CN108114283A

CN108114283A - Applications of the biomarker MUC21 in osteosarcoma diagnose and treat

Info

Publication number: CN108114283A
Application number: CN201810031225.1A
Authority: CN
Inventors: 刘扬; 陈笑天; 官建中
Original assignee: First Affiliated Hospital of Bengbu Medical College
Current assignee: First Affiliated Hospital of Bengbu Medical College
Priority date: 2018-01-12
Filing date: 2018-01-12
Publication date: 2018-06-05

Abstract

The invention discloses applications of the biomarker MUC21 in osteosarcoma diagnose and treat, and the present invention provides a kind of means for diagnosing osteosarcoma, the means are the expression of detection MUC21.Invention also provides a kind of pharmaceutical composition and means for treating osteosarcoma, described pharmaceutical composition includes the inhibitor of MUC21 functional expressions.

Description

Applications of the biomarker MUC21 in osteosarcoma diagnose and treat

Technical field

The invention belongs to biomedicine fields, are related to applications of the biomarker MUC21 in osteosarcoma diagnose and treat.

Background technology

Osteosarcoma is most common bone primary malignant neoplasm, and incidence accounts for the 20% of primary malignant bone tumor, originates from Mesenchymal tissue, the mesenchymal cell of long bone, spindle cell and osteoid are formed as its Pathologic Characteristics, and tumor cell growth is proliferated soon Rapidly, growth pattern destroys normal surrounding tissue in infiltrative growth.Osteosarcoma can all fall ill in each age level, but with youngster Virgin and juvenile onset is common, and the high-incidence season, male was more than women at 15-19 Sui, and the predilection site of osteosarcoma is patient's long bone The dry dirty end of active growth, wherein more common with distal femur and proximal tibia, followed by humerus and fibula near-end.Osteosarcoma is disliked Property degree it is high and progress is fast, early stage easily occurs Blood route metastasis and Lung metastases easily occurs, high recurrence rate, and prognosis is also poor. It is to threaten most common primary malignant bone tumor at present.

Although we have newer understanding to the study of incident mechanism aspect of osteosarcoma in recent years, face for improvement In terms of bed therapeutic effect or inadequate, the treatment of human bone osteosarcoma is still the problem that orthopaedics is currently faced.Although current bone and flesh The comprehensive treatment that knurl combines other auxiliary treatments again based on surgical resection has made great progress, but osteosarcoma Grade malignancy is generally higher, even there is no DISTANT METASTASES IN Patients with Osteosarcoma after treatment existence in its 5 years be 60%~ 65%.Patients with Osteosarcoma mostly occurs Lung metastases, with 5 years survival rates after the Patients with Osteosarcoma treatment of DISTANT METASTASES IN be only 20~ 29%.

The early diagnosis of osteosarcoma relies primarily on foundation, and effectively easily Noninvasive diagnosis technology and osteosarcoma specificity are divided The screening of son mark；And treating bone and flesh tumor metastasis bone and flesh tumor metastasis relative specific molecule clear and definite first can simultaneously be screened.Mesh The two preceding aspects have the research of clinical value not yet.The research of effect of the gene of rising in recent years in terms of tumour is upper The exploration for stating another field provides very promising research strategy, such as patent CN 201510075917.2, CN 201510075920.4th, CN201510075918.7, CN201510075919.1 just pair with the relevant base of osteosarcoma occurrence and development Because being studied.But the biomarker for the osteosarcoma reported so far is still less, it is impossible to meet clinical diagnosis and The needs for the treatment of, therefore new effective biomarker is found, it diagnoses and controls for the accurate of individuation for realizing osteosarcoma Treatment has great importance.

The content of the invention

In order to make up for the deficiencies of the prior art, it is an object of the invention to provide a kind of relevant with osteosarcoma occurrence and development Biomarker, and then applied to the clinical diagnosis and treatment of osteosarcoma, realize the accurate medical treatment of patient, improve the life of patient And life quality.

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

The present invention provides a kind of purposes of MUC21 genes, are used to prepare prevention or treat the pharmaceutical composition of osteosarcoma.

Further, described pharmaceutical composition includes the inhibitor of MUC21 functional expressions.The inhibitor is selected from：With MUC21 or its transcript for target sequence and can inhibit the disturbing molecule of MUC21 gene expressions or genetic transcription, including： ShRNA (children purpura nephritis), siRNA (siRNA), dsRNA, Microrna, antisensenucleic acids can be expressed or formed and is described ShRNA, siRNA, dsRNA, Microrna, the construction of antisensenucleic acids；Or the protein binding that specificity is encoded with MUC21 Binding molecule (antibody or ligand that can such as inhibit MUC21 protein actives).

Further, the inhibitor is siRNA.

Further, the sequence of the siRNA is as shown in SEQ ID NO.7 and SEQ ID NO.8.

