidity be 90% incubator in cultivate.It changes within 2-3 days liquid 1 time, makes With the 0.25% trypsase conventional digestion passage containing EDTA.

2, the extraction of RNA

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

3, reverse transcription specific steps are the same as embodiment 2

4, QPCR amplifications examine specific steps with embodiment 2

5, result

The results are shown in Figure 2, and compared with normal liver cell system, DUXAP8 genes are in hepatocellular carcinoma cells HepG2, Huh7 Expression is raised, and difference has statistical significance (P<0.05), consistent with RNA-sep results.

The silence of 4 DUXAP8 genes of embodiment

1, cell culture

Human hepatocellular carcinoma Cell Line HepG2, with the culture medium DMEM containing 10% fetal calf serum and 1%P/S 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.

2, siRNA is designed

For the siRNA sequence of DUXAP8 genes：

Negative control siRNA sequence (siRNA-NC)：

Positive-sense strand：5 '-UUCUCCGAACGUGUCACGU-3 ' (SEQ ID NO.5),

Antisense strand：5'-ACGUGACACGUUCGGAGAA-3'(SEQ ID NO.6)；

siRNA1：

Positive-sense strand：5 '-UAAAGAGAUCAAGAAACUCUC-3 ' (SEQ ID NO.7),

Antisense strand：5'-GAGUUUCUUGAUCUCUUUAUC-3'(SEQ ID NO.8)；

siRNA2：

Positive-sense strand：5 '-AAACCUUUGGGAAAUCCUGCC-3 ' (SEQ ID NO.9),

Antisense strand：5'-CAGGAUUUCCCAAAGGUUUCU-3'(SEQ ID NO.10)；

siRNA3：

Positive-sense strand is 5 '-ACAUACACAAAGAGAAUAGUC-3 ' (SEQ ID NO.11),

Antisense strand is 5 '-CUAUUCUCUUUGUGUAUGUCC-3 ' (SEQ ID NO.12)

Cell is pressed 2 × 10⁵/ hole is inoculated into six porocyte culture plates, in 37 DEG C, 5%CO₂Cell culture in incubator 24h；In without dual anti-DMEM culture mediums containing 10%FBS, transfection (is purchased from according to lipofectamine 3000 Invitrogen companies) specification transfection.

Experiment be divided into blank control group (HepG2), negative control group (siRNA-NC) and experimental group (20nM) (siRNA1, SiRNA2, siRNA3), the sequence of wherein negative control group siRNA and DUXAP8 genes is without homology, a concentration of holes 20nM/, together When transfected respectively.

3, QPCR detects the expression of DUXAP8 genes

The extraction specific steps of 3.1 cell total rnas are the same as embodiment 4.

3.2 reverse transcription steps are the same as embodiment 2.

3.3 QPCR amplification steps are the same as embodiment 2.

4, result

As a result such as Fig. 3 is shown, compared to HepG2, transfection zero load siRNA-NC groups, experimental group can reduce the expression of DUXAP8 Level, and the reduction level of siRNA1 groups is the most notable, therefore siRNA1 is selected to carry out subsequent experiment.

5 CCK8 of embodiment detects cell proliferation experiment

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

2, CCK8 detects cell Proliferation

By the HepG2 cell inoculations of logarithmic proliferation phase in 96 orifice plates, per hole 2 × 10³Cell is divided into three groups by a cell, It is blank control group, transfection siRNA-NC groups and transfection siRNA1 respectively, every group sets 6 multiple holes；Respectively transfection 0h, for 24 hours, 10 holes μ l/ CCK8 reagents are added after 48h, 72h, 108h, 120h, is put into incubator to be incubated after 1h and detects A450 using microplate reader Light absorption value.

3, result

Result shown in Fig. 4 is shown：Blank control group transfects the cell life of siRNA1 groups with unloaded group no significant difference The vitro growth rates of the apparent low control group of long speed, difference have statistical significance (P<0.05), the above results show DUXAP8 is related with the proliferation of liver cancer cells.

6 Cell migration assay of embodiment

1, after each group cell transfecting for 24 hours, conventional digestion, centrifugation is resuspended with the serum-free DMEM culture solutions containing 10g/L, It is 1 × 10 to adjust cell concentration⁵/ml；

2, take the cell suspension inoculation of 200 μ 1 to not spreading in the cells the Transwell upper chamber of Matrigel glue；

3, room adds 1ml to contain the DMEM culture solutions of 10% fetal calf serum under 24 orifice plates, and the cells Transwell are placed in 24 holes In plate hole, avoid forming bubble between culture solution and cell；37 DEG C, 5%CO₂, routine culture 48h；

4, cell, PBS elution are taken out, cotton swab carefully cleans the cell in cell upper chamber face, and lower room face is fixed with methanol 15min, 1% violet staining 5min；10 visuals field are randomly selected under 200 times of inverted microscopes, are counted across microporous barrier lower layer Cell number, be averaged, every group of 3 cells are repeated 3 times.

5, result

The results are shown in Figure 5, after hepatocellular carcinoma cells transfect RNA interfering, compared with the control group, the migration of experimental group Ability decreased significantly, and as a result illustrate that DUXAP8 participates in the transition process of liver cancer cells.

