CN109609653B

CN109609653B - The molecular marked compound and its application of diagnosis or treatment glioma

Info

Publication number: CN109609653B
Application number: CN201910111415.9A
Authority: CN
Inventors: 李刚; 薛皓; 于锐; 孙中正; 郭钦栋; 申杰
Original assignee: Qilu Hospital of Shandong University
Current assignee: Shandong Huachain Medical Technology Co ltd
Priority date: 2019-02-12
Filing date: 2019-02-12
Publication date: 2019-07-19
Anticipated expiration: 2039-02-12
Also published as: CN109609653A

Abstract

The present invention provides diagnosis or controls molecular marked compound and its application of glioma.It is proved through experimental study, the expression of microRNA-588 more normal oxygen under hypoxia obviously raises in glioma, Vitro Experimental Results show that miR-588 can inhibit the invasion of glioma cell to migrate and VM Forming ability, for molecular mechanism clearly therein, further experiment discovery Robo1 is the direct and function related target of miR-588 in glioma.Knocking out Robo1 can inhibit matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) to express, and then inhibit invasion migration and the VM Forming ability of glioma.Therefore, miR-588 and Robo1 can be used as the molecular marked compound of diagnosis and treatment glioma.

Description

The molecular marked compound and its application of diagnosis or treatment glioma

Technical field

The invention belongs to biological medicines and technical field of molecular biology, and in particular to the molecule of diagnosis or treatment glioma Marker 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.

Glioma is the most common malignant tumour of encephalic, and disease incidence is 3-8/10 ten thousand, accounts for the 40-50% of intracranial tumors.Glue The Biological characteristics of matter tumor mainly include high mortality and high relapse rate, strong invasive ability, very strong angiogenesis ability and wide General hypoxia.

The growths such as solid tumour such as breast cancer, liver cancer, glioma are rapid and corresponding blood supply is insufficient, and hypoxemia microenvironment exists It is generally existing in these tumours.The hypoxemia of tumour is directly related with low life cycle.There are many researchs to confirm hypoxemia and tumour at present Proliferation, invasion migration, the progress correlation such as angiogenesis.In addition, glioma blood supply is abundant, anti-angiogenic medicaments treat colloid The application prospect of tumor attracts attention.However, it is found by the inventors that anti-angiogenic Therapy is of short duration using rear generally existing curative effect and treats Imitate not good enough problem.The it is proposed of vascular mimicry (Vasculogenic mimicry VM) concept in 1999, make it was recognized that In tumor tissues other than the blood vessel being made of endothelial cell, there are also other blood supply forms.Vascular mimicry is by tumour A kind of cell pipeline for being functionally similar to normal blood vessels that simulated blood vessel endothelial cell is formed under specific circumstances, it is micro- with tumour Circulation interlinks, and the webbed blood conveying circulation of structure is supported for tumor with nutrition.It has confirmed at present, including glioma There is vascular mimicry in Several Kinds of Malignancy inside, and its positive rate and the poor prognosis of tumour rank and patient are close Correlation, hypoxemia microenvironment form closely bound up with vascular mimicry.

Although there are several mechanism to illustrate the invasion migration of the tumour cell of hypoxia inducible at present, still there are Many researchers Inquiring into the effect of miR (microRNA) in tumor hypoxia effect.MicroRNA is a series of endogenic 20-22 nucleosides The sub-thread non-coding RNA of acid sequence adjusts gene expression in post-transcriptional level negative.Before, there is research to confirm in lung cancer, stomach MiR-588 plays cancer suppressing action in the diseases such as cancer, breast cancer, and miR-588 is found to have promotion tumour cell in prostate cancer Proliferation function and show as promotion sensitivity gene.

Summary of the invention

In view of the above shortcomings of the prior art, the present invention provides diagnosis or controls the molecular marked compound of glioma and its answer With.It is proved through experimental study, the expression of microRNA-588 more normal oxygen under hypoxia obviously raises in glioma, external real Testing miR-588 as the result is shown can inhibit the invasion of glioma cell to migrate and VM Forming ability, for molecular mechanism clearly therein, Further experiment discovery Robo1 is the direct and function related target of miR-588 in glioma.Matrix can be inhibited by knocking out Robo1 Metalloproteinases 2 (MMP2) and matrix metalloproteinase 9 (MMP9) expression, and then the invasion migration of glioma and VM is inhibited to be formed Ability.Therefore, miR-588 and Robo1 can be used as the molecular marked compound of diagnosis and treatment glioma.

The present invention is achieved through the following technical solutions:

The first aspect of the invention provides any one or more following substance in preparation glioma molecular marked compound Application, the substance include miR-588, Robo1 gene and its expression product, matrix metalloproteinase 2 (MMP2) gene and Its expression product and matrix metalloproteinase 9 (MMP9) gene and its expression product.