The present invention provides a kind of for preventing or treating the pharmaceutical composition of osteosarcoma, described pharmaceutical composition includes The inhibitor and/or pharmaceutically acceptable carrier of MUC21 functional expressions.

Further, the inhibitor is selected from：

Nucleic acid inhibitor, protein inhibitor, proteolytic enzyme, protein binding molecule can be in albumen or gene level The expression of the upper albumen for lowering MUC21 genes or its coding or activity.

In the present invention, the inhibitor of the MUC21 can be additionally used in the multiplication for inhibiting osteosarcoma cell.

The present invention provides a kind of purposes of MUC21 genes, for screening the candidate compound of prevention or treatment osteosarcoma.

Further, the step of candidate compound of screening prevention or treatment osteosarcoma includes：

In test group, the addition test compound in the cultivating system of cell, and observe in the cell of the test group The expression quantity and/or activity of MUC21；In control group, do not add test compound in the cultivating system of same cell, and see Examine the expression quantity and/or activity of MUC21 in the cell of control group；

Wherein, if the expression quantity of the MUC21 of cell and/or activity indicate that the test less than control group in test group Compound is expression to MUC21 and/or activity have inhibitory action treating cancer candidate compound.

In the present invention, the step further includes：The candidate compound of acquisition is carried out further cell experiment and/ Or animal experiment, further to select and determine from candidate compound for preventing, alleviating or treating the useful object of osteosarcoma Matter.

In the present invention, the system of the candidate compound of screening prevention or treatment osteosarcoma is not limited to cell system, also wraps Cell system, subcellular fraction system, solution system, organizational framework, organ systems or animal system etc. are included, the system is not limited to Above-mentioned form, as long as the system can detect test compound and can reduce expression and the/activity of MUC21.

The candidate compound includes but not limited to：Albumen or its upstream or downstream for MUC21 genes or its coding The disturbing molecule of gene or protein design, nucleic acid inhibitor, binding molecule (such as antibody or ligand), micromolecular compound.

The present invention provides a kind of reagent for detecting MUC21 gene expression doses in the product for preparing diagnosis osteosarcoma Using.The product includes but is not limited to chip, preparation or kit.

Further, the reagent is selected from：

The probe of specific recognition MUC21；Or

The primer of specific amplification MUC21；Or

Specifically bind the antibody or ligand of the albumen of MUC21 codings.

Further, the primer sequence of the specificity amplification MUC21 is as shown in SEQ ID NO.1~2.

Description of the drawings

Fig. 1 is the expression figure in osteosarcoma tissue using QPCR detection MUC21 genes；

Fig. 2 is the expression figure in osteosarcoma tissue using Western Blot detection MUC21 albumen；

Fig. 3 is the influence figure transfected using QPCR detections siRNA to MUC21 gene expressions in osteosarcoma cell；

Fig. 4 is the influence transfected using western blot detections siRNA to MUC21 protein expressions in osteosarcoma cell Figure；

Fig. 5 is the influence figure with mtt assay detection MUC21 gene pairs human osteosarcoma cell proliferations.

Specific embodiment

The present invention, by high-flux sequence method, detects gene in osteosarcoma samples and exists by in-depth study extensively Tumor tissues and the expression of cancer beside organism find wherein there is the genetic fragment of apparent differential expression, inquire into itself and osteosarcoma Relation between generation, so as to which the early detection for osteosarcoma and targeted therapy find better approaches and methods.By screening, Present invention firstly discovers that MUC21 conspicuousnesses raise in osteosarcoma.It is demonstrated experimentally that the expression by reducing MUC21, it can The multiplication of osteosarcoma cell is effectively inhibited, osteosarcoma early diagnosis can be become by prompting the expression of detection MUC21 genes One of auxiliary diagnostic index, interference MUC21 gene expressions can become the new way prevented or treat osteosarcoma.

Marker

Marker (is used alone or such as bone and flesh tumor markers, osteosarcoma specificity marker is combined with other qualitative terms Object, control marker, external source marker, endogenous marker) refer to it is measurable, can calculate or can otherwise obtain, with appointing What molecule or molecular combinations are related, can be used as the parameter of the indicant of biology and/or chemical state.In the present invention, " mark Object " refers to the core with one or more biomolecule relevant parameter (i.e. " biomarker ") for example natural or artificial synthesized generations Acid (i.e. genes of individuals and coding and noncoding DNA and RNA) and albumen (such as peptide, polypeptide)." marker " in the present invention Further include the list for referring to and can calculating or otherwise obtain by considering the expression data from two or more unlike signal objects A parameter.

Bone and flesh tumor markers, which refer to, may be used as and (combining individually or with other markers) osteosarcoma indicant in subject Certain types of marker, the present invention specific embodiment in, bone and flesh tumor markers can be used for provide (individually or and other Marker combines) marker of osteosarcoma clinical assessment in subject.

