CN108165624A - Application of the biomarker in osteosarcoma diagnosis and treatment - Google Patents
Application of the biomarker in osteosarcoma diagnosis and treatment Download PDFInfo
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- CN108165624A CN108165624A CN201810031964.0A CN201810031964A CN108165624A CN 108165624 A CN108165624 A CN 108165624A CN 201810031964 A CN201810031964 A CN 201810031964A CN 108165624 A CN108165624 A CN 108165624A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The invention discloses application of the biomarker in osteosarcoma diagnosis and treatment, the biomarker is PNPLA5.The present invention has found PNPLA5 up-regulated expressions in Patients with Osteosarcoma by high-flux sequence, further study show that, the expression of PNPLA5 is interfered, the proliferation rate of osteosarcoma cell can be reduced, prompts PNPLA5 that can be applied to the clinical diagnosis and treatment of osteosarcoma as molecular indexes.
Description
Technical field
The invention belongs to biomedicine fields, are related to application of the biomarker in osteosarcoma diagnosis and treatment, specific biology
Indicate for PNPLA5.
Background technology
Osteosarcoma is most common Primary Malignant Bone Tumor, and the grade malignancy for being derived from embryo's mesenchymal tissue is very high
Solid tumor.Most of osteosarcoma are primary tumor, and only small part is secondary, and it is abundant that predilection site is located at blood fortune
The metaphysis of Limb bone, it is most common with lower limb distal femur and proximal tibia, secondly moon bright bone and fibula proximal end.Osteosarcoma is mostly
Bone resorption sexually revises, and minority is sexually revised for skeletonization, bone cartilage, fibrous connective tissue and increasing containing heterogeneity in osteosarcoma tissue
Raw bone sample bone tissue.Osteosarcoma Pathologic Characteristics are that tumor tissues can generate high malignancy fusiformis stroma cell, and form bone sample
Tissue, tumour cell can also directly skeletonization and generation bone matrix, it is wettable to diffuse to cortex of bone and pulp cavity.Also have at present new
Research shows that the malignant cell of osteosarcoma, not entirely from bone matrix cell, the multipotential stem cell of bone interstitial is pernicious
Proliferation can result in the generation of osteosarcoma.
Osteosarcoma incidence occupies first place in Primary Malignant Bone Tumor, osteosarcoma age of onset mostly at 15~25 years old,
Incidence is 4.5/100 ten thousand, and in the U.S., new Patients with Osteosarcoma is about 900 every year.Osteosarcoma how early phase DISTANT METASTASES IN, and
And quickly grow, the rate of transform and case fatality rate are very high.Simple row amputation treatment or row limb-sparing surgery and tumorous type prosthetic replacement
Operation can not effectively control the transfer of tumour, and patient often dies of neoplasm lung metastasis in a short time, how treat osteosarcoma with lung transfer
As facing one of most significant problems in clinical treatment of osteosarcoma.With the continuous improvement for the treatment of means, at present using new adjuvant chemotherapy
The comprehensive therapies such as limb-sparing surgery is added to have become the major way for the treatment of osteosarcoma, the short-term existence of patient can be effectively improved
Rate has been achieved for apparent progress to the treatment of osteosarcoma.With the fast development of the technologies such as operation and new adjuvant chemotherapy, do not send out
5 years survival rates of Patients with Osteosarcoma of raw transfer are increased to 70% or so, but still do not have to the problems such as bone and flesh tumor metastasis and recurrence
It is effectively solved, the short-term survival rate of Patients with Osteosarcoma shifted with lung does not significantly improve.
More and more treatment beginnings are close in terms of molecular genetics and molecular biology, from gene and stem cell angle
It goes to attempt the developing direction that treatment neoplastic lesion is medicine in future.The treatment of tumour at present includes interventional treatment, immunization therapy, does
The multiple means such as cell transplantation, the treatment for malignant tumour provides reliable and safety treatment, and gene is in osteosarcoma
Effect be also more taken seriously, as patent CN 201510075917.2, CN 201510075920.4,
WWP1, PNPLA5, CCT γ, PLEKHA5 and osteosarcoma are disclosed in CN201510075918.7, CN201510075919.1
Occurrence and development it is related.Although the research of osteosarcoma achieves significant progress, the life for the osteosarcoma reported so far
Object marker is still less, and not yet effective biomarker is applied to the clinical diagnosis and treatment of osteosarcoma.Therefore it finds
New effective biomarker, has great importance for the early diagnosis and targeted therapy of osteosarcoma.