Further, in the application, the marker is used to diagnose, detect, monitor or predict the progress of glioma.

The progress of the glioma includes migration, invasion and/or the vascular mimicry (Vasculogenic of glioma cell Mimicry, VM) it is formed；

Wherein, miR-588, Robo1 gene and its expression product, MMP2 gene and its expression product, MMP9 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 MiR-588, Robo1 gene and its expression product, MMP2 gene and its expression product and/or MMP9 gene and its table in tumor sample Up to product；Or based on immunologic detection method detection glioma samples in miR-588 regulation target gene expression, The substance of Robo1 expression product situation, MMP2 protein expression situation and/or MMP9 expression product situation.

Wherein, the target gene of the miR-588 regulation includes Robo1 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-588, Robo1, MMP2 and/or MMP9 in hybridization check glioma samples；Using ELISA, colloidal gold detection, egg The expression of the target gene of miR-588 regulation, Robo1 expression product situation, MMP2 egg in white chip detection glioma samples White expression and/or MMP9 expression product situation；

Further, the present invention provides a kind of kit, and the kit includes for diagnosing, detecting, monitor or predict The composition of the progress of glioma；

The progress of the glioma includes migration, invasion and/or the vascular mimicry (Vasculogenic of glioma cell Mimicry, VM) it is formed.

The third aspect of the invention provides the substance, inhibition that can promote that miR-588 is expressed and/or activity improves Substance, inhibition MMP2 gene and its expression product and/or activity drop that Robo1 gene and its expression product and/or activity reduce Low substance, and/or the substance for inhibiting MMP9 gene and its expression product and/or activity to reduce following (I)-(III) at least A kind of application in:

(I) inhibit the migration of glioma cell, or product of the preparation for inhibiting glioma cell to migrate；

(II) inhibit the invasion of glioma cell, or prepare the product for inhibiting invasion of glioma cells；

(III) inhibit the vascular mimicry of glioma cell, or prepare the product for inhibiting growth of glioma cells.

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 improved into miR-588 expression and/or activity, the object for inhibiting Robo1 gene and its expression product and/or activity to reduce Matter, the substance for inhibiting MMP2 gene and its expression product and/or activity to reduce, and/or inhibition MMP9 gene and its expression product And/or the substance that activity reduces；

The substance for promoting miR-588 expression and/or activity to improve, including using the microRNA function based on RNA Property obtain technology and/or gene specific miR Mimics technology up-regulation miR-let-7i-5p expression and/or promote its activity； Further preferably artificial synthesized miR-588 short hairpin RNA (short hairpin RNA, shRNA) or up-regulation miR-588 The promoter of expression.

The substance for inhibiting Robo1 gene and its expression product and/or activity to reduce, special including Robo1 albumen Antibody, rnai molecule or antisense oligonucleotides, micromolecular inhibitor, siRNA for Robo1 mRNA；Further, institute Stating antibody is human antibody.

The substance for inhibiting MMP2 gene and its expression product and/or activity to reduce, including MMP2 albumen resisting specifically Body, rnai molecule or antisense oligonucleotides, micromolecular inhibitor, siRNA for MMP2 mRNA；Further, described Antibody is human antibody.

The substance for inhibiting MMP9 gene and its expression product and/or activity to reduce, including MMP9 albumen resisting specifically Body, rnai molecule or antisense oligonucleotides, micromolecular inhibitor, siRNA for MMP9 mRNA；Further, described Antibody is human antibody.

In the present invention, " treatment or prevention " refer to that being able to suppress the migration of glioma cell, invasion and/or blood vessel intends State.

Further, described pharmaceutical composition can also include usually used appropriate carrier, excipient and diluent.And And according to usual way, pulvis, granule, tablet, capsule, suspension, emulsion, syrup, spray can be fabricated to Deng oral agents, external preparation, suppository and aseptic injectable solution form dosage form use.The carrier that may include, excipient and dilute Releasing agent has lactose, glucose, sucrose, D-sorbite, mannitol, xylitol, antierythrite, maltitol, starch, Arab Glue, alginates, gelatin, calcium phosphate, calcium silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, Methyl hydroxybenzoate, nipasol, talcum powder, magnesium stearate and mineral oil etc..