" endogenous marker " refers to the marker (such as nucleic acid or polypeptide) from the subject identical with sample to be analysed.More Specifically, " endogenous control marker " refer to it is available compares marker (individually or with other control markers combining), with treating point Analyse the marker that sample is derived from same subject.In one embodiment, endogenous control marker may include one or more Endogenous gene (i.e. " crt gene " or " reference gene "), expression are stablized relatively, such as in osteosarcoma and non-osteosarcoma sample In and/or between subjects.

" external source marker " refers to the marker (such as nucleic acid or polypeptide) from the subject different from sample to be analysed.More Specifically, " external source control marker ", which refers to can use, compares marker (individually or with other control markers combining), is not derived from The marker of the subject identical with sample to be analysed.In the present invention, on the one hand the external source marker or external source control indicate Object is seen from different subject's separation or can synthetically be produced, and may be added to that sample to be analyzed.On the other hand, the external source pair Can be the molecule that addition or mark-on are used as internal positive or negative reference material into sample to be analysed according to marker.External source compares Marker can be used in conjunction with distinguishing " true negative " result (such as non-osteosarcoma with the detection of one or more bone and flesh tumor markers Diagnosis) and " false negative " or " information is not provided " result (such as the problem of due to amplified reaction).

" control marker " or " reference marker " refers to may for (individually or with other control markers combining) control Disturbing factor and/or provide on sample quality, effective sample preparation and/or appropriate reaction combination/progress (such as RT- PCR react) one or more indexs concrete type marker.In some embodiments, control marker can be Endogenous control marker, external source control marker and/or osteosarcoma Specificity control marker as described herein.To sighting target Will object can altogether be detected with the bone and flesh tumor markers of the present invention or detected respectively.Control marker can be one or more endogenous bases Cause, such as the combination of house-keeping gene or osteosarcoma Specificity control marker or gene.

MUC21 genes

In the present invention, MUC21 is to be located at the gene that takes of 2 area 1 of No. 6 the short arm of a chromosome of people, MUC21 include wild type, Saltant type or its segment.A kind of representative MUC21 genes have in database GeneBank represented by NC_000006.12 Nucleotide sequence or amino acid sequence.

People's MUC21 nucleotide full length sequence of the present invention or its segment can usually use PCR amplification method, recombination method or artificial Synthetic method obtains.For PCR amplification method, can according to published related nucleotide sequence, and with commercially available cDNA storehouses or CDNA storehouses as prepared by conventional method well known by persons skilled in the art expand as template and obtain related sequence.Work as sequence When longer, it is often necessary to carry out twice or multiple PCR amplification, the segment for then again amplifying each time are spliced by proper order Together.

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 appropriate nucleotides inserted or missing), nucleotide base at least about 60%, 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 with another nucleic acid (or its complementary strand), a chain or its complementary series 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.

Inhibitor and pharmaceutical composition

Discovery based on the present inventor, the present invention provides a kind of purposes of the inhibitor of MUC21, are used to prepare inhibition bone The pharmaceutical composition of sarcoma.As used herein, the inhibitor of the MUC21 includes but not limited to inhibitor, antagonist, retardance Agent, blocking agent, nucleic acid inhibitor etc..

The MUC21 genes or the inhibitor of albumen refer to any activity for reducing MUC21 albumen, reduce MUC21 The stability of gene or albumen, the expression for lowering MUC21 albumen reduce MUC21 albumen effective acting times or inhibit MUC21 The substance of the transcription and translation of gene, these substances are used equally for the present invention, as lowering the useful substances of MUC21, from And it can be used for preventing or treating osteosarcoma.For example, the inhibitor is：Nucleic acid inhibitor, protein inhibitor, antibody, ligand, Proteolytic enzyme, protein binding molecule, as long as it can lower MUC21 albumen or its encoding gene on albumen or gene level Expression.

As a kind of selection mode of the present invention, the inhibitor of the MUC21 is that a species specificity is combined with MUC21 Antibody.The antibody can be monoclonal antibody or polyclonal antibody.MUC21 protein immune animals can be used, such as rabbit is small Mouse, rat etc. produce polyclonal antibody；A variety of adjuvants can be used for enhancing immune response, include but not limited to Freund's adjuvant etc.. Similar, it expresses MUC21 or its cell with antigenic segment can be used to immune animal to produce antibody.Described Antibody can also be monoclonal antibody, and such monoclonal antibody can be prepared using hybridoma technology." specificity " of antibody Refer to that antibody can be incorporated into MUC21 gene outcomes or segment.It is preferred that referring to those can be combined with MUC21 gene outcomes or segment But nonrecognition and the antibody for being incorporated into other non related antigen molecules.The antibody of the present invention can pass through those skilled in that art It is prepared by known various technologies.The present invention not only includes complete monoclonal or polyclonal antibody, but also including having Immunocompetent antibody fragment, such as Fab ' or (Fab) 2 segment；Heavy chain of antibody；Antibody light chain；Genetically engineered scFv Molecule；Or chimeric antibody.The antibody of anti-MUC21 albumen can be used in immunohistochemistry technology, detect in biopsy specimen MUC21 protein contents are also used as preventing the specific treatment agent of osteosarcoma.MUC21 albumen in blood sample or urine Directly measure can as tumour auxiliary diagnosis and more after observation index, also can be as the foundation of early diagnosis of tumor. Antibody can be coupled by ELISA, WesternBlot engram analysis or with detection moiety, pass through chemiluminescence, isotope The methods of tracer, is detected.