Invention content
One of the objects of the present invention is to provide a diagnosis marker, which is PNPLA5.
The second object of the present invention provides a kind of product, can realize the early diagnosis of osteosarcoma, improves Patients with Osteosarcoma
Survival rate and quality of life.
The third object of the present invention provides a kind of therapeutic targets, realizes the personalized treatment of Patients with Osteosarcoma.
The fourth object of the present invention provides a kind of means for the drug for screening prevention and treatment osteosarcoma, when being screened medicine
When object can effectively reduce the expression of biomarker, it is potential drug candidate to illustrate the drug.
To achieve these goals, present invention employs following technical solutions：
Application in the product for diagnosing osteosarcoma in preparation the present invention provides the reagent of detection PNPLA5 expressions.Its
In, PNPLA5 up-regulated expressions in Patients with Osteosarcoma.
Further, the reagent is included by using qRT-PCR, marking hybridization, in situ hybridization, hybridization array, gene core
Piece or new-generation sequencing detect the reagent of PNPLA5 expressions.
Further, the reagent is selected from：
The probe of specific recognition PNPLA5 genes；Or
The primer of specific amplification PNPLA5 genes；Or
Specifically bind the antibody or ligand of the albumen of PNPLA5 codings.
Further, the reagent is selected from the primer of specific amplification PNPLA5 genes, the sequence such as SEQ ID of the primer
Shown in NO.1~2.
The present invention provides a kind of product for diagnosing osteosarcoma, the product includes PNPLA5 expressions in detection sample
Preparation, chip or kit, chips include genetic chip, protein chip；Kit includes gene detecting kit, egg
White detection kit.
Further, the gene detecting kit includes the primer of specific amplification PNPLA5.Preferably, the primer
Sequence is as shown in SEQ ID NO.1~2.
The present invention provides applications of the PNPLA5 in the compound of screening prevention or treatment osteosarcoma.
Inhibitor the present invention provides PNPLA5 functional expressions is in the pharmaceutical composition for preparing prevention or treatment osteosarcoma
Application in object.
Further, the inhibitor is selected from：Nucleic acid inhibitor, protein inhibitor, proteolytic enzyme, protein binding molecule；
The preferred inhibitor is nucleic acid inhibitor siRNA.
Preferably, the sequence of the siRNA is as shown in SEQ ID NO.7~8.
The present invention provides a kind of pharmaceutical compositions for treating osteosarcoma, and it is functional that described pharmaceutical composition includes PNPLA5
The inhibitor of expression.
The inhibitor is selected from：Using PNPLA5 or its transcript as target sequence and can inhibit PNPLA5 gene expressions or
The disturbing molecule of genetic transcription, including：ShRNA (children purpura nephritis), siRNA (siRNA), dsRNA, Microrna, antisense
Nucleic acid or the construction that can express or be formed the shRNA, siRNA, dsRNA, Microrna, antisense nucleic acid；It is or special
Property with PNPLA5 coding protein bound binding molecule (antibody or ligand that can such as inhibit PNPLA5 protein actives).
Further, described pharmaceutical composition further includes pharmaceutically acceptable carrier.The carrier includes but is not limited to
Diluent, excipient, adhesive, wetting agent, sorbefacient, surfactant, Humectant, absorption carrier, lubricant, buffering
Agent, stabilizer, bacteriostatic agent, isotonic agent, chelating agent, pH controlling agents.
The advantages of the present invention：
Present invention firstly discovers that the expression of PNPLA5 is to the occurrence and development of osteosarcoma related, by detecting PNPLA5
Level, it can be determined that whether patient with osteosarcoma or suffers from the risk of osteosarcoma.