The invention has the advantages that: first demonstration that in glioma the expression of microRNA-588 under hypoxia compared with Normal oxygen obviously raises, and Vitro Experimental Results show that miR-588 can inhibit the invasion of glioma cell to migrate and form energy with VM Power, while being further discovered that Robo1 is the direct and function related target of miR-588 in glioma.Base can be inhibited by knocking out Robo1 Matter metalloproteinases 2 (MMP2) and matrix metalloproteinase 9 (MMP9) expression, and then inhibit the invasion migration and VM shape of glioma At ability.Therefore, miR-588 and Robo1 can be used as the molecular marked compound of diagnosis and treatment glioma, have good practical application it Value.

Detailed description of the invention

Fig. 1 is hypoxemia glioma cell chip results and the expression series of drawing of miR-588, wherein Figure 1A is to pass through The analysis of volcano figure method, the microRNAs that full-length genome chip of expression spectrum filters out significant difference expression has 84, wherein raising 67, lower 17；Wherein miR-588 is significantly raised under hypoxemia；Figure 1B is the microRNAs significantly raised, and miR-588 is low Multiple is raised under oxygen is located at first 10；For RT-qPCR, miR-588 is significantly raised Fig. 1 C under hypoxemia as the result is shown, and and hypoxemia Incubation time is related；Fig. 1 D is that CCK8 knocks out and be overexpressed as the result is shown miR-588 to cell activity without influence.

Fig. 2 is that miR-588 is knocked out and overexpression is migrated to invasion of glioma cells and the influence series of drawing of VM Forming ability； Wherein, Fig. 2A is that the transfer ability of U251 cell is obvious under hypoxemia as the result is shown for normal oxygen and the lower 36 hours scratch experiments of hypoxia condition Enhancing knocks out the transfer ability of miR-588 enhancing glioma cell, is overexpressed the migration that miR-588 then inhibits glioma cell Ability；Fig. 2 I is statistical chart, as a result there is significant difference；Fig. 2 B is that A172 glioma cell repeats scratch reality under normal oxygen and hypoxemia It tests, it is as a result consistent with U251 cell experiment；Fig. 2 J is statistical chart；Fig. 2 C and Fig. 2 D are respectively the U87 under normal oxygen and hypoxia condition Experiment is migrated with the Transwell of A172 cell, knocks out the migration energy that miR-588 significantly increases glioma cell as the result is shown Power is overexpressed the transfer ability that miR-588 then inhibits glioma cell；Fig. 2 K and Fig. 2 L are respectively statistical chart, are as a result had significant Sex differernce；Fig. 2 E and Fig. 2 F are respectively that the Matrigel matrigel invasion of the U87 and A172 cell under normal oxygen and hypoxia condition is real It tests, knocks out the invasive ability that miR-588 significantly increases glioma cell as the result is shown, be overexpressed miR-588 and then inhibit glioma The invasive ability of cell, hypoxemia can enhance this invasive ability；Fig. 2 M and Fig. 2 N are respectively statistical chart, as a result have conspicuousness poor It is different；Fig. 2 G and Fig. 2 H are respectively that VM of the U87 and A172 cell in Matrigel matrigel under normal oxygen and hypoxia condition is formed Experiment knocks out the VM Forming ability that miR-588 significantly increases glioma cell as the result is shown, is overexpressed miR-588 and then inhibits glue The VM Forming ability of matter oncocyte, hypoxemia can significantly increase this VM Forming ability；Fig. 2 O and

Fig. 2 P is respectively statistical chart, as a result there is significant difference.

Fig. 3 is the direct target validation series that miR-588 regulates and controls the migration of hypoxemia invasion of glioma cells and VM Forming ability Figure；There are the target spots that binds directly of miR-588 for the 3 ' UTR regions that Fig. 3 A is prediction result display Robo1, and construct the combination The luciferase reporter plasmid of site mutation；Fig. 3 B is that luciferase reporter gene experimental result shows the binding site Mutation can eliminate miR-588 to the inhibiting effect of Robo1；

Fig. 3 C shows that high expression ROBO1 person's life cycle is shorter in patients with gliomas；Fig. 3 D is the expression of ROBO1 in glioma Amount is apparently higher than normal cerebral tissue；Fig. 3 E is the active nothing of glioma cell after CCK8 applies siRNA to knock out Robo1 as the result is shown Significant changes；After Fig. 3 F and Fig. 3 G is Western Blot as the result is shown U87 and A172 cell overexpression miR-588, Robo1's Expression is obvious suppressed, knocks out the expression that miR-588 then increases Robo1, the expression and the expression trend of Robo1 of MMP2 and MMP9 Unanimously；Fig. 3 H is that Western Blot experimental result shows that the knockout efficiency of Robo1 siRNA is satisfied, knocks out MMP2 after Robo1 Expression with MMP9 also declines therewith.