As a kind of preferred embodiment of the present invention, the inhibitor of the MUC21 is a kind of small interference of MUC21 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 (RNAinterference) 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 syntheses, by anneal, can generate the double-strand of synthesis thereafter RNA compounds.

When screening effective siRNA sequence, the present inventor is optimal effective so as to find out by largely comparing analysis Segment.The present inventor's design has synthesized a variety of siRNA sequences, and they are transfected osteosarcoma cell line by transfection reagent respectively It is verified, select the optimal siRNA of interference effect, they are respectively provided with the sequence shown in SEQIDNO.7, SEQIDNO.8, into It is tested in cellular level to one step, as a result proves that inhibition efficiency is very high for test cell line.

As a kind of optional mode of the present invention, the inhibitor of the MUC21 can also be a kind of " children purpura nephritis (SmallhairpinRNA, shRNA) " is the non-coding small RNA molecular that can form hairpin structure, and children purpura nephritis can By RNA interference channels come the expression of suppressor.As above-mentioned, shRNA can be expressed by double-stranded DNA template.Double-stranded DNA mould Plate is inserted into a carrier, such as plasmid or viral vectors, is then connected to a promoter in vitro or in vivo and is expressed. ShRNA under the action of DICER enzymes, can be cut into siRNA molecule in eukaryocyte, hence into RNAi approach. " shRNA expression vectors " refers to plasmid of some this fields conventionally used for building shRNA structures, exist on the usual plasmid " Every sequence " and positioned at " intervening sequence " both sides multiple cloning sites or for replace sequence, so as to people can by shRNA (or Analog) corresponding DNA sequence dna be inserted by way of forward and reverse multiple cloning sites or replace thereon for replacing sequence, RNA after DNA sequence dna transcription can form shRNA (ShortHairpin) structure." the shRNA expression vectors " is current It is obtained through that can be bought completely by commercially available approach, such as some viral vectors.

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 appropriate transfection reagent quilt It is transported into the cell or multiple technologies known in the art also can be used and be transported into the cell.

Pharmaceutical composition

The present invention also provides a kind of pharmaceutical compositions, it contains the inhibitor and medicine of a effective amount of MUC21 Acceptable carrier on.The composition can be used for inhibiting osteosarcoma.The inhibitor of any foregoing MUC21 is used equally for The preparation of composition.

As used herein, described " effective quantity " refer to that people and/or animal can be generated function or activity and can by people and/ Or the amount that animal is received.The effective quantity of inhibitor can become with the pattern of administration and the severity of disease to be treated etc. Change.Preferred a effective amount of selection can be determined (such as to pass through clinic by those of ordinary skill in the art according to various factors Experiment).The factor includes but not limited to：The pharmacokinetic parameter of the inhibitor of the MUC21 genes is for example biological Utilization rate, metabolism, half-life period etc.；Patient the severity of disease to be treated, the weight of patient, patient immune state, Approach of administration etc..

" pharmaceutically acceptable carrier " refers to the carrier for Therapeutic Administration, including various excipient and dilution Agent.The term refers to some such medicament carriers：Themselves it is not necessary active ingredient, and does not have undue poison after applying Property.Suitable carrier is well known to those of ordinary skill in the art.Pharmaceutically acceptable carrier can contain in the composition 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 (host cell) transfection examination can also be contained in the carrier Agent.

The present invention may be employed 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.It for example, can be directly by the inhibitor of MUC21 by such as noting The methods of penetrating delivers medicine to subject；Alternatively, can will be carried by certain approach the inhibitor of MUC21 ceneme (such as Expression vector or virus etc. or siRNA or shRNA) it is delivered on target spot, and the MUC21 inhibitor of expression activity is allowed to, specifically Situation need to be depending on the type of the inhibitor, these are well-known to those skilled in the art.

Term " host cell " can be prokaryotic cell, such as bacterial cell；Or low eukaryocyte, such as yeast cells； Or higher eucaryotic cells, such as mammalian cell.Representative example has：Escherichia coli, the bacterial cell of streptomyces；Fungi Cell such as yeast；Plant cell；The insect cell of drosophila S2 or Sf9；Zooblast of CHO, COS or 293 cells etc..