The present invention provides a kind of product for diagnosing osteosarcoma, which can fall ill in osteosarcoma is diagnosed in early days,
Doctor is instructed to carry out early intervention, improves the survival rate and quality of life of patient.
The present invention provides a kind of pharmaceutical compositions and drug targets for treating osteosarcoma, can be realized by targeted therapy
Precision is treated.
Description of the drawings
Fig. 1 is to detect PNPLA5 genes expression figure in osteosarcoma tissue using QPCR；
Fig. 2 is the expression figure in osteosarcoma tissue using Western Blot detection PNPLA5 albumen；
Fig. 3 is the influence figure transfected using QPCR detections siRNA to PNPLA5 gene expressions in osteosarcoma cell；
Fig. 4 is the influence figure to PNPLA5 albumen in osteosarcoma cell using Western Blot detection siRNA transfections；
Fig. 5 is the influence figure with mtt assay detection PNPLA5 gene pairs human osteosarcoma cell proliferations.
Specific embodiment
The present invention after extensive and in-depth study, by high-flux sequence method, detects gene in osteosarcoma samples and exists
Tumor tissues and the expression of cancer beside organism find wherein there is the gene of apparent differential expression, inquire into its generation with osteosarcoma
Between relationship, so as to which the early detection for osteosarcoma and targeted therapy find better approaches and methods.Pass through screening, this hair
It is bright to be found that in osteosarcoma that PNPLA5 conspicuousnesses raise for the first time.It is demonstrated experimentally that the expression by reducing PNPLA5, Neng Gouyou
Inhibit to effect the growth and invasion of osteosarcoma cell, prompt the expression of detection PNPLA5 genes that can become osteosarcoma and examine in early days
One of disconnected auxiliary diagnostic index, interference PNPLA5 gene expressions can become prevent or treatment osteosarcoma or bone and flesh tumor metastasis it is new
Approach.
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, in specific embodiments of the present invention, bone and flesh tumor markers can be used for provide (individually or and other
Marker combines) marker of osteosarcoma clinical assessment in subject.
PNPLA5 genes
PNPLA5 (NC_000022.11) is taken positioned at 1 area 3 of No. 22 chromosome long arms of people, the PNPLA5 packets in the present invention
Include wild type, saltant type or its segment.The people PNPLA5 nucleotide full length sequences or its segment of the present invention can usually be expanded with PCR
Increasing method, recombination method or artificial synthesized method obtain.
Detection technique (method)
The gene of the present invention is detected using a variety of detection techniques known to persons of ordinary skill in the art, these technologies
Including but not limited to：Nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification technologies, immunoassay technology.
The exemplary, non-limitative example of Nucleic acid sequencing techniques includes but not limited to chain terminator (Sanger) sequencing and dye
Expect terminator sequencing.Those of ordinary skill in the art it will be recognized that due to RNA in cell less stable and in an experiment
Be more vulnerable to nuclease attack, thus before sequencing usually by RNA reverse transcriptions into DNA.
The another exemplary non-limiting examples of Nucleic acid sequencing techniques include next-generation sequencing, and (deep sequencing/high pass measures
Sequence), high throughput sequencing technologies be it is a kind of based on unimolecule cluster in synthesis sequencing technologies, based on proprietary reversible termination chemistry
Reaction principle.The random fragment of the DNA of genome is attached to optically transparent glass surface during sequencing, these DNA fragmentations warp
After crossing extension and bridge amplification, hundreds of millions of clusters is formed in glass surface, each cluster is the list for having thousands of parts of same templates
Then molecular cluster utilizes four kinds of special deoxyribonucleotides with fluorophor, skill is sequenced in synthesis by reversible
Template DNA to be measured is sequenced in art.