Fig. 4 is that Robo1 specificity siRNA can block miR-588 to form invasion of glioma cells migration and VM The regulating and controlling effect series of drawing of ability；Fig. 4 A transfects Robo1 siRNA for scratch experiment as the result is shown can significantly inhibit glioma The transfer ability of cell, and miR-588 inhibitor can be blocked to promote the effect of glioma cell migration, while enhancing miR- Inhibiting effect of 588 mimics to cell migration；Fig. 4 E is statistical chart, as a result there is significant difference；Fig. 4 B and Fig. 4 C is Transwell migration and Matrigel, the glioma after Robo1 siRNA can significantly block miR-588 to knock out as the result is shown are thin Born of the same parents invade transfer ability up-regulation, and can enhance miR-588 overexpression and act on the invasion inhibition of metastasis of glioma cell；Fig. 4 F and Fig. 4 G is respectively statistical chart, as a result there is significant difference；Fig. 4 D is VM shape of the U87 glioma cell in Matrigel matrigel At experiment, Robo1 siRNA can significantly inhibit the VM Forming ability of glioma cell as the result is shown, and miR-588 can be blocked to strike Invasion of glioma cells transfer ability up-regulation after removing can be enhanced the invasion that miR-588 is overexpressed to glioma cell and migrate Inhibiting effect；Fig. 4 H is statistical chart, as a result there is significant difference.

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:

Solution hybridization (solution hbridization): keep the determined nucleic acid of denaturation single-stranded and radiation bamboo isotope labeling Known acid single-stranded (probe) keep the temperature in the solution, be allowed to form hybridization complex.After reaction, with hydroxyapatite method or Enzymatic isolation method separates the single-stranded and heteroduplex not being hybridized, then the probe amount after knot diaphragm in miscellaneous adornment molecule, so that it may calculate Tested nucleic acid amount out.

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.

Glioma is cultivated under low oxygen conditions, it may occur that special biologically, including regulation proliferation, angiogenesis, The activation of the signal path of transfer and apoptosis.Tumor hypoxia environment and prognosis be poor, radiotherapy resistance, chemoresistance etc. are related.? In the samples of human glioma of excision of performing the operation, it is seen that cell is around the necrotic tissue at center, and to external migration, this might mean that low Oxygen plays regulating and controlling effect in this process.This confirms that tumor hypoxia leads to the invasion of glioma cell by inside and outside experiment Transfer ability enhancing, but specific mechanism is still unclear, and recent study shows that different miR participates in this process.However, needle The relationship being in progress to miR-588 and glioma is unclear；It is therefore desirable to the specific molecule machines to miR-588 in glioma System is researched and analysed.

Glioma is a kind of brain tumor of very vascular, but anti-angiogenic therapy occurs treating after clinical application at present Imitate the of short duration and not good enough problem of curative effect.With the proposition of vascular mimicry concept in 1999, it was recognized that being removed in tumor tissues Outside the blood vessel that is made of endothelial cell, there are also other blood supply forms.Vascular mimicry is by tumour cell in specific feelings A kind of pipeline for being functionally similar to normal blood vessels that condition Imitating vascular endothelial cell is formed, interlinks with tumor microcirculation, The webbed blood conveying circulation of structure, is supported for tumor with nutrition.It has the following characteristics that (1) pipeline by tumour cell and It is not that endothelial cell is arranged to make up, is communicated with tumor microcirculation system；(2) DECM is remolded；(3) PAS stained positive and CD31 dye Color is negative.It has been confirmed that, there are vascular mimicry, positive rate and tumours in the Several Kinds of Malignancy including glioma at present The poor prognosis of rank and patient are closely related, and the high-caliber patient of vascular mimicry is more prone to metastases occur and have lower Survival rate.Thus, the therapeutic strategy of target vascular therapy mimicry is likely to a kind of very promising Tumor vasculature targeting hand Section.VM, which is formed, mainly passes through glioma stem cells, TGF β, VEGFR-2/FLK-1, VE-cadherin, RTK/PI3K/Akt/ The accesses such as mTOR, 2 chain pathway of MMP-laminin5 γ carry out, and Epithelial and stromal conversion and hypoxemia microenvironment are all wherein It plays an important role.The study found that miR-588 is obviously raised under hypoxemia, and it is overexpressed miR-588 and inhibits glioma cell Invasion migration, it is related that these all prompt miR-588 that may be formed with the VM of glioma.VM by carrying out glioma cell is formed As a result experiment confirms that miR-588 inhibits the VM of glioma cell to be formed.These discoveries illustrate that miR-588 plays cancer suppressing actions, can be with Biomarker as potential glioblastoma.