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

Pharmaceutical composition in the present invention includes the inhibitor of MUC21, and/or other medicine classes with the inhibitor compatibility And pharmaceutically acceptable carrier and/or auxiliary material.

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

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

Drug screening

The present invention provides it is a kind of screen prevention or treatment bone and flesh tumor medicine method, i.e.,：

In experimental group, testing compound is added in into cell culture system, and measures the expression of MUC21；Right According in group, testing compound is added without into same cultivating system, and measures the expression of MUC21；Wherein, if experiment The expression of MUC21 is more than control group in group, then illustrates the inhibitor that the candidate compound is MUC21.

In the present invention, the method further includes：The candidate compound obtained to previous step further tests its suppression The effect of osteosarcoma processed if test compound has osteosarcoma significant inhibition, illustrates the compound for prevention or controls Treat the potential substance of osteosarcoma.

Detection method

The present invention can utilize any method known in the art to measure gene expression.Those skilled in the art should manage Solution, the means for measuring gene expression are not the importances of the present invention.The example of rna expression in quantitative sample known in the art Property method include but not limited to Southern traces, Northern traces, microarray, PCR (PCR), NASBA, TMA, RT-PCR, real-time fluorescence quantitative PCR.

In the present invention, term " up-regulation " or " overexpression " refer in cancerous tissue (such as in osteosarcoma tissue) with compared with Horizontal high expression (such as RNA and/or protein expression) gene in other corresponding tissues.The gene raised in cancer with than Other organize the expression height at least 10%, preferably at least 25% in (such as normal or non-cancerous bone tissue) accordingly, More preferably at least 50%, more preferably at least 100%, more preferably at least 200%, most preferably at least 300% level Expression.

Chip, kit

The genetic chip of the present invention includes：Solid phase carrier；And the widow on the solid phase carrier is fixed in order Nucleotide probe, the oligonucleotide probe specifically correspond to the part or all of sequence shown in MUC21.

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

" probe " be intended to cover in promotion hybridization under conditions of with the target sequence specific hybrid in nucleic acid or its complement, So as to allow the nucleic acid oligomer or aptamer that detect target sequence or the nucleic acid of its amplification.Detection can be directly (i.e. by direct Generated with the probe that target or the sequence of amplification hybridize) or indirectly (i.e. by in the sequence of linking probe and target or amplification Between molecular structure hybridize probe generate)." target " of probe is often referred in the nucleotide sequence of amplification and at least part probe The sequence that sequence passes through standard hydrogen bond or " base pairing " specific hybrid.The sequence of " substantially complementary " allow probe sequence with Target sequence stablizes hybridization, even if two sequences are not fully complementary.Probe can be labeled or not mark.Probe can be by specific The molecular cloning production of DNA sequence dna, can also be synthesized.Those skilled in the art in the invention can easily determine can be in this hair A variety of primer and probes of design and use under bright background.

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

The conventional manufacturing method of biochip known in the art can be used in the preparation of the MUC21 chips.For example, such as Fruit solid phase carrier is gone here and there using modification slide or silicon chip, 5 ' ends of probe containing amido modified poly- dT, can be by oligonucleotides Probe is configured to solution, then using point sample instrument by its point modification slide or silicon chip on, be arranged in predetermined sequence or array, Then fixed by standing overnight, so that it may obtain the genetic chip of the present invention.

The present invention provides a kind of kit, the kit can be used for the expression of detection MUC21 genes or albumen.It is preferred that , also containing being useful for the label of labeled RNA sample and corresponding with the label in the preparation or kit Substrate.In addition, may also include to extract the various reagents needed for RNA, PCR, hybridization, colour developing etc. in the kit, wrap It includes but is not limited to：Extract, amplification liquid, hybridization solution, enzyme, comparison liquid, developing solution, washing lotion etc..In addition, in the kit also Include the use of specification and/or chip image analysis software.

The component of kit can pack in the form of aqueous medium or in the form of lyophilized.Appropriate container in kit Typically at least include a kind of bottle, test tube, flask, PET bottle, syringe or other containers, wherein a kind of component can be placed, and And preferably, suitably decile can be carried out.There are during more than one component in kit, will generally also be included in kit Second, third or other additional containers, wherein being positioned separately additional component.However, the component of various combination can be wrapped It is contained in a bottle.The kit of the present invention for accommodating the container of reactant, will generally also be sealed including a kind of for Commercial distribution.This container may include the plastic containers of injection molding or blowing mould, wherein can retain required bottle.

The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this It invents rather than limits the scope of the invention.The experimental method of actual conditions is not specified in embodiment, usually according to conventional strip Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in or according to the condition proposed by manufacturer.