The exemplary, non-limitative example of nucleic acid hybridization technique 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
A position tissue part or slice (original position) are the spy in entire tissue (full organization embedding ISH) if tissue is sufficiently small
The hybridization of different in nature DNA or RNA sequence.DNA ISH can be used for determining the structure of chromosome.RNA ISH are used to measuring and positioning group
Knit the mRNA and other transcripts (for example, ncRNA) in slice or full organization embedding.At usually to sample cell and tissue
Reason increases the entrance of probe with fixation in situ target transcript.Probe hybridizes at high temperature with target sequence, then by extra spy
Needle is washed off.Respectively using autoradiograph, fluorescence microscopy or immunohistochemistry, to using radiation, fluorescence or antigen in tissue
The probe of the kilobase marker of label is positioned and is quantified.Two or more can also be used by radioactivity by ISH or other are non-
The probe of radioactive label substance markers, to detect two or more transcripts simultaneously.
The present invention simultaneously can expand nucleic acid (for example, ncRNA) before detection or with detection.Nucleic acid amplification technologies
Exemplary, non-limitative example include but not limited to：PCR (PCR), reverse transcriptase polymerase chain reaction (RT-
PCR), the amplification (TMA) of transcriptive intermediate, ligase chain reaction (LCR), strand displacement amplification (SDA) and based on nucleic acid sequence
It expands (NASBA).Those of ordinary skill in the art will be it will be recognized that certain amplification techniques (for example, PCR) needs will before amplification
RNA reverse transcriptions are into DNA (for example, RT-PCR), and other amplification techniques then direct cloning RNA (for example, TMA and NASBA).
The PCR of commonly referred to as PCR uses annealing and the primer extend of denaturation, primer pair and opposite strand
Multiple cycles, exponentially increase target nucleic acid sequence copy number；The amplification of the transcriptive intermediate of TMA is (in substantial constant
Temperature, multiple copies of target nucleic acid sequence are autocatalytically synthesized under conditions of ionic strength and pH, wherein target sequence is more
A RNA copies autocatalytically generate other copy；The ligase chain reaction of LCR uses miscellaneous with the adjacent area of target nucleic acid
The two groups of complementary DNA oligonucleotides handed over；Other amplification methods are included for example：The expansion based on nucleic acid sequence of commonly referred to as NASBA
Increase；Use rna replicon enzyme (the commonly referred to as Q β replicase) amplification of amplification probe molecule in itself；Amplification method based on transcription；
And the sequence amplification of self―sustaining.
The nucleic acid of non-amplification or amplification can be detected by any conventional means in the present invention.
Chip, kit
In the present invention, " chip ", " microarray ", " array " can be with equivalent substitutes, including but not limited to：DNA microarray
(for example, cDNA microarrays and oligonucleotide microarray), protein microarray, micro-array tissue, transfection or cell microarray, change
Chemical combination object microarray and Antibody microarray.The DNA microarray of commonly referred to as genetic chip, DNA chip or biochip is micro-
The set of DNA points is seen, these points are connected on the surface of solids (for example, glass, plastics or silicon chip), are formed for thousands of kinds
Gene is carried out at the same time expression pattern analysis or the array of expression monitoring.Fixed DNA fragmentation is known as probe, thousands of available
In single DNA microarray.Microarray can be used for identifying disease base by comparing the gene expression in disease and normal cell
Cause or transcript (for example, ncRNA).Multiple technologies can be used to be manufactured for microarray, including but not limited to：It is printed with apicule needle
Photoetching is carried out on to glass slide, using prefabricated mask, carries out photoetching, ink jet printing or microelectrode battle array using dynamic micro mirror element
Electrochemical method on row.
Kit in the present invention can be used for the expression of detection PNPLA5, it is preferred that the kit, which includes detection, to be had
The reagent of the detection PNPLA5 genes of effect amount, one or more substances selected from the group below：Container, operation instructions, positive control
Object, negative control object, buffer, auxiliary agent or solvent.Such as the solution for being suspended or fixing cell, detectable label or mark
Note makes nucleic acid be easy to the solution of hybridization, the solution for lytic cell or the solution for nucleic acid purification.
The present invention kit in can also have kit operation instructions, be described how using kit into
Row is detected and how tumor development to be judged using testing result, therapeutic scheme is selected.
Kit using the present invention can detect PNPLA5 by various methods (including but not limited to) selected from the group below：
Real Time RT-PCR, biochip test method, southern blotting technique method or RNA blottings or hybridization in situ.This field is common
Technical staff can be according to physical condition and needing to be adjusted detection mode and change.