The access report for participating in miR-588 mechanism of tumor suppressor is seldom, present invention demonstrates that the relationship between miR-588 and hypoxemia. Experiment and research based on early period the result shows that, miR-588 can antagonism hypoxia inducible glioma malignant progression, suppression can be played The ability that tumor invasion migration processed and VM are formed.The motility of the previous experiments cell that miR-588 mimics is transfected as the result is shown And VM Forming ability is remarkably decreased, and is knocked out miR-588 and is then obtained adverse consequences, and these of miR-588 act on hypoxemia shape Amplified under state.MiR-588 is up-regulation to the reaction of hypoxemia, and plays cancer suppressing action.MiR-588 may take part in as a result, Protectiveness compensatory mechanism plays important work in terms of inhibiting the invasion of glioma cell of hypoxia inducible migration and VM Forming ability With.By predicting that miR-588 downstream movement related gene Robo1 has been found as potential promotion sensitivity gene target spot in website, and pass through Experimental verification.Robo1 is a kind of conservative transmembrane receptor protein, is mainly expressed in nervous system, participates in conduction biological information. Still without defining, the effect of oncogene or tumor suppressor gene has performance for effect of the Robo1 in tumor development.

Analyze TCGA database the results show that in glioma the expression of Robo1 obvious liter with normal cerebral tissue compared with Height, and Robo1 expression quantity high person's life cycle is obviously shortened in patients with gliomas.Luciferase reporter gene experimental verification The direct target spot relationship of miR-588 and Robo1.Western Blot experiment confirms that miR-588 can inhibit the expression of Robo1, together When also inhibit invasion migration and VM marker MMP2 and MMP9 expression.After further cell experiment confirmation knocks out Robo1 The invasion of glioma cell migrate and the decline of VM Forming ability, while the rush of miR-588 inhibitor invades transfer ability It is suppressed.Therefore the mechanism of action for considering miR-588 is under low-oxygen environment, and miR-588 binds directly the downward of the 3 ' areas-UTR Robo1 expression, reduces the expression of MMP2 and MMP9, to inhibit, the invasion of cell are migrated and VM Forming ability, miR-588 are A kind of tumor suppressor of hypoxia inducible can be acted on the rush invasion generated under antagonism hypoxia condition.

In view of this, providing any one or more following substance in a specific embodiment of the invention and preparing glue Application in matter tumor molecular marked compound, the substance include miR-588, Robo1 gene and its expression product, MMP2 gene and its Expression product and MMP9 gene and its expression product.

The progress of the glioma includes that migration, invasion and/or the vascular mimicry of glioma cell are formed；

In still another embodiment of the invention, provide it is a kind of for diagnose, detect, monitor or predict glioma into The composition of exhibition, it includes based on high-flux sequence method and/or based on quantifying PCR method and/or based on probing procedure Detect miR-588, Robo1 gene and its expression product, MMP2 gene and its expression product and/or MMP9 base in glioma samples Cause and its expression product；Or the expression based on the target gene of miR-588 regulation in immunologic detection method detection glioma samples The substance of situation, Robo1 expression product situation, MMP2 protein expression situation and/or MMP9 expression product situation.

Wherein, the target gene of the miR-588 regulation includes Robo1 gene；

In still another embodiment of the invention, using solution hybridization, Northern hybridizing method, miRNA express spectra Chip, ribozyme protection analytical technology, RAKE method, in situ hybridization detection glioma samples in miR-588, Robo1, MMP2 and/or The transcription of MMP9；Using the target gene of miR-588 regulation in ELISA, colloidal gold detection, protein chip detection glioma samples Expression, Robo1 expression product situation, MMP2 protein expression situation and/or MMP9 expression product situation；

In still another embodiment of the invention, the present invention provides a kind of kit, and the kit includes for examining The composition of the progress of disconnected, detection, monitoring or prediction glioma；

In still another embodiment of the invention, the object that can promote that miR-588 is expressed and/or activity improves is provided Matter, the substance for inhibiting Robo1 gene and its expression product and/or activity to reduce, inhibit MMP2 gene and its expression product and/or The substance, and/or inhibit MMP9 gene and its expression product and/or the substance of activity reduction at following (I)-that activity reduces (III) application at least one:

In still another embodiment of the invention, provide it is a kind of for treating or preventing the pharmaceutical composition of glioma, It includes the substance for promoting miR-588 expression and/or activity to improve, inhibit Robo1 gene and its expression product and/or activity drop The substance, and/or inhibit MMP9 gene and its table that low substance, inhibition MMP2 gene and its expression product and/or activity reduce The substance reduced up to product and/or activity；

In still another embodiment of the invention, described pharmaceutical composition can also include usually used appropriate load Body, excipient and diluent.Moreover, pulvis, granule, tablet, capsule, suspension can be fabricated to according to usual way The oral agents of agent, emulsion, syrup, spray etc., external preparation, suppository and aseptic injectable solution form dosage form use.

