CN109609651A - A kind of molecular marker of diagnosis and treatment glioma and its application - Google Patents
A kind of molecular marker of diagnosis and treatment glioma and its application Download PDFInfo
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Abstract
The present invention provides molecular marker and its application of a kind of diagnosis and treatment glioma.Present invention firstly discloses GALE (UDP- galactolipin -4- epimerase) gene and its expression product up-regulated expressions in glioma, and are positively correlated with the increase of glioma grading；Meanwhile research is it has furthermore been found that miR-let-7i-5p has the function of Targeted-control GALE gene, miR-let-7i-5p passes through in conjunction with through GALE 3'-UTR, inhibit GALE albumen to generate, to reduce glioma and migration, induces human glioma cells Cycle Arrest and apoptosis.Therefore, miR-let-7i-5p and GALE gene and its expression product can be used as the molecular marker of diagnosis and treatment glioma.
Description
Technical field
The invention belongs to biological medicines and technical field of molecular biology, and in particular to a kind of molecule mark of diagnosis and treatment glioma
Will object and its application.
Background technique
Disclosing the information of the background technology part, it is only intended to increase understanding of the overall background of the invention, without certainty
It is considered as recognizing or implying in any form that information composition has become existing skill well known to persons skilled in the art
Art.
Human glioma is most common and most fatal primary brain tumor, accounts for about the 75% of all primary brain tumors.Root
According to the World Health Organization (World Health Organization), glioma is divided into 4 tumour ranks (I-IV) in histopathology.World Health Organization IV
Grade or many types of glioblastoma (GBM) prognosis are worst, and after initial diagnosis, the mean survival time is only 12 to 15 months.
MiRNA is the long single-stranded non-coding rna of a kind of endogenous 20-22 nucleotide (nt), has been confirmed to be water after transcription
The negative regulatory factor of flat gene expression.MicroRNA (miRNA) is played in the occurrence and development of human cancer, progress and recurrence
Important function.Let-7 is accredited as a kind of gene in Caenorhabditis elegans first, due to larva growth failure, mutation pair
These biologies are fatal.Bioinformatic analysis shows that let-7microRNA is highly conserved in different animals species, supports
Its key effect in early development stage.Let-7 ensures and exits the correct of cell cycle and the relevant event of terminal differentiation
Time.Inventors have found that miR-let-7 family low expression level is reported in many gliomas, including cancer of pancreas, people
Class colon cancer, Human Lung Cancer and breast cancer cell.
Summary of the invention
In view of the above shortcomings of the prior art, the present invention provides molecular marker and its application of a kind of diagnosis and treatment glioma.
It is proved through experimental study, the upper mileometer adjustment in glioma of GALE (UDP- galactolipin -4- epimerase) gene and its expression product
It reaches, and is positively correlated with the increase of glioma grading；Meanwhile research is it has furthermore been found that miR-let-7i-5p has Targeted-control
The effect of GALE gene, miR-let-7i-5p is by inhibiting GALE albumen to generate, to drop in conjunction with through GALE 3'-UTR
Low glioma and migration induce human glioma cells Cycle Arrest and apoptosis.Therefore, miR-let-7i-5p and
GALE gene and its expression product can be used as the molecular marker of diagnosis and treatment glioma.
The present invention is achieved through the following technical solutions:
The first aspect of the invention provides miR-let-7i-5p and/or GALE gene and its expression product and is preparing glue
Application in matter tumor molecular marker.
Further, miR-let-7i-5p and/or GALE gene and its expression product are in preparation glioma molecular marker
In application, the marker is used to diagnosing, detect, monitor or predicting the progress of glioma.
The progress of the glioma includes proliferation, growth and/or the migration of glioma cell；
The glioma includes Low grade glioma (I-II grades) and High Grade Gliomas (III-IV grades)；
Wherein, miR-let-7i-5p the and/or GALE gene and its expression product can be source of people.
The second aspect of the invention provides a kind of combination of progress for diagnosing, detecting, monitor or predict glioma
Object, it includes detect colloid based on high-flux sequence method and/or based on quantifying PCR method and/or based on probing procedure
The transcription of miR-let-7i-5p and/or GALE in tumor sample；Or based on miR- in immunologic detection method detection glioma samples
The expression of the target gene of let-7i-5p regulation or the substance of GALE expression product situation.
Wherein, the target gene includes GALE gene；
It is preferred that protecting analytical technology, RAKE method, original position using Northern hybridizing method, miRNA chip of expression spectrum, ribozyme
The transcription of miR-let-7i-5p and/or GALE in hybridization check glioma samples；Using ELISA, colloidal gold strip, albumen
Chip detects the expression or GALE expression product situation of the target gene of miR-let-7i-5p regulation in glioma samples；
Further, the present invention provides a kind of kit, and the kit includes for diagnosing, detecting, monitor or in advance
Survey the composition of the progress of glioma.
The third aspect of the invention provides the substance that can promote that miR-let-7i-5p is expressed and/or activity improves
And/or the substance for inhibiting GALE gene and its expression product and/or activity to reduce answering in following (a')-(c') is at least one
With:
(a') inhibit the migration of glioma cell, or product of the preparation for inhibiting glioma cell to migrate；
(b') inhibit the proliferation of glioma cell, or prepare the product for inhibiting glioma；
(c') inhibit the growth of glioma cell, or prepare the product for inhibiting growth of glioma cells.
The glioma includes Low grade glioma (I-II grades) and High Grade Gliomas (III-IV grades)；
The fourth aspect of the invention, provides a kind of for treating or preventing the pharmaceutical composition of glioma, and it includes rush
The substance and/or inhibition GALE gene and its expression product and/or activity improved into miR-let-7i-5p expression and/or activity
Reduced substance；
For the substance for promoting miR-let-7i-5p expression and/or activity to improve, including using based on RNA's
MicroRNA functionality obtain technology and/or gene specific miR Mimics technology up-regulation miR-let-7i-5p expression and/or
Promote its activity；The short hairpin RNA (short hairpin RNA, shRNA) of preferably artificial synthesized miR-let-7i-5p or
Raise the promoter of miR-let-7i-5p expression.