Screening prevention or the compound for the treatment of osteosarcoma
The present invention can utilize the compound of biomarker PNPLA5 screenings prevention or treatment osteosarcoma, the present invention's
In a kind of embodiment, including step：In test group, the addition test compound in cultivating system, and observe the test group
Cell in PNPLA5 expression quantity and/or activity；In control group, test chemical combination is not added in identical cultivating system
Object, and observe the expression quantity and/or activity of PNPLA5 in the cell of control group；
Wherein, if the expression quantity of the PNPLA5 of cell and/or activity indicate that the test less than control group in test group
Compound is expression to PNPLA5 and/or activity have inhibiting effect treating cancer candidate compound.
As one embodiment of the present invention, the step further includes：To the candidate compound of acquisition into advancing one
The cell experiment and/or animal experiment of step, further to select and determine for preventing, alleviating or treat from candidate compound
The useful substance of osteosarcoma.
As embodiments of the present invention, the system of the candidate compound of screening prevention or treatment osteosarcoma is not limited to cell
System further includes cell system, subcellular system, solution system, organizational framework, organ systems or animal system etc., the body
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 ASPRV1
.
Inhibitor and pharmaceutical composition
Discovery based on inventor, the present invention provides a kind of purposes of the inhibitor of PNPLA5, are used to prepare inhibition bone
The pharmaceutical composition of sarcoma.As used herein, the inhibitor of the PNPLA5 includes but not limited to inhibitor, antagonist, resistance
Stagnant dose, blocking agent, nucleic acid inhibitor etc..
The PNPLA5 genes or the inhibitor of albumen refer to any activity for reducing PNPLA5 albumen, reduce
The stability of PNPLA5 genes or albumen, the expression for lowering PNPLA5 albumen reduce PNPLA5 albumen effective acting times or suppression
The substance of the transcription and translation of PNPLA5 genes processed, these substances are used equally for the present invention, as useful for lowering PNPLA5
Substance, so as to be used to prevent or treat osteosarcoma.For example, the inhibitor is：Nucleic acid inhibitor, protein inhibitor,
Antibody, ligand, proteolytic enzyme, protein binding molecule, as long as it can lower PNPLA5 albumen on albumen or gene level
Or the expression of its encoding gene.
As a kind of selection mode of the present invention, the inhibitor of the PNPLA5 is that a species specificity is combined with PNPLA5
Antibody.The specific antibody includes monoclonal antibody, polyclonal antibody；The present invention not only includes complete antibody molecule,
Also any segment including antibody or modification, for example, chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc..As long as the segment energy
Enough binding abilities retained with PNPLA5 albumen.For the antibody of protein level preparation when those skilled in the art it is public
Know, and the present invention may use any method to prepare the antibody.
As a kind of preferred embodiment of the present invention, the inhibitor of the PNPLA5 is a kind of small interference of PNPLA5 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 by anneal, can generate the double-strand of synthesis thereafter
RNA compounds.
When screening effective siRNA sequence, the present inventor is best effective so as to find out by largely comparing analysis
Segment.The present inventor's design has synthesized a variety of siRNA sequences, and they are transfected osteosarcoma cell line by transfection reagent respectively
It is verified, select the best siRNA of interference effect, they are respectively provided with the sequence shown in SEQ ID NO.7, SEQ ID NO.8
Row are further tested in cellular level, as a result prove to inhibit efficiency very high for test cell line.
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.
As a kind of optional mode of the present invention, the inhibitor of the PNPLA5 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.ShRN under the action of DICER enzymes, can be cut into siRNA molecule in eukaryocyte, hence into RNAi approach.
" shRNA expression vectors " refers to plasmid of some this fields conventionally used for building shRNA structures, exist on the usual plasmid "
Every sequence " and positioned at " intervening sequence " both sides multiple cloning sites or for replace sequence, so as to people can by shRNA (or
Analog) corresponding DNA sequence dna be inserted by way of forward and reverse multiple cloning sites or replace thereon for replacing sequence,
RNA after DNA sequence dna transcription can form shRNA (Short Hairpin) structure." the shRNA expression vectors " is current
It can be bought and obtained, such as some viral vectors by commercially available approach completely.