7 cell invasion of embodiment is tested

1, it is coated with basilar memebrane：Matrigel glue is placed in 4 DEG C of refrigerator overnight liquefaction, it on ice will with the pipette tips of precooling Matrigel glue presses 1:It is small that 3 dilution proportions in the DMEM culture solutions of serum-free, by 50 holes μ l/ are equably covered in Transwell On the film of room, room temperature natural air drying；

2, residual liquid in culture plate is sucked out, 50 μ l are then added per hole and contain the serum-free medium of l0g/L BSA, 37 DEG C be incubated 30min；

3, after each group cell transfecting for 24 hours, conventional digestion, centrifugation, with the serum-free DMEM culture solution weights containing 10g/L BSA It is outstanding.Adjustment cell concentration is 1 × l 0⁵ml；

4, take the cell suspension inoculation of 200 μ l to being coated in the cells the Transwell upper chamber of Matrigel glue；In 24 holes Room adds 1ml to contain the DMEM culture solutions of 10% fetal calf serum under plate, and the cells Transwell are placed in 24 orifice plates, culture solution is avoided Bubble is formed between cell；37 DEG C, 5%CO₂, routine culture 48h；

5, cell, PBS elution are taken out, cotton swab carefully cleans the Matrigel glue and cell in cell upper chamber face, and lower room face is used Methanol fixes 15min, 1% violet staining 5min；It is rinsed 2 times with PBS, randomly selecting 10 under 200 times of inverted microscopes regards Open country counts the cell number across microporous barrier lower layer, is averaged, every group of 3 cells are repeated 3 times.

6, result

The results are shown in Figure 6, and compared with the control group, the invasion cell number of experimental group is reduced, and illustrates that DUXAP8 is thin with liver cancer The invasion of born of the same parents are related.

The influence of 8 DUXAP8 gene pairs hepatocellular carcinoma cells apoptosis of embodiment

Use the influence of flow cytomery DUXAP8 gene pairs Apoptosis.

1, cell culture step is the same as embodiment 4.

2, cell transfecting step is the same as embodiment 5.

3, Apoptosis detects

1) 10 × sample-loading buffers of 3m1 27ml distilled water is diluted；

2) collection of cellular samples and with precooling PBS clean, by cell be added 1 × sample-loading buffers of lml, 300g centrifuge Buffer solution is sucked out in 10min；

3) 1 × sample-loading buffer is added again, cell concentration in cell suspension is adjusted to 1 × 10⁶A/ml；

4) cell suspension is taken out into 100 μ 1, be added in EP pipes；The Annexin V FITC of 5 μ l are added in EP pipes, mixing Liquid in EP pipes is protected from light is incubated 10min at room temperature；

5) 5 μ 1PI dye liquors are continuously added, are protected from light 5min at room temperature；

6) PBS solution of 500 μ l is added, gently mixing, flow cytometer is detected in 1h.

4, result：

The results show that experimental group is compared with the control group, apoptosis rate is without significant change (P<0.05), which illustrates, The expression of DUXAP8 is unrelated with the apoptosis of cancer cell.

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 also be fallen into the protection domain of the claims in the present invention.

Sequence table

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

Chinese Academy of Medical Sciences Beijing Union Medical College Hospital

<120>Applications of the DUXAP8 in diagnosis of hepatoma and treatment

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<170> SIPOSequenceListing 1.0

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<212> DNA

<213>Artificial sequence (Artificial Sequence)

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<210> 3

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<213>Artificial sequence (Artificial Sequence)

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<213>Artificial sequence (Artificial Sequence)

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<212> RNA

<213>Artificial sequence (Artificial Sequence)

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<212> RNA

<213>Artificial sequence (Artificial Sequence)

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<212> RNA

<213>Artificial sequence (Artificial Sequence)

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Claims (14)

1. detecting application of the reagent of DUXAP8 expressions in the product for preparing diagnosing hepatocellular carcinoma.

2. application according to claim 1, which is characterized in that the reagent is selected from：

The probe of specific recognition DUXAP8；Or

The primer of specific amplification DUXAP8.

3. application according to claim 2, which is characterized in that the primer sequence such as SEQ of the specific amplification DUXAP8 Shown in NO.3~4 ID.

4. a kind of product of diagnosing hepatocellular carcinoma, which is characterized in that the product includes the reagent described in claim 1-3.

Application of the 5.DUXAP8 genes in hepatocellular carcinoma drug candidate is treated in screening.

6. a kind of method of the drug candidate of screening treatment hepatocellular carcinoma, which is characterized in that the method includes：

The system expressed or containing DUXAP8 genes is handled with substance to be screened；With

Detect the expression of DUXAP8 genes in the system；

Wherein, if the substance to be screened can reduce the expression of DUXAP8 genes, show that the substance to be screened is treatment The drug candidate of hepatocellular carcinoma.

7. a kind of composition, which is characterized in that described pharmaceutical composition includes：

The inhibitor of DUXAP8 functional expressions；And pharmaceutically acceptable carrier.

8. composition according to claim 7, which is characterized in that the inhibitor is siRNA；Preferably, the siRNA Sequence as shown in NO.7~8 SEQ ID.

Applications of the 9.DUXAP8 in the pharmaceutical composition for preparing treatment hepatocellular carcinoma.

Applications of the 10.DUXAP8 in the pharmaceutical composition for preparing treatment hepatocellular carcinoma invasion.

Applications of the 11.DUXAP8 in the pharmaceutical composition for preparing treatment hepatocellular carcinoma transfer.

12. application of the composition in the drug for preparing treatment hepatocellular carcinoma described in claim 7 or 8.

13. application of the composition in the drug for preparing treatment hepatocellular carcinoma invasion described in claim 7 or 8.

14. application of the composition in the drug for preparing treatment hepatocellular carcinoma transfer described in claim 7 or 8.