Embodiment 1 and the screening of the relevant gene marker of osteosarcoma

1st, sample collection

6 osteosarcoma tissues and cancer beside organism's sample are respectively collected, the acquirement of tissue samples obtains the informed consent of patient, and And it obtains and passes through the agreement of the committee of organizational ethics.

2nd, the preparation of RNA sample

RNA is extracted using the tissue RNA extracts kits of Invitrogen companies, concrete operations are with reference to specification.

3rd, the quality analysis of RNA sample

The RNA concentration and purity extracted are detected using Nanodrop2000, agarose gel electrophoresis detection RNA Integrality, Agilent2100 measure RIN values.Concentration >=200ng/ μ l, OD260/280 is between 1.8~2.2.

4th, rRNA is removed

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

5th, construction cDNA library

The structure of cDNA library is carried out using the Truseq RNA sample Prep Kit using Illumina, it is specific to grasp Make by specification progress.

6th, upper machine sequencing

CDNA library is sequenced using Hiseq4000 microarray datasets, concrete operations by specification carries out.

7th, high-throughput transcript profile sequencing data analysis

Bioinformatic analysis is carried out to sequencing result, trim, trim are carried out to the 5 ' of reads and 3 ' ends with cutadapt Fall quality<20 base, and the reads that N is more than 10% is deleted, tophat is compared onto reference gene group, is used The expression quantity of cuffquant quantification of mrna, cuffdiff compare differential expression of the control group with tumor group, the screening of differential gene Standard is with fdr<0.05, the difference of two groups of fpkm average values is more than 5.

8th, result

RNA-seq is the results show that MUC21 expressions significantly raise in Patients with Osteosarcoma.

The differential expression of 2 QPCR sequence verification MUC21 genes of embodiment

1st, large sample QPCR verifications are carried out to MUC21 gene differential expressions.It is selected according to the sample collection mode in embodiment 1 Select Patients with Osteosarcoma cancer beside organism and each 50 of osteosarcoma tissue.

2nd, RNA extracts specific steps as described in Example 1.

3rd, reverse transcription

Using FastQuant cDNA the first chain synthetic agent box (article No.s：KR106 mRNA reverse transcriptions) are carried out.Specific steps It is as follows：

(1) 5 × gDNA Buffer 2.0 μ l, 1 μ g of total serum IgE are added in and adds Rnase Free ddH₂O makes total volume to 10 μ L, 42 DEG C of heating 3min in water-bath；

(2) 20 μ l reaction systems are built：10 × Fast RT Buffer, 2.0 μ L, RT Enzyme Mix 1.0 μ l, FQ- 2.0 μ l, RNase Free ddH of RT Primer Mix₂Mixing in the mixed liquor in (1) is added in after 5.0 μ l of O mixing；

(3) 42 DEG C of heating 15min in water-bath, 95 DEG C of heating 3min, -20 DEG C store for future use.

4th, QPCR is expanded

(1) design of primers

QPCR amplimers are designed according to the coded sequence of MUC21 genes in Genebank and house-keeping gene GAPDH genes, It is synthesized by Bo Maide companies.

The primer sequence of MUC21 genes：

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

Reverse primer sequences are 5 '-GATCCAGTGTTGGCAGAG-3 ' (SEQ ID NO.2).

The primer sequence of GAPDH genes：

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

Reverse primer sequences are 5 '-GGCTGTTGTCATACTTCTCATGG-3 ' (SEQ ID NO.4).

(2) PCR reaction systems：Forward primer and each 0.6 μ l, 2 × SuperReal PreMix Plus, 10 μ of reverse primer L, DNA profiling 2 μ l, ddH₂7.4 μ l, 50 × ROX Reference Dye of O^△2 μ l, 4.8 μ l of sterile purified water.

(3) PCR reaction conditions：95 DEG C of 15min, (95 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 32s) × 40 Xun Huans, 95 DEG C of 15s, 60 DEG C of 60s, 95 DEG C of 15s.PCR reactions are carried out on 7300 type fluorescence quantitative PCR instruments of ABI, pass through melt curve analysis analysis and electrophoresis Determine purpose band, Δ Δ CT methods carry out relative quantification.

5th, statistical method

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

6th, result

The results are shown in Figure 1, and compared with cancer beside organism, MUC21 up-regulated expressions in osteosarcoma tissue, difference has statistics Learn meaning (P<0.05) it is, consistent with high-flux sequence result.

The differential expression of 3 protein immunization imprinting of embodiment experiment detection MUC21 albumen

1st, the extraction of total protein is organized

It puts it into and is placed in the glass homogenizer in ice after shredding tissue with scissors, RIPA lysates and PMSF are with 100: 1 ratio mixing adds in the RIPA lysates of the ratio addition respective amount of 100 μ l lysates, glass according to every 20mg tissue specimens Glass homogenizer pulverize tissue until its fully crack, the liquid after cracking is drawn in EP pipes, at 4 DEG C 14000rpm centrifuge 5min collects supernatant.