The present invention also provides a kind of pharmaceutical composition, it contain a effective amount of PNPLA5 inhibitor and
Pharmaceutically acceptable carrier.The composition can be used for inhibiting osteosarcoma.The inhibitor of any aforementioned PNPLA5
For the preparation of composition.The carrier includes but is not limited to diluent, excipient, adhesive, disintegrant, absorption enhancement
Agent, surfactant, Humectant, absorption carrier, lubricant, buffer, stabilizer, bacteriostatic agent, isotonic agent, chelating agent, pH controls
Preparation.
As used in the present invention, described " effective quantity " refer to that people and/or animal can be generated function or activity and can be by people
And/or the amount that animal is received.The effective quantity of inhibitor can with the pattern of administration and the severity of disease to be treated etc. and
Variation.Preferred a effective amount of selection can be determined according to various factors (such as by facing by those of ordinary skill in the art
Bed experiment).The factor includes but not limited to：The pharmacokinetic parameter of the inhibitor of the PNPLA5 genes is for example raw
Object utilization rate, metabolism, half-life period etc.；Patient the severity of disease to be treated, the weight of patient, patient immune shape
Condition, approach of administration etc..
Pharmaceutical composition Orally-administrable of the present invention, parenteral administration, by suck spray delivery, part to
Medicine, rectally, nasal administration, cheek administration, vagina administration are administered by the storage medicine device of implantation.It is preferred that oral medication or injection
Administration.Pharmaceutical composition of the present invention contains any commonly employed nontoxic pharmaceutical acceptable carrier, auxiliary material or excipient.
The pharmaceutical composition of the present invention can also be with the drug combination of other treatment osteosarcoma, and other therapeutic compound can
To be administered simultaneously with main active constituent or even be administered simultaneously in same composition.Can also with individual composition or
The dosage form different from main active constituent individually gives other therapeutic compounds.
Preferably, the means that gene therapy can be used carry out.It for example, can be directly by the inhibitor of PNPLA5 by such as noting
The methods of penetrating delivers medicine to subject；Alternatively, can will be carried by certain approach the inhibitor of PNPLA5 ceneme (such as
Expression vector or virus etc. or siRNA or shRNA) it is delivered on target spot, and be allowed to the PNPLA5 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.
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 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 PNPLA5 expressions significantly raise in Patients with Osteosarcoma.
The differential expression of embodiment 2QPCR sequence verification PNPLA5 genes
1st, large sample QPCR verifications are carried out to PNPLA5 gene differential expressions.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 amplifications are designed according to the coded sequence of PNPLA5 genes in Genebank and house-keeping gene GAPDH genes to draw
Object is synthesized by Bo Maide companies.
The primer sequence of PNPLA5 genes：
Forward primer sequence is 5 '-ATGTGCAAGTCAAGGATG-3 ' (SEQ ID NO.1)；
Reverse primer sequences are 5 '-TCGTACATGCTTTCTTCAG-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 cycles, 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,
Using SPSS18.0 statistical softwares come for statistical analysis, 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, PNPLA5 up-regulated expressions in osteosarcoma tissue, difference has system
Meter learns meaning (P<0.05) it is, consistent with high-flux sequence result.
The differential expression of 3 protein immunization imprinting of embodiment experiment detection PNPLA5 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 corresponding 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
Using SPSS18.0 statistical softwares come for statistical analysis, 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 expression of the PNPLA5 albumen in osteosarcoma tissue is significantly higher than cancer beside organism.
The silence of embodiment 4PNPLA5 genes
1st, cell culture
Cell line of human osteosarcoma U-2OS, with contain the DMEM culture mediums of 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, cell growth is good, is grown in monolayer adherence.Make
It is passed on the 0.25% trypsase conventional digestion containing EDTA.