In still another embodiment of the invention, carrier, excipient and the diluent that may include have lactose, grape Sugar, sucrose, D-sorbite, mannitol, xylitol, antierythrite, maltitol, starch, Arabic gum, alginates, gelatin, phosphorus Sour calcium, calcium silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, hydroxyl Yl benzoic acid propyl ester, talcum powder, magnesium stearate and mineral oil etc..When by the composite preparation, common filler, increasing are used Amount agent, adhesive, wetting agent, disintegrating agent, the diluent of surfactant etc. or excipient and manufacture.Oral solid formulation includes Tablet, pill, pulvis, granule, capsule etc., this solid pharmaceutical preparation are to be mixed more than at least one in the composition Excipient, such as starch, calcium carbonate (calcium carbonate), sucrose, lactose, gelatin etc. and manufacture.Moreover, in addition to letter Other than single excipient, such as magnesium stearate, talcum powder lubricant are also used.Oral liquid phase preparation has suspension, liquor, cream Agent, syrup etc. may include a variety of excipient other than common simple diluent, such as water, liquid paraffin, such as Wetting agent, sweetener, aromatic, preservative agent etc..Non-oral formulation include aseptic aqueous solution, nonaqueous solvents, suspending agent, emulsion, Lyophilized preparation, suppository.Propylene glycol (propylene glycol), polyethylene glycol, as olive can be used in nonaqueous solvents, suspending agent The vegetable oil of oil, as the injectable esters etc. of ethyl acetate.Witepsol, polyethylene glycol, tween can be used in suppository base (tween) 61, cocoa butter, laurin fat, glycerin gelatine etc..

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

One, experimental material

Human glioma cells strain U251, U87, A172 are purchased from Cell Bank of Chinese Academy of Sciences, and grade scale is IV grades, adherent life It is long.DMEM complete medium is purchased from Gibco company, and Tissue Culture Flask and culture plate are purchased from Corning company, and trypsase is purchased from Invitrogen company, fetal calf serum are purchased from Hyclone company, and dual anti-to be purchased from Sigma company, it is all that PBS dry powder is purchased from Shanghai song Biology, Trizol are purchased from TAKARA company, and full genome chip of expression spectrum and full microRNA expression chip are by upper Haikang at biology Detection is bought and entrusted in Co., Ltd on behalf, and RT-qPCR kit is purchased from TAKARA company, RIPA lysate, 5 × SDS-PAGE loading Buffer, Tris-HCL is filled with gum cover and primary antibody dilution is purchased from green skies company, protease inhibitors, Tris, SDS, sweet ammonia Acid, Tween20, Lipofectamine2000 are purchased from Sigma company, and 0.45 μm of pvdf membrane and Western luminescence reagent box are purchased from Millipore company, it is rich still biological that microRNA mimics and inhibitor are purchased from Shanghai, miR-588 surely turn slow virus and Luciferase reporter plasmid wins still biosynthesis by Shanghai.

Two, experimental method

1, the normal oxygen of cell and hypoxemia culture

Glioma cell line U251, U87, A172 routine culture is in 5%CO₂, in 37 DEG C of cell incubators, and choose state Good cell, in hypoxemia incubator (5%CO₂, 1%O₂, 37 DEG C) in culture.

2, mRNA extraction and RT-qPCR

The glioma cell of culture prepares suspension, and precipitating is left and taken after centrifugation, and 1ml Trizol, lysis at room temperature 5min is added. 200 μ l chloroforms are added, are stored at room temperature 2min, is centrifuged 15min, goes 400 μ l of upper strata aqueous phase into new EP pipe.400 μ l isopropyls are added Alcohol stands 20min.It is centrifuged 15min, supernatant is abandoned and keeps on file the white precipitate of portion mRNA, and after being precipitated with ethanol washing, centrifugation 5min.It discards supernatant, back-off EP pipe, drying precipitated rear DEPC water dissolves mRNA, and NanoDrop surveys concentration.Sample is used The RT-PCR kit of TAKARA carries out cDNA reverse transcription.Reaction condition be 30 DEG C of 10min, 42 DEG C of 30min, 99 DEG C of 5min, 5 DEG C 5min.The 0.5 μ l of miR-RT primer that specificity is added also is needed in the RT-PCR system of microRNAs, instead of double steamings of 0.5 μ l Water.Then the preparation of real-time quantitative fluorescence PCR system is carried out using SYBR Green PCR Master Mix kit, reacts item Part is 95 DEG C of activation 5min, 95 DEG C of denaturation 10s, 60 DEG C of annealing 20s, 72 DEG C of extension 20s, is recycled 30-35 times, solubility curve 95 DEG C 5s, 65 DEG C of 1min, 97 DEG C -40 DEG C cooling 10s.After reaction, Roche real-time fluorescence quantitative PCR instrument automatically generates report, And result is exported, calculated result.

3, miR-588 mimics and inhibitor is transiently transfected

Glioma cell in good condition is uniformly inoculated in 6 orifice plates, 12h is cultivated.Lipo mixed liquor is prepared first, so Afterwards match microRNA transfection agents mixed liquor, every hole 2ml serum free medium add 4 μ g microRNA inhibitor or Mimics is added in lipo3000 mixed liquor after evenly mixing, stands 15min.Former culture medium is absorbed, PBS is rinsed, and configuration is added The good hole transfection cocktail 2ml/.It is incubated for 6 hours in 37 DEG C of incubators, replaces the fresh complete DMEM culture medium containing serum, after Continuous culture carried out subsequent experimental after 24 hours.

4, protein extraction and Western Blot

Cell protein extracts

It will cultivate to 1 × 10⁷Six orifice plates of/hole cell, PBS are washed 3 times, and cell scraping scrapes cell, and cell is collected by centrifugation. 15~30min is cracked on ice using dedicated 500 μ l of western cell pyrolysis liquid.It is centrifuged 10min.Draw 400 μ l supernatants.Add Enter 5 × appropriate sample-loading buffer, be denaturalized 10 minutes in boiling water, is detected after cooling.

PAGE gel electrophoresis

With preparing gel on glue frame, after solidification, micro sample adding appliance loading, 80V constant pressure electrophoresis 1.5 hours or so, to purpose Protein band separation stops when obvious.Remove gel, be placed in transferring film liquid, be sequentially placed sponge, filter paper, gel strips, pvdf membrane, Filter paper, sponge are fixed.200mA constant current carries out transferring film.TBST solution washes film on shaking table, closes 1 hour in confining liquid.TBST is washed It washs, pvdf membrane is incubated overnight for 4 DEG C in primary antibody working solution.TBST solution is incubated for secondary antibody working solution, room temperature shaker after washing film 30min.TBST solution washes film.The colour developing of HRP luminescence method, Bio-Rad infrared imaging system scan pvdf membrane, save and export figure Picture.

5, scratch experiment

It collects the good glioma cell of growth conditions to be inoculated in six orifice plates, culture to cell fusion degree 70% or so, It replaces serum free medium and carries out the pretreatment of 12h starvation.Every hole scratch 3 using sterile 10 μ l pipette tips.Free serum culture is added Base takes pictures under inverted microscope and (is denoted as 0h).Then 12h, for 24 hours, 36h equi-time point take pictures, observation cell migration fusion Degree.ImageG software calculates degrees of fusion.

6, Transwell is tested

It collects the good glioma cell of growth conditions to be inoculated in six orifice plates, cell is resuspended simultaneously in digestion after cultivating and handling It counts, adjusts to same cell concentration.Every group of 200 μ l of cell suspension is added in the cell Transwell upper chamber, lower room uses Complete medium of the 600 μ l containing 20% fetal calf serum carries out chemotactic.Upper chamber film is taken after culture 12h, 4% methanol fixes 10min, she Red colouring 15min after cotton swab wipes top surface cell away, is placed in observation on glass slide and takes pictures.

The experiment of Matrigel matrigel invasion is then to use cell in advance on above-mentioned transwell experiment basis Matrigel matrigel coating.The 100 μ l of Matrigel matrigel diluted, is uniformly laid on cell bottom, in 37 DEG C of incubators After solidifying 30min, for testing.

7, VM forms experiment

Take the every hole of 96 orifice plates of pre-cooling that 50 μ l Matrigel matrigels are added, then in 5%CO₂, put in 37 DEG C of incubators It sets 30 minutes, solidifies matrigel again.Single cell suspensions are made in digestion process each group test cell and resuspension, and every hole is inscribed 100 μ l of kind, are placed in 37 DEG C of normal oxygen or hypoxemia incubator and continue to cultivate.It is seen at culture 2h, 4h, 6h, 8h, 10h, 12h time point Examine the VM formational situation and integrated degree of each test group cell.The visual field is randomly selected in every area Kong Ge to take pictures, and is counted into pipe number Mesh, every group of experiment are repeated 3 times.decagram

8, luciferase reporter gene is tested

According to the target gene title of selection and site, the gene sequence of its 3 '-UTR region is inquired in pubmed database Column, find specific miR Complementary binding sites, and synthesis has the luciferase reporter plasmid of wild type 3 '-UTR sequence (WT), the luciferase reporter plasmid of the saltant type 3 '-UTR sequence after and miR Complementary binding sites being mutated (MUT).Carry out the cotransfection of wild type and mutant luciferase reporter plasmid and microRNA mimics.Use 6 holes After plate cotransfection 48 hours, PBS is rinsed, and carries out cell cracking 30 minutes at room temperature using 1 × PLB lysis buffer, then plus The Stop Buffer for entering 100 μ l terminates cracking.It is added in 96 hole elisa Plates, the sample into each hole is added 100 μ l's Luciferase Assays Reagent II, sufficiently shakes up, while it is blank control wells that 1 × PLB lysis buffer, which is arranged, is made It is detected with microplate reader.Fluoroscopic examination parameter is set as: minute 10s, measuring interval 2s.Excitation wavelength 480nm, detection Fluorescence intensity at 560nm wavelength carries out statistics and computing.

Three, statistical method

All experiments are repeated 3 times above, and all data are all made of mean ± standard deviation to indicate.Data analysis uses SPSS17.0 is carried out, and statistical chart is drawn to be completed using GraphPad Prism 5.0.Two groups of experimental datas are used double in experiment in vitro Side Student t is examined, and multi-group data is examined using the Kruskal-Wallis of non-matching.Two comparison among groups of experiment in vivo use Mann Whitney U is examined.Survival analysis uses Kaplan-Meier function and log-rank rank sum test.Use One-way ANOVAs carries out multiple-group analysis.Pearson ' s correlation analysis (quantitative data) and Spearman ' s correlation analysis (ranked data) For the correlation between analyzing two groups.All data * p < 0.05, * * think statistically significant in p < 0.01.

Four, result

1, it the miR to variable expression in glioma cell line under hypoxia and is trained under normal oxygen, hypoxia respectively The analysis for the difference that miR-588 is expressed in feeding glioma cell

Because hypoxemia is the independent prognostic factor of patients with gliomas low life cycle, sieved using miR micro-array chip fluxoid Selected the full-length genome miRs of glioma cell of U251 under hypoxemia and normal oxygen, come study hypoxia inducible miR table, then determine The miRs obviously raised is expressed under some hypoxia conditions.Microarray results show 84 miRs expression, and there were significant differences.Its The highest miR-210 of middle up-regulation it is verified that be hypoxemia miR marker, miR-588 be preceding ten highest miR of up-regulation it One.Higher miR is raised under 8 hypoxemia such as experimental verification miR-1207, miR-597, wherein miR-588 shows obviously to press down The ability of glioma invasion migration processed.Using volcano map analysis array experiment data, show that miR-588 is significant under hypoxia Up-regulation.NC, miR-588 mimics and miR-588 inhibitor are applied in an experiment.NC is negative control RNA, mimics It is the double stranded rna molecule of synthesis, the function of endogenous miR can be simulated, plays the role of being overexpressed correlation RNA after transfection.miR Inhibitor is artificial synthesized single stranded RNA molecule, can interfere related miR's by the irreversible block function that connected Function plays a part of to knock out miR-588.First using RT-qPCR technical identification NC, miR-588 mimics, miR-588 Transfection efficiency of the inhibitor in glioma cell line, the expression of miR-588 does not influence after transfection NC as the result is shown, turns Dye mimics can significantly improve the expression of miR-588, and transfection inhibitor obviously inhibits the expression of miR-588.Then, Again using the expression water of RT-qPCR technical checking miR-588 in U251 and U87 under normal oxygen and different duration hypoxias Flat, discovery miR-588 under hypoxia is expressed obviously to raise under more normal oxygen condition, and the glioma of hypoxemia culture about 24 hours The miR-588 of cell is expressed as top.

Influence for observation transfection to cell activity has carried out CCK8 experimental verification, experimental result display transfection NC, miR- 588 mimics, miR-588 inhibitor glioma cell activity there was no significant difference, illustrate to turn 80nM mimics wink Endogenous miR-588 expression can significantly be regulated and controled with inhibitor, while not influencing cell viability.

2, miR-588 inhibits invasion migration and the VM Forming ability of glioma cell.It is thin that miR-588 knocks out enhancing glioma The invasion of born of the same parents migrate and VM Forming ability, and can enhance the invasion migration of hypoxia inducible.MiR-588, which is overexpressed, inhibits glioma thin The invasion of born of the same parents migrate and VM Forming ability.

For study miR-588 upper reconciliation lower whether can regulate and control hypoxia inducible glioma cell migration invasion and VM is formed, and U87, U251 and the A172 for turning NC, miR-588 mimics, miR-588 inhibitor wink are applied in test Glioma cell line.To probe into the influence that miR-588 