2nd, total protein concentration measures

The measure of protein concentration is carried out according to the specification of BCA determination of protein concentration kits.

3rd, SDS-PAGE electrophoresis

According to the separation gel of the specification preparation 8% of PAGE gel reagent preparation box and 5% concentration glue and carry out Electrophoresis.

4th, western is detected

1) electrotransfer

Pvdf membrane is put into methanol solution and activates 5min, is put into transferring film buffer solution and balances 20min.PAGE glue is taken out to put Enter in transferring film buffer solution, cut corresponding PAGE glue, according to be followed successively by from down to up filter paper, pvdf membrane, PAGE glue, filter paper it is suitable Sequence is put into half-dried transferring film instrument, constant pressure 25V transferring films 1.5h；

2) immuning hybridization

Pvdf membrane is taken out, PBS flushings are placed in 5%BSA solution shakes closing 2h at room temperature, and pvdf membrane is put into hybridization In bag, add in primary antibody and stay overnight, wash pvdf membrane with TBST buffer solutions, add corresponding secondary antibody, be incubated 2h at room temperature, TBST delays Fliud flushing is washed.

3) DAB develops the color

The slightly dry rear DAB developing solutions that Fresh is added dropwise of pvdf membrane, record is scanned after pvdf membrane colour developing.Made with β-actin For internal reference, sxemiquantitative gray analysis carries out band using Quantity One Labworks image acquisition and analysis softwares, experiment repeats 3 It is secondary, as a result take average gray value；

5th, statistical analysis

Statistical analysis is carried out using SPSS18.0 statistical softwares, result data is all in a manner of mean+SD Come what is represented, analyzed using the variance test (ANOVA) of various sample average, it is believed that work as P<There is statistics meaning when 0.05 Justice.

6th, result

The results are shown in Figure 2, and in osteosarcoma tissue, MUC21 protein expression levels are significantly higher than cancer beside organism.

The silence of 4 MUC21 genes of embodiment

1st, cell culture

Cell line of human osteosarcoma U-2OS, with contain the DMEM culture mediums of 10% hyclone and 1%P/S 37 DEG C, 5% CO₂, relative humidity be 90% incubator in cultivate.Change within 2-3 days liquid 1 time, cell growth is good, is grown in monolayer adherence.Make It is passed on the 0.25% trypsase conventional digestion containing EDTA.

2nd, transfect

1) precellular processing is transfected

The day before transfection plants 3~5 × 10 on 6 well culture plates⁵A cells/well cultivates one in antibiotic-free culture medium My god, cell density is 30~50% during transfection, changes serum free medium into before transfection.

2) design of siRNA

Negative control siRNA sequence (siRNA-NC)：

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

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

siRNA1：

Positive-sense strand is 5 '-ACAUAAGGAGAACAUUUCCUU-3 ' (SEQ ID NO.7)

Antisense strand is 5 '-GGAAAUGUUCUCCUUAUGUUU-3 ' (SEQ ID NO.8)

siRNA2：

Positive-sense strand is 5 '-UCUCAUUGGAAUUUGUUGCAG-3 ' (SEQ ID NO.9)

Antisense strand is 5 '-GCAACAAAUUCCAAUGAGACU-3 ' (SEQ ID NO.10)

siRNA3：

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

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

Experiment is divided into three groups：Control group (U-2OS), negative control group (siRNA-NC) and experimental group (siRNA1, SiRNA2, siRNA3), the sequence of wherein negative control group siRNA and MUC21 genes is without homology.

3) transfect

It is transfected using the liposome Lipofectamine 2000 of Invitrogen companies, by specification is grasped Make, be as follows：

A. the 3 μ l of siRNA that concentration is 50pmol is taken to add in the serum free medium of 47 μ l, gently mixing, incubation at room temperature 5min；

B. 1 μ l Lipofectamine 2000 is taken to add in 49 μ l serum free mediums.Gently mixing is incubated at room temperature 5min；

C. above two mixture is mixed into (100 μ l of total volume), gently mixing, is incubated 25min at room temperature, so that compound Body is formed；

D. the compound of 100 μ l and appropriate culture medium are added in per hole in 6 orifice plates, gently mixing；

E. the silence effect of gene is observed after 48~96h of incubation.

5th, QPCR detects the transcriptional level of MUC21 genes

The extraction of 5.1 cell total rnas

The RNA in cell is extracted using the cell RNA extracts kit of Qiagen, experimental implementation is to specifications It carries out.

5.2 reverse transcription steps are the same as embodiment 2.

5.3QPCR amplification steps are the same as embodiment 2

6th, statistical method

Experiment is all completed according to being repeated 3 times, and result data is represented in a manner of mean+SD, Statistical analysis is carried out using SPSS18.0 statistical softwares, the difference between MUC21 Gene Experiments group and control group is examined using t It tests, it is believed that work as P<There is statistical significance when 0.05.