2nd, it transfects
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 '-UCGUACAUGCUUUCUUCAGUG-3 ' (SEQ ID NO.7)
Antisense strand is 5 '-CUGAAGAAAGCAUGUACGAGG-3 ' (SEQ ID NO.8)
siRNA2：
Positive-sense strand is 5 '-UCUAUGAGGAGCUAUCUGGGG-3 ' (SEQ ID NO.9)
Antisense strand is 5 '-CCAGAUAGCUCCUCAUAGAGA-3 ' (SEQ ID NO.10)
Experiment is divided into three groups：Control group (U-2OS), negative control group (siRNA-NC) and experimental group (siRNA1,
SiRNA2), the sequence of wherein negative control group siRNA and PNPLA5 genes is without homology.
3) it transfects
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 of a concentration of 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 PNPLA5 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,
Using SPSS18.0 statistical softwares come for statistical analysis, the difference between PNPLA5 Gene Experiments group and control group uses t
It examines, 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 expressions reduce
Significantly, difference has statistical significance (P<0.05).
Influences of the embodiment 5Western blot detection transfections siRNA to PNPLA5 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
The measure of protein concentration is carried out according to the specification of BCA determination of protein concentration kits.
4th, 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.
5th, western detecting steps detailed in Example 3.
6th, statistical analysis
Using SPSS18.0 statistical softwares come for statistical analysis, 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.
7th, result
The results are shown in Figure 4, and compared with the control group, the PNPLA5 expressing quantities transfected in the cell of siRNA1 groups are notable
It lowers.
The influence of 6 PNPLA5 gene pairs human osteosarcoma cell proliferations of embodiment
PNPLA5 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, it 1 after inoculation, 2,3,4,5 days daily OD values taken out 3 hole cells and its 490nm is detected with mtt assay, is counted
Number calculates average value.
4th, liquid is discarded supernatant before detecting, culture solution is 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 horizontal axis, OD value draws cell growth curve for the longitudinal axis.
6th, statistical analysis
Experiment is all completed according to being repeated 3 times, using SPSS18.0 statistical softwares come for statistical analysis, 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 proliferation of cell significantly receives suppression
System, difference have statistical significance (P<0.05) illustrate that PNPLA5 has the function 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>Application of the biomarker in osteosarcoma diagnosis and treatment
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Claims (10)
1. detect application of the reagent of PNPLA5 expressions in the product for preparing diagnosis osteosarcoma.
2. application according to claim 1, which is characterized in that the reagent includes miscellaneous by using qRT-PCR, the marking
Friendship, in situ hybridization, hybridization array, genetic chip or new-generation sequencing detect the reagent of PNPLA5 expressions.
3. application according to claim 2, which is characterized in that the reagent is selected from：
The probe of specific recognition PNPLA5 genes；Or
The primer of specific amplification PNPLA5 genes；Or
Specifically bind the antibody or ligand of the albumen of PNPLA5 codings.
4. application according to claim 3, which is characterized in that the reagent is selected from drawing for specific amplification PNPLA5 genes
Object, the sequence of the primer is as shown in SEQ ID NO.1~2.
5. a kind of product for diagnosing osteosarcoma, which is characterized in that the product includes PNPLA5 expressions in detection sample
Preparation, chip or kit, chips include genetic chip, protein chip；Kit includes gene detecting kit, albumen
Detection kit.
6. product according to claim 5, which is characterized in that the gene detecting kit includes specific amplification
The primer of PNPLA5.
Applications of the 7.PNPLA5 in the drug of screening prevention or treatment osteosarcoma.
Application of the inhibitor of 8.PNPLA5 functional expressions in the pharmaceutical composition for preparing prevention or treatment osteosarcoma.
9. application according to claim 8, which is characterized in that the inhibitor is selected from：Nucleic acid inhibitor, albumen inhibit
Agent, proteolytic enzyme, protein binding molecule；The preferred inhibitor is nucleic acid inhibitor siRNA.
10. a kind of pharmaceutical composition for treating osteosarcoma, which is characterized in that described pharmaceutical composition includes PNPLA5 functionality tables
The inhibitor reached.
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Cited By (1)
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| CN110373457A (en) * | 2019-06-20 | 2019-10-25 | 镇江市第一人民医院 | A kind of mRNA marker and its application for ulcerative colitis diagnosis |
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