For the substance for inhibiting GALE gene and its expression product and/or activity to reduce, including GALE albumen is special
Antibody, rnai molecule or antisense oligonucleotides, micromolecular inhibitor, siRNA for GALE mRNA；Further,
The antibody is human antibody.
In the present invention, " treatment or prevention " refer to being able to suppress the migration of glioma cell, proliferation and/or growth.
Further, described pharmaceutical composition also includes at least one or more of acceptable auxiliary pharmaceutically or in bromatology
Material.Auxiliary material used can be solid-state or liquid.
Preferably, the drug is solid orally ingestible, liquid oral medicine or injection.
It is further preferred that the drug be tablet, dispersible tablet, enteric coatel tablets, chewable tablets, oral disintegrating tablet, capsule, sugar-coat agent,
The small water needle of granule, dry powder doses, oral solution, injection, injection freeze-dried powder, big infusion or primary infusion.
The invention has the advantages that: first demonstration that GALE (UDP- galactolipin -4- epimerase) gene and its table
Up to product in glioma up-regulated expression, and be positively correlated with the increase of glioma grading；Meanwhile research it has furthermore been found that
MiR-let-7i-5p has the function of Targeted-control GALE gene, and miR-let-7i-5p with by GALE 3'-UTR by tying
It closes, GALE albumen is inhibited to generate, to reduce glioma and migration, induce human glioma cells Cycle Arrest and wither
It dies.Therefore, miR-let-7i-5p and GALE gene and its expression product can be used as the molecular marker of diagnosis and treatment glioma, be
The diagnosing and treating of the related diseases such as glioma provides new target spot, so that clinician be instructed to provide prevention side for subject
Method or therapeutic scheme just have the value of good practical application.
Detailed description of the invention
Fig. 1 is the expression series of drawing related to the tumor grade of glioma of GALE；Wherein, Fig. 1 a is quantitative analysis TCGA number
According to expression figure of the concentration GALE mRNA in glioma；Fig. 1 b and Fig. 1 c are in TCGA (n=667) database respectively
Analyze prognosis meaning figure of the GALE in Low grade glioma LGG and glioblastoma GBM patient；Fig. 1 d is different stage glue
The representative diagram of matter tumor and normal brain activity sample GALE immunohistochemical staining, top figure amplification factor × 200, lower section figure are put in Fig. 1 d
Big multiple × 400, separation are located at the intermediate value of GALE expression quantity.*P<0.05；**P<0.01；***P<0.001.
Fig. 2 is the proliferation series of drawing that GALE silencing can reduce glioma cell；Wherein, Fig. 2 a and Fig. 2 b is fixed for real-time fluorescence
Measure PCR analysis shows that, si-GALE transfection after GALE opposite mRNA level in-site significantly reduce, with GAPDH be compare, immunoblotting
Lysate (12 μ g) from U87 and U251 cell transfecting and negative control (NC) and GALE siRNAs hatching with GALE antibody；
β-Actin is used as internal reference；Fig. 2 c and Fig. 2 d are that CCK-8 detects U87 the and U251 cell growth curve figure based on OD450；Fig. 2 e
It is that EdU analysis carries out figure after 48 hours of transfection with Fig. 2 f；Fig. 2 g is that si-GALE handles the Clonal drop of glioblastoma cells
Low figure.*P<0.05；**P<0.01；***P<0.001.
Fig. 3 is the migration series of drawing that GALE silencing reduces glioma cell；Wherein, Fig. 3 a is that U87 cell is used in 24 hours
It carries out VM and forms experiment；Fig. 3 b is that the cell migration after transwell method is incubated for 6 hours of U87 cell is transfected with NC or si-GALE
Figure；Fig. 3 c is to transfect U87 with NC or si-GALE, carries out wound healing assay figure, and recovery time is for 24 hours；Fig. 3 d be with NC or
Si-GALE transfects U251, carries out wound healing assay figure, and recovery time is for 24 hours；Fig. 3 e is that U87 warp is transfected with NC or si-GALE
Migrating cell number histogram after transwell method is incubated for 6 hours；Fig. 3 f is that U87 and U251 cell wound is transfected with NC or si-GALE
Mouth healing rate histogram, recovery time are for 24 hours.*P<0.05；**P<0.01；***P<0.001.
Fig. 4 is that the downward of GALE leads to human glioma cells Cycle Arrest and apoptosis series of drawing；Wherein, Fig. 4 a with NC or
Si-GALE transfects U87 and U251 cell, carries out cell cycle analysis figure using flow cytometry；Fig. 4 b is with NC or si-GALE
U87 and U251 cell is transfected, using Annexin V-FITC antibody in flow cytometry and PI dyeing detection Apoptosis figure；Figure
4c is to transfect U87 cell with NC or si-GALE, carries out cell cycle analysis histogram using flow cytometry；Fig. 4 d is to use NC
Or si-GALE transfects U251 cell, carries out cell cycle analysis histogram using flow cytometry；Fig. 4 e is with NC or si-
GALE transfects U87 and U251 cell, using Annexin V-FITC antibody in flow cytometry and PI dyeing detection Apoptosis
Percentage histogram；Fig. 4 f-i is that known Cell cycle regulatory proteins, apoptosis, EMT marker are detected using Western blot
And possible cell-signaling pathways protein expression level figure.β-Actin is used as internal reference.*P<0.05；**P<0.01；***P<
0.001。
Fig. 5 is that miR-let-7i-5p is directly targeted GALE；Wherein, Fig. 5 a is miR-let-7i-5p binding site in GALE
Sequence and 3'-UTR luciferase assay results；It is combined using miR-let-7i-5p in 3 '-UTR of Bioinformatics Prediction GALE
Site；By 3 '-UTR carriers and miR-let-7i-5p or miRNA negative control cotransfection U251 cell, measured carefully after transfecting 48h
The relative activity of luciferase in cellular lysate liquid；The cell of luciferase activity levels and the transfection of miRNA negative control is carried out
It is normalized to 1；Fig. 5 b-e be real-time fluorescence quantitative PCR analysis shows that, transfect miR-let-7i-5p mimics after GALE phase
MRNA level in-site is significantly reduced, is control with GAPDH, the downward of GALE is expressed after miR-let-7i-5p mimics transfection,
It is confirmed through western blotting, β-Actin is used as internal reference.*P<0.05；**P<0.01；***P<0.001.
Fig. 6 is to be overexpressed miR-let-7i-5p to reduce the proliferation series of drawing of glioma cell；Wherein, Fig. 6 a and Fig. 6 b
It is carried out after 48 hours of transfection for EdU analysis, treats glioblastoma cells, Fig. 6 c drop with miR-let-7i-5p analogies
Low cell clonal figure；Fig. 6 d is to compare NC group and miR-let-7i-5p mimics group EMT mark by western blot
Object expression, the equiprobable cell-signaling pathways protein expression figure of Cell cycle regulatory proteins.*P<0.05；**P<0.01；***P<
0.001。
Fig. 7 is that the overexpression of miR-let-7i-5p reduces the migration series of drawing of glioma cell；Wherein, Fig. 7 a is 24 small
When it is interior with U87 cell carry out VM formed experiment；Fig. 7 b is to transfect U87 cell with NC or miR-let-7i-5p mimics；Through
Transwell method assesses cell migration situation map after being incubated for 6 hours；Fig. 7 c is to be transfected with NC or miR-let-7i-5p mimics
U87 cell carries out wound healing assay figure, and recovery time is for 24 hours；Fig. 7 d is to be transfected with NC or miR-let-7i-5p mimics
U251 cell carries out wound healing assay figure, and recovery time is for 24 hours；Fig. 7 e is to be turned with NC or miR-let-7i-5p mimics
Contaminate U87 cell；Cell migration histogram is assessed after transwell method is incubated for 6 hours；Fig. 7 f is with NC or miR-let-7i-
5p mimics transfects U87 and U251 cell, wound healing rate histogram, and recovery time is for 24 hours.*P<0.05；**P<
0.01；***P<0.001.
Fig. 8 is to knock out GALE to inhibit in-vivo tumour that series of drawing occurs；Wherein, Fig. 8 a uses bioluminescence imaging
The tumour of (biolescence imaging, BLI) system evaluation each group BALB/c nude mouse original position heterograft glioma into
Exhibition；Fig. 8 b is measurement tumor size histogram (mm3)；Fig. 8 c is the animal life for being implanted into U87MG negative control or sh-GALE cell
Depositing analysis, (log-rank examines P < 0.01；Every group 5)；Fig. 8 d is the mouse brain U87MG NC group or sh-GALE xenogenesis of HE dyeing
The slice of transplantation group；Fig. 8 e-h is the immunohistochemistry figure of GALE, BCL-2, Ki-67 and MMP2 in heterograft slice；Times magnification
Number: 200 times and 400 times.*P<0.05；**P<0.01；***P<0.001.
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to the illustrative embodiments of the application.As used herein, unless the context clearly indicates otherwise, otherwise singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.
Term explanation and illustration in the present invention:
MiRNA chip of expression spectrum: principle is using the target molecule on label probe detection solid support.Pass through design
MiR-96 gene and internalcontrol sequence on chip, can Accurate Analysis go out the expression of corresponding miRNA in sample.Genetic chip has
High-throughput advantage can once detect whole expression of several hundred a genes in same sample.What Luminex company developed
Liquid-phase chip (Liquid chip) is also known as multifunctional suspending dot matrix (Multi analyte suspension array, MASA),
It is biochip technology of new generation out.Liquid-phase chip system is made of many spherulas for main matrix, on every kind of spherula
It is fixed with different probe molecules, in order to distinguish different probes, each sphere matrix for being used for label probe all has one
A unique color numbers constitute liquid-phase chip system as soon as these spherulas are suspended in a liquid-phase system.The system
Quick qualitative and quantitative analysis, this detection technique can be carried out simultaneously to multiple and different molecules in the same trace sample
Referred to as FMAP (Flexible multianalyte profiling) technology.Molecule hybridization carries out in aaerosol solution, detects
Speed is exceedingly fast.
Ribozyme protects analytical technology (RPA): the detection of miRNA can also protect analytical technology using ribozyme, will mark
Probe and RNA sample to be measured mixing, hybridize after thermal denaturation, non-hybridized RNA and the single-chain nucleic acid enzymic digestion of extra probe,
Shielded RNA molecule is purified after heat inactivation nuclease, and probe, colour developing is separated by electrophoresis finally by denaturation PAGE.It is this to be based on
The new method of solution hybridization is simple and quick, high sensitivity.
RAKE method (RNA primed array based Klenow emzyme): being the base in miRNA microarray
The Klenow segment of DNA polymerase i, the method for hybridizing miRNA with fixed DNA probe are utilized on plinth.RAKE can be sensitive
MiRNA is specifically detected, suitable for largely quickly screening all known miRNA.It can be in specific cell and tumour
Detect miRNA express spectra situation.Moreover, RAKE method can also be from the tissue of the paraffin embedding secured by formalin
It isolates miRNA and analyzes it.
In situ hybridization (in situ hybridization): hybridization in situ technique can intuitively understand miRNA expression way,
It is a kind of easier method for observing miRNA spatial and temporal expression, normal mark mode includes digoxin, biotin, fluorescent marker etc..
In situ hybridization (Locked Nucleic Acid (LNA) based in situ hybridization on the basis of locked nucleic acid
It (LNA-ISH)) is the more probe mode of current application.
High-flux sequence (High-throughput sequencing): also known as next-generation sequencing technologies (next
Generation sequencing) it is the change for tradition being sequenced revolution, once to hundreds of thousands to millions of DNA
Molecule carries out sequencing, greatly improves sequencing efficiency.This kind of large scale sequencing technology greatly improves multiple species and loses
The solution reading rate of communication breath, for the sequence information for obtaining all miRNA, decryption miRNA map provides guarantee.It is high-throughput simultaneously
Sequencing to carry out the analysis of careful overall picture to the transcript profile and genome of species, so the depth that is otherwise known as
It is sequenced (deep sequencing).The representative of high-flux sequence platform is 454 sequenator (Roch of Roche Holding Ag (Roche)
GSFLX sequencer), the Solexa genome analysis instrument (Illumina Genome Analyzer) of Illumina company and
The SOLiD sequenator (ABI SOLiD sequencer) of ABI.
Immunologic detection method: is quantified or is determined to determinand using a kind of antibody or Multiple Antibodies as analytical reagent
Property analysis detection method.The basic principle is that the interaction between antibody and antigen.To improve antigen and antibody test
It is anti-to reflect that whether there is or not antigen-antibodies by detecting marker for sensibility, the substance that will easily show in known antibodies or antigenic mark
It answers, to measure micro antigen or antibody indirectly.Common marker have enzyme, fluorescein, radioactive isotope, colloidal gold and
Electron dense substances etc..The specific reaction for showing that object is carried out on this antigen or antibody label is known as immunolabelling technique
(immunolabelling technique).Immunoassay technology most widely used at present mainly has: enzyme-linked immunosorbent assay
(enzyme-linked immunosorbent assay, ELISA), colloidal gold immunity chromatography etc..
Enzyme-linked immunosorbent assay principle is to combine antigen or antibody and substrate (enzyme), it is made to keep immune response and enzyme
Activity.The antigen or antibody of label and the ligand binding that is coated on solid phase carrier, then it is allowed to and corresponding colorless substrate
It acts on and display color, determines result according to the range estimation of colour developing depth degree or with microplate reader measurement OD value.
Colloidal gold strip is generally made of sample pad, gold-labelled pad, chromatographic film, four part of water absorption pad.Chromatographic material has nitre
Change tunica fibrosa, polyester film, nylon membrane and pvdf membrane etc., needs may be selected the film of different requirements according to test, wherein nitrocellulose
Film is the most commonly used, can determine the need for activating according to test concrete condition before or handle, in most cases without handling,
It can be used directly.By gold mark protein solution even application in gold-labelled pad, dry at room temperature spare.Nitrocellulose membrane can be caught
A certain amount of coating (antibody) and secondary antibody are obtained as detection line and nature controlling line.Finally by sample pad, gold-labelled pad, nitrocellulose membrane and
Blotting paper is in turn secured to PVC board, test strips.
The acquired technology of microRNA function based on RNA: the precursor substance synthesized by exogenous supplement miRNAs
To increase the level of miRNAs.For example, can the artificial synthesized and consistent short hair clip sample RNA (short of endogenous miRNA sequence
Hairpin RNA, shRNA), promoter is done by polymerase II or III, is that carrier transfects cell with virus, by Dicer enzyme modification
It is loaded into RISC afterwards to play a role, is equivalent to the level for increasing pre-miRNA, function and effect are stable and lasting.
Gene specific miR Mimics technology: this technique avoids the nonspecific actions of miRNA and gene.This people
The specific oligonucleotide chain of the combination complementary with 3 ' UTR of target gene of work synthesis, is adjusted after capable of playing transcription identical with miRNA
Section effect.
As previously mentioned, miR-let-7i-5p and the relationship that glioma is in progress are unclear；It is therefore desirable to miR-
Specific molecular mechanism of the let7i-5p in glioma is researched and analysed.
Meanwhile disclosing GALE in the prior art is a kind of bifunctional enzyme, the Leloir access as galactose metabolism
A part, the reversible transition of dp- galactolipin (UDP-Gal) to udp- glucose (UDP-Glc) is catalyzed, and in NAD+Presence
Under by udp-n- acetylgalactosamine (UDP-GalNAc) generate udp-n- acetylgalactosamine (UDP-GalNAc), this be generate
The steps necessary of glycosyl glycoprotein synthesis glycosyl.
GALE is dynamically adjusted in various metabolism environments.It is reported that udp- galactolipin is converted into lacking for udp- glucose
Fall into the general features for leading to galactosemia, including vomiting, low pass gas, epilepsy, jaundice, galactosuria and hepatomegaly.
And in the present invention, research finds that GALE (UDP- galactolipin -4- epimerase) gene and its expression product exist
Up-regulated expression in glioma, and be positively correlated with the increase of glioma grading；Meanwhile research is it has furthermore been found that miR-let-7i-
5p has the function of Targeted-control GALE gene, and miR-let-7i-5p is by inhibiting GALE in conjunction with through GALE 3'-UTR
Albumen generates, to reduce glioma and migration, induces human glioma cells Cycle Arrest and apoptosis.
In view of this, in a specific embodiment of the invention, provide miR-let-7i-5p and/or GALE gene and its
Application of the expression product in preparation glioma molecular marker.
In still another embodiment of the present invention, miR-let-7i-5p and/or GALE gene is provided and its expression product exists
The application in glioma molecular marker is prepared, the marker is used to diagnose, detect, monitor or predict the progress of glioma.
In still another embodiment of the present invention, the glioma progress include glioma cell proliferation, growth and/
Or migration；
In still another embodiment of the present invention, the glioma includes Low grade glioma (I-II grades) and high-level glue
Matter tumor (III-IV grades)；
In still another embodiment of the present invention, wherein miR-let-7i-5p the and/or GALE gene and its expression
Product can be source of people.
In still another embodiment of the present invention, provide a kind of for diagnosing, detecting, monitor or predicting the progress of glioma
Composition, it includes based on high-flux sequence method and/or based on quantifying PCR method and/or based on probing procedure examine
It surveys the transcription of miR-let-7i-5p and/or GALE in glioma samples or is detected in glioma samples based on immunologic detection method
The expression of the target gene of miR-let-7i-5p regulation or the substance of GALE expression product situation.
Wherein, the target gene includes GALE gene；
In still another embodiment of the invention, using Northern hybridizing method, miRNA chip of expression spectrum, ribozyme
Protect analytical technology, RAKE method, in situ hybridization detection glioma samples in miR-let-7i-5p and/or GALE transcription；Using
ELISA, colloidal gold strip, protein chip detect the expression feelings of the target gene of miR-let-7i-5p regulation in glioma samples
Condition or GALE expression product situation；
In still another embodiment of the present invention, the present invention provides a kind of kit, the kit include for diagnosing,
The composition of the progress of detection, monitoring or prediction glioma.
In still another embodiment of the present invention, provide can promote miR-let-7i-5p express and/or activity improve
Substance and/or the substance for inhibiting GALE gene and its expression product and/or activity to reduce are in following (a')-(c') at least one
Application:
(a') inhibit the migration of glioma cell, or product of the preparation for inhibiting glioma cell to migrate；
(b') inhibit the proliferation of glioma cell, or prepare the product for inhibiting glioma；
(c') inhibit the growth of glioma cell, or prepare the product for inhibiting growth of glioma cells.
The glioma includes Low grade glioma (I-II grades) and High Grade Gliomas (III-IV grades)；
In still another embodiment of the present invention, provide it is a kind of for treating or preventing the pharmaceutical composition of glioma,
Comprising the substance for promoting miR-let-7i-5p expression and/or activity to improve and/or inhibit GALE gene and its expression product and/
Or the substance that activity reduces；
For the substance for promoting miR-let-7i-5p expression and/or activity to improve, including using based on RNA's
MicroRNA functionality obtain technology and/or gene specific miR Mimics technology up-regulation miR-let-7i-5p expression and/or
Promote its activity；The short hairpin RNA (short hairpin RNA, shRNA) of preferably artificial synthesized miR-let-7i-5p or
Raise the promoter of miR-let-7i-5p expression.
For the substance for inhibiting GALE gene and its expression product and/or activity to reduce, including GALE albumen is special
Antibody, rnai molecule or antisense oligonucleotides, micromolecular inhibitor, siRNA for GALE mRNA；Further,
The antibody is human antibody.
In the present invention, " treatment or prevention " refer to being able to suppress the migration of glioma cell, proliferation and/or growth.
In still another embodiment of the present invention, described pharmaceutical composition also include it is at least one or more of pharmaceutically or food
Acceptable auxiliary material in conduct and learning.Auxiliary material used can be solid-state or liquid.
In still another embodiment of the present invention, the drug is solid orally ingestible, liquid oral medicine or injection.
In still another embodiment of the present invention, the drug be tablet, dispersible tablet, enteric coatel tablets, chewable tablets, oral disintegrating tablet,
Capsule, sugar-coat agent, granule, dry powder doses, oral solution, the small water needle of injection, injection freeze-dried powder, big infusion or small defeated
Liquid.
Explanation is further explained to the present invention by the following examples, but is not construed as limiting the invention.It should be understood that
These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.In addition, unspecified in embodiment
Molecular biology method is the method for this field routine, and concrete operations can be referring to molecular biosciences guide or product description.
Embodiment
1.1Cox proportional hazard model
Gene relevant to survival of patients is selected using Cox proportional hazard model, and establishes one for following prediction
Prediction model.As a result timing definition is the disease-free survival moon.Select N number of gene constructed Cox regression model；For each gene Gj (j
=1,2 ..., N), following Cox model is established, to consider the harm of t moment life state or death.
WhereinFor the baseline hazard function of gene Gj, X1, X2 ..., XP be covariant.The covariant of adjustment includes
Race, age, or sex, KPM (Karnofsky performance) scoring, neoplastic state and lower rectal cancer history.Pass through
Above method has selected GALE.
1.2 tissue samples and data bank
The present embodiment includes from the glioma sample of Shandong Qilu Hospital's neurosurgery and normal cerebral tissue.
This research approach is ratified through hospitalism examination board, and through the written informed consent of every subject.Patient's routine operation is controlled
It treats and obtains 4 normal brain activities, 4 WHO II grades, 7 of WHO I grades, 8 III levels, 9 IV grades of (GBM) tissue specimens.Glioma mark
This is verified and is classified according to WHO staging by two veteran CIin Paths.Cancer gene group map is ground
The clinical information for studying carefully network sample (n=667: is analyzed using TCGA, http://cancergenome.nih.gov), is seen
Table 1.
Expression correlation of 1 GALE of table in the human glioma patient with kinds of clinical pathological feature；
P value is obtained by card side and accurate examine of expense snow
1.3 cell culture and reagent
People's glioblastoma cells strain U87 and U251 are purchased from Cell Bank of Chinese Academy of Sciences.All cells, which use, contains 10%
The DMEM culture medium culture of fetal calf serum is simultaneously incubated at 37 DEG C and in the cell incubator containing 5% carbon dioxide.
1.4 cell transfecting
It is designed by GenePharma (Shanghai, China) and provides mature miR-let7i-5p analogies, miR-
Let7i-5p inhibitor and negative control double-strand (NC) SiRNA antagonism GALE (abbreviation siGALE).Use Lipofectamine
3000 (Invitrogen, Carlsbad, the U.S., CA, CA) illustrate progress cell transfecting and cotransfection according to manufacturer.It is real
When fluorescence quantitative PCR detection transfection efficiency.After transfection 48 hours, glioma cell is taken to carry out subsequent experimental.
The analysis of 1.5 cell viabilities
For U251 and U87 cell in 96 hole Tissue Culture Dish, density is 3000 cells/wells.Transfection 24,48, after 72h with thin
Born of the same parents count Kit-8 and analyze cell proliferative conditions.10 μ L CCK-8 solvents are added in every hole, and 1h is then incubated in cell incubator.
Then optical density is measured at 450nm using Ensight (PerkinElmer).In siGALE, miR-Let7i-5p appropriate or
After NC processing, the cell cultivated in 24 hole culture dishes is measured using the external kit of EdU Apollo567 (Cell-Light)
DNA synthesis.EdU measurement result is detected using 8 microscope of card DMi is come.
1.6 healing assay
Every hole paving 1 × 10 in 6 orifice plates5A cell is simultaneously incubated for after a night, using siGALE, miR-Let7i-5p or yin
Property control to transfect cell.With 90% degree of polymerization, aseptic straw point scratches cell monolayer, and PBS removes free cell.Scratch plate
It is cultivated in 1% fetal calf serum DMEM.In the case where coming card microscope, image was shot along kerf at 0 to 12 hours.According to relative to
The original distance for scratching ranging offset obtains opposite scuffing width.
1.7 Cell migration assay
Cell migration assay uses the cell Transwell (6.5 millimeters of diameter of measurement, 8 μm of pore sizes, Corning
Company's production).Serum free medium paving 5 × 10 is used in the upper chamber of the cell Transwell4Cell after transfection.It is added 10%
The culture medium of fetal calf serum is into lower room.12, after 24 and 48 hours, the cell that small indoor surface does not migrate is removed using swab stick.It moves
The cell for moving to lower surface fixes 15min using violet staining, counts under microscope.Cell is counted using 5 random views
Number.
1.8 Bioinformatics Predictions and luciferase reporting analysis
Correlation analysis is carried out to GALE using available gene expression profile in TCGA data set.Utilize multi-objective predictive journey
Sequence, including online miRNA forecasting tool TargetScan Human 7.2 and miRBase, it is pre- to identify computer aided algorithm
The common miR-let-7i-5p target surveyed.From Jinan, BioSune obtains miR-let-7i-5p-GALE and miR-let-7i-5p-
The reporter sequence of mutGALE (mutation of target seed sequence).Glioma cell luciferase reporting object and
Lipofectamine 3000 detects substance or carrier cotransfection.After transfection cell 48 hours, illustrated according to manufacturer, using double
Luciferase reporting kit (Promega) analysis of fluorescence element enzymatic activity.Firefly luciferase activity passes through purple perilla fluorescein
Enzymatic activity normalization.
1.9RNA is extracted and real-time quantitative PCR
Using TRIzol reagent, total serum IgE is extracted according to manufacturer's agreement.Then, according to the manufacturer's instructions, use
MiRNA stem-loop RT primer or U6RT primer reverse total serum IgE using ReverTra Ace qPCR RT kit
Record generates cDNA.Real-time PCR is carried out using SYBR Premix Ex TaqTM kit, primer is shown in Table 2.This experiment uses480II instrument carries out.U6 and GAPDH expression is as endogenous control.Absolute expression is calculated
Roche is used at concentration rate480II system.
2 oligonucleotide sequence of table
A F, forward primer；R, reverse primer；
B wild type target area is indicated using italic overstriking；Saltant type target area indicates using underscore
1.10 immunoblotting assay
Total protein is extracted using the RIPA buffer containing 1%PMSF.The protein of equivalent is packed into 10%
In SDSpolyacrylamide gel.Next, protein blot uses primary antibody in 4 DEG C of refrigerator overnights, horseradish peroxidating is then used
The secondary antibody of enzyme label is in incubation at room temperature 1h.Finally, protein band uses ChemiDoc XRS+ (BIO-RAD) and image lab
Software images.It the use of antibody is mainly GALE antibody (1:5000Abcam) and other relevant protein antibodies, internal reference uses
β-Actin。
1.11 immunohistochemical staining
The Human Brain Gliomas sample or solid tumor for being derived from mouse are fixed with 4% formaldehyde.Tumour paraffin-embedded tissue is cut
It 5 μ m thick of piece and is fixed on glass slide, carries out antigen retrieval using the EDTA of 1mMol.In the first containing 3% hydrogen peroxide
Slice is incubated in alcohol with deactivating endogenous peroxydase activity, then slice, which is placed in PBS, rinses 6 minutes.Next, cut
Piece hatches 2h using lowlenthal serum at room temperature, and then plus 4 DEG C of primary antibody are incubated overnight.Then it is rinsed and is sliced with PBS, with horseradish mistake
The goat anti-rabbit antibodies or anti-mouse antibody of oxide enzyme connection are incubated for, and are then reacted with diaminobenzidine, with the Soviet Union of Mayer
Another name for is counter to be contaminated.
1.12 flow cytometry
Flow cytometry cell cycle distribution and level of apoptosis.Cell cycle analysis transfects neuroglial cytoma
It is collected by trypsinized, is flushed three times using cold PBS, stayed overnight for 4 DEG C after then being fixed using 70% ethyl alcohol.After PBS washing, turn
15min is incubated in the cell of dye and the third ammonium of iodate at room temperature dark.Finally, using FACScan flow cytometer (BD, USA)
Cell is analyzed.
According to manufacturer specification, annexin V-FITC apoptosis detection kit (Invitrogen Life is used
Technologies, CA) analysis Apoptosis.The cell of transfection is collected, at room temperature in the dark with annexin V-FITC and PI
It is incubated for 15min.Then, these cells are analyzed using FACScan flow cytometer (BD, USA).Each experiment carries out three
It is secondary.
1.13 mouse brains plant Glioma Model in situ
In order to carry out animal experiment in vivo, human glioma cells heterograft is that implantation transfection is oriented by instrument
Luciferase-labeled U87 cell (1 × 106A cell/mouse) enter brain (Jiangsu of male BALB/c nude mice
Jicuiyaokang Biotechnology Co., Ltd, Nanjing).It is right using slow virus carrier LV3-GALE-home-899 or lv3 feminine gender
Standard scheme infection is carried out according to the U87 cell of (Genepharma, Shanghai) to the luciferase label of culture.By mouse with
Machine is divided into two groups (NC, shGALE), and every group 5.Animal was carried out in PerkinElmer IVIS every 5 days Polaroid.According to human relations
Criterion is managed, when final image shows that two groups of mouse had significant difference enough between the 20th day, mouse is condemned to death；It is rounded
Brain, 4% paraformaldehyde are fixed, dehydration, paraffin embedding.Serial section (5 μ m-thick) simultaneously carries out H&E dyeing, then under the microscope
Observation.The processing of mouse and experimental arrangement are carried out according to experimental animals guide.Experiment meets ministry of Health of China the care of animal regulation,
Experimental program is ratified through Shandong University's animal protection and using the committee.
1.14 statistical analysis
All experiments are repeated three times.Using SPSS 22.0 and GraphPad Prism Software Create statistical analysis and
Generate experiment figure.Descriptive statistic, including mean value ± SD, t inspection, kaplan meier analysis, log-rank are examined, Dan Yin
Plain variance analysis and Spearman correlation test, for analyzing the conspicuousness of difference, p < 0.01 * p < 0.05, * *, there are also * * * p
< 0.001 is considered to have statistical significance.
2. result
2.1GALE expression increases to be positively correlated with glioma classification levels
It is research GALE in the developmental effect of glioma, GALE is analyzed from TCGA data set in GBMs, low level
Gene expression dose in glioma (LGGs) and normal cerebral tissue.GALE mRNA level in-site is apparently higher than in TCGA in GBMs
LGGs and normal cerebral tissue (Fig. 1 a).Human brain colloid from Jinan City Shandong hospital is had detected using immunohistochemical method
The GALE protein level of tumor (32) and normal cerebral tissue (4).Consistent with mRNA expression of results, GALE albumen is high in GBMs
In LGGs or Normal brain (Fig. 1 d).Therefore, in disclosed database and primary tumor sample queue, GALE and tumour point
The increase of grade is positively correlated.
2.2GALE expression is related to prognosis mala
Examine GALE expression in Response in Patients with Gliomas overall survival (OS) in Kaplan-Meier survivorship curve
Prognostic value.In TCGA (n=667), in LGG (P < 0.001) and GBM (P < 0.05) patient, GALE expression is significant with prognosis
It is worse than GALE expression (Fig. 1 b, 1c).Therefore, GALE may be a kind of new glioma biomarker.
2.3GALE silencing can reduce the proliferation and migration of glioma cell in vitro
In order to directly detect effect of the GALE in glioma cell is survived and is proliferated, cell transfecting siRNA is knocked out
GALE, and carry out CCK-8 and EdU experiment.The downward of GALE leads to transfect after 48h OD450 value and edu in U87 and U251 cell
The statistically significant decline of positive cell percentage (Fig. 2 c-2f).Whether will affect cell in order to further determine GALE
Growth, has used a clonogenic assay to assess the influence of GALE cell proliferation.It is worth noting that, in siRNA processing group
In, GBM cell strain has apparent inhibiting effect (Fig. 2 g) to cell clone increment.Result above prompts GALE to regulate and control GBM cell
Proliferation.
Using wound healing test and effect of the transwell experimental observation GALE in GBM cell migration.U87 and
U251 cell uses NC or siGALE to handle respectively, and compared with NC group, siGALE group is in wound healing experiment and transwell experiment
Middle surface of a wound closure effect is poor, and staining cell is less (Fig. 3).The result shows that the inhibition of GALE results in GBM cell line migration energy
Power significantly inhibits.Secondly, being studied using western blot GALE downstream targets.GALE gene knockout significantly drops
The level of some gene/accesses necessary to low N-CAD, mmp-2 and various types cancer progression including glioma
(Fig. 4 h, 4i).
The downward of 2.4GALE can induce human glioma cells Cycle Arrest and apoptosis
Flow cytometry cell cycle analysis also indicates that GALE strikes the low quantity (figure for increasing G1 phase U87 and U251 cell
4a,4c,4d).In addition, GALE silencing promotes U87 and U251 cell line cell apoptosis, compared with the control (Fig. 4 b, 4e).?
In western blot analysis, after GALE silencing, cyclin-dependent kinase 2 (CDK2), CDK4, cyclin
A2 and bcl-2 expression reduces.In contrast, bax and c-caspase3 is considered as tumor suppressor, the table in siGALE group
Up to increase (Fig. 4 f, 4g).In conclusion the missing of GALE can inhibit cell cycle progression and the induction of neuroglial cytoma
Apoptosis.
2.5miR-let-7i-5p Targeted-control GALE
It is generally believed that miRNA is combined by the 3 '-UTR with target gene carries out post-transcriptional control.In order to find GALE regulation
The target spot of cell movement and invasive ability, using Bioinformatics Prediction software Targetscan (http: //
www.targetscan.org/).In this thousands of candidate target, it is of interest that miR-let-7i-5p.In order to find
Whether miR-let-7i-5p passes through the movement of GALE3 '-UTR regulating cell and invasive ability, has carried out luciferase reporting test.
Luciferase detection (Fig. 5 A) is carried out using U251 glioma cell.Transfect fluorescein enzyme activity in the cell of mir-let-7i-5p
Property falls to approximately 76.8%.In order to detect inhibiting effect of the miR-let-7i-5p on protein level, in miR-let-7i-
48h carries out western blot analysis after 5p inhibitor, and simulates and be transfected into U251 and U87 cell.As can be seen that with negative
The glioma cell of control miRNA transfection is compared, and the GALE protein level of U251 and U87 cell is decreased obviously.
2.6GALE participates in the GBM cell Proliferation of mir-let-7i-5p induction and the inhibition of migration
Whether there is the function of similar GALE in order to probe into miR-let-7i-5p in GBM cell, using the side of RNA
Method.The overexpression of miR-let-7i-5p confirms (Fig. 5 b-5e) by real-time RT-PCR and western blot.Crossing table
Up to after miR-let-7i-5p, EDU is carried out to U87 and U251 cell line and bacterium colony forms detection.MiR-let-7i-5p crosses table
Up to group, there were significant differences (Fig. 6 a-6c) in two groups of experiments with NC group.
Then, the effect of wound healing and transwell Germicidal efficacy miR-let-7i-5p in GBM cell migration.
U87 and U251 cell uses miR-let-7i-5p mimics to handle respectively, and miR-let-7i-5p mimics group is in wound healing
In experiment and transwell experiment, compared with NC group, in wound healing experiment and transwell experiment, the surface of a wound is shown as respectively
It is less (Fig. 7) to be closed slow and staining cell, western blot experimental result is similar to siGALE group (Fig. 6 d).These results
Show that the overexpression of miR-let-7i-5p results in significant transfer ability and inhibits, it is essentially identical with the mode of GALE silencing.
The generation of 2.7GALE gene silencing inhibition in-vivo tumour
In order to further study effect of the GALE albumen in human glioma, internal assessment has been carried out to tumour growth.
Fluorescein-labeled U87 glioma cell is expressed gale targeting shRNA or controls the lentiviruses transduction of shRNA.By sh-GALE
Or in negative control cell implantation nude mouse, establish heterograft in situ.Every group passes through weekly biodiversity resources (BLI) system
Assess the development of tumour.Compared with the control group, the animal tumor size for carrying sh-GALE cell significantly reduces (Fig. 8 a, 8b), side
Definitely (Fig. 8 d), survival rate increases (Fig. 8 c) on boundary.Immunohistochemistry confirms the xenograft GALE that sh-GALE cell generates
Protein level reduces.Sh-GALE xenograft Bcl-2, mmp-2 and proliferation index Ki-67 also declines (Fig. 8 e-8h).These
The result shows that the reduction that the inhibition of GALE causes glioma cell to grow and invade in vivo.
To sum up, the present embodiment result demonstrates GALE height in glioma especially High Grade Gliomas and expresses, and miR-
The generation of GALE albumen can be effectively suppressed in let-7i-5p, to reduce glioma and migration, shows that GALE can be used as glue
The biomolecule marker of matter tumor especially High Grade Gliomas, and miR-let-7i-5p can be used as a kind of new glioma suppression
MiRNA processed.In addition, GALE can be the potential therapy target of glioblastoma, especially GBM patient.
It should be noted that above example is only used to illustrate the technical scheme of the present invention rather than is limited.Although ginseng
It is described the invention in detail according to given example, but those skilled in the art can be as needed to this hair
Bright technical solution is modified or replaced equivalently, without departing from the spirit and scope of the technical solution of the present invention.
SEQUENCE LISTING
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Claims (10)
1.miR-let-7i-5p and/or the application of GALE gene and its expression product in preparation glioma molecular marker.
2. application as described in claim 1, which is characterized in that the glioma molecular marker for diagnosing, detect, monitoring or
Predict the progress of glioma.
3. application as claimed in claim 2, which is characterized in that the progress of the glioma includes the proliferation of glioma cell, life
Long and/or migration；
Preferably, the glioma includes Low grade glioma (I-II grades) and High Grade Gliomas (III-IV grades).
4. a kind of composition of the progress for diagnosing, detecting, monitor or predict glioma, which is characterized in that including based on height
Throughput sequencing methodology and/or based on quantifying PCR method and/or based on probing procedure detection glioma samples in miR-let-
The transcription of 7i-5p and/or GALE；Or the target based on miR-let-7i-5p regulation in immunologic detection method detection glioma samples
The expression of gene or the substance of GALE expression product situation；
Wherein, the target gene includes GALE gene；
Preferably, analytical technology, RAKE method, original position are protected using Northern hybridizing method, miRNA chip of expression spectrum, ribozyme
The transcription of miR-let-7i-5p and/or GALE in hybridization check glioma samples；Using ELISA, colloidal gold strip, albumen
Chip detects the expression or GALE expression product situation of the target gene of miR-let-7i-5p regulation in glioma samples.
5. a kind of kit, which is characterized in that the kit includes described in claim 4 for diagnosing, detecting, monitoring or in advance
Survey the composition of the progress of glioma.
6. the substance and/or inhibition GALE gene and its expression product that promote miR-let-7i-5p expression and/or activity to improve
And/or application of the substance of activity reduction in following (a')-(c') is at least one:
(a') inhibit the migration of glioma cell, or product of the preparation for inhibiting glioma cell to migrate；
(b') inhibit the proliferation of glioma cell, or prepare the product for inhibiting glioma；
(c') inhibit the growth of glioma cell, or prepare the product for inhibiting growth of glioma cells；
Preferably, the glioma includes Low grade glioma (I-II grades) and High Grade Gliomas (III-IV grades).
7. a kind of for treating or preventing the pharmaceutical composition of glioma, which is characterized in that including promoting miR-let-7i-5p table
It reaches and/or substance that activity improves and/or the substance for inhibiting GALE gene and its expression product and/or activity to reduce.
8. pharmaceutical composition as claimed in claim 7, which is characterized in that the promotion miR-let-7i-5p expresses and/or lives
Property the substance that improves, short hairpin RNA including artificial synthesized miR-let-7i-5p or up-regulation miR-let-7i-5p expression are opened
Mover；
The substance for inhibiting GALE gene and its expression product and/or activity to reduce, including the special antibody of GALE albumen, needle
Rnai molecule or antisense oligonucleotides, micromolecular inhibitor, siRNA to GALE mRNA.
9. pharmaceutical composition as claimed in claim 7, which is characterized in that described pharmaceutical composition also includes at least one or more
Acceptable auxiliary material kind pharmaceutically or in bromatology.
10. pharmaceutical composition as claimed in claim 8 or 9, which is characterized in that the drug is tablet, dispersible tablet, enteric
Piece, chewable tablets, oral disintegrating tablet, capsule, sugar-coat agent, granule, dry powder doses, oral solution, the small water needle of injection, injection freeze-drying
Powder needle, big infusion or primary infusion.
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| CN110396544A (en) * | 2019-06-19 | 2019-11-01 | 山东大学齐鲁医院 | Application of the CUL7 in diagnosis of glioma, treatment and prognosis |
| CN110396544B (en) * | 2019-06-19 | 2021-12-24 | 山东大学齐鲁医院 | Application of CUL7 in glioma diagnosis, treatment and prognosis |
| CN112129938A (en) * | 2019-06-25 | 2020-12-25 | 中国科学院分子细胞科学卓越创新中心 | Application of UDP-Glc in lung cancer metastasis assessment |
| CN112129938B (en) * | 2019-06-25 | 2023-08-18 | 中国科学院分子细胞科学卓越创新中心 | Use of UDP-Glc in lung cancer metastasis assessment |
| CN112322731A (en) * | 2020-10-22 | 2021-02-05 | 山东大学 | Application of LIGHT in glioma diagnosis and treatment |
| CN116732183A (en) * | 2023-07-27 | 2023-09-12 | 南通大学 | Molecular marker for glioma diseases and application thereof |
| CN117721204A (en) * | 2023-12-26 | 2024-03-19 | 山东第一医科大学第一附属医院(山东省千佛山医院) | ceRNA regulatory mechanism of circ0104727 and application thereof in glioma |
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