7th, result

As a result such as Fig. 3 is shown, with non-transfection group compared with transfecting siRNA-NC groups, experimental group siRNA1's and siRNA2 MRNA level in-site is all declined, and siRNA1 interference effect highests, therefore siRNA1 is selected to carry out subsequent experiment.

Influences of 5 Western blot of the embodiment detection transfections siRNA to MUC21 protein expressions

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

2nd, the extraction of total protein of cell

The cell of the different disposal group in logarithmic phase is collected, cell is washed with the PBS of precooling.By RIPA cell pyrolysis liquids With PMSF with 100:1 ratio mixing adds in above-mentioned 150 μ l of lysate into cell, places 30min on ice, use cell scraper Knife scrapes off the cell of cracking, and the liquid after cracking is drawn in EP pipes using pipettor, and 14000rpm is centrifuged at 4 DEG C 5min.Supernatant after careful collection centrifugation.

3rd, total protein concentration measures

4th, SDS-PAGE electrophoresis

5th, western detecting steps detailed in Example 3.

6th, statistical analysis

7th, result

The results are shown in Figure 4, compared with the control group, transfects the MUC21 albumen tables in the cell of siRNA1 and siRNA2 groups It is all lowered up to amount, wherein siRNA1 groups are the most notable, therefore selection siRNA1 does the research of subsequent experimental.

The influence of 6 MUC21 gene pairs human osteosarcoma cell proliferations of embodiment

MUC21 gene pairs human osteosarcoma cell proliferation capacities are detected using MTT experiment

1st, the good cell of upgrowth situation is taken, conventional digestion counts cell, cell is diluted to conjunction into after single cell suspension The cell suspension of suitable concentration.

2nd, in 96 well culture plates, the different disposal group cell per well after dilution is inoculated with 2000 cells, 3 is at least set and puts down Row hole and cell-free medium control, 37 DEG C, 5%CO₂Culture is for 24 hours.

3rd, 1 after inoculation, 2,3,4,5 days daily OD values taken out 3 hole cells and its 490nm is detected with mtt assay, counted Number calculates average value.

4th, abandoning supernatant before detecting, culture solution are washed 3 times, and MTT free serum cultures based sols (5mg/ml) 10 μ is added in per hole L continues in 37 DEG C of incubators to cultivate 4h, terminates culture.

5th, 100 μ l Formanzan lysates are added in per hole, shaking table shakes 1min slowly.With wavelength it is 490nm in microplate reader Optical density (OD) value is measured, using the time as transverse axis, OD value draws cell growth curve for the longitudinal axis.

6th, statistical analysis

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

7th, result

The results are shown in Figure 5, and compared with the control, for experimental group after siRNA1 is transfected, the multiplication of cell substantially receives suppression System, difference have statistical significance (P<0.05), illustrate that MUC21 plays an important role of to promote cell Proliferation.

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

Sequence table

<110>The first affiliated hospital of Bengbu Medical College（Attached tumour hospital of Bengbu Medical College）Liu Yang

<120>Applications of the biomarker MUC21 in osteosarcoma diagnose and treat

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Claims

The purposes of 1.MUC21 genes, which is characterized in that be used to prepare prevention or treat the pharmaceutical composition of osteosarcoma.
2. purposes according to claim 1, which is characterized in that described pharmaceutical composition includes MUC21 functional expressions Inhibitor.
3. purposes according to claim 2, which is characterized in that the inhibitor is siRNA.
4. purposes according to claim 3, it is characterised in that the sequence of the siRNA such as SEQ ID NO.7 and SEQ ID Shown in NO.8.
5. a kind of pharmaceutical composition for being used to preventing or treating osteosarcoma, which is characterized in that described pharmaceutical composition includes MUC21 The inhibitor and/or pharmaceutically acceptable carrier of functional expression.
6. pharmaceutical composition according to claim 5, which is characterized in that the inhibitor is selected from：

Nucleic acid inhibitor, protein inhibitor, proteolytic enzyme, protein binding molecule can be above and below albumen or gene level Adjust expression or the activity of the albumen of MUC21 genes or its coding.
7. a kind of purposes of MUC21 genes, which is characterized in that for screening the candidate compound of prevention or treatment osteosarcoma.
8. detect application of the reagent of MUC21 gene expression doses in the product for preparing diagnosis osteosarcoma.
9. application according to claim 8, which is characterized in that the reagent is selected from：

The probe of specific recognition MUC21；Or

The primer of specific amplification MUC21；Or

Specifically bind the antibody or ligand of the albumen of MUC21 codings.
10. application according to claim 9, which is characterized in that the primer sequence of the specificity amplification MUC21 is such as Shown in SEQ ID NO.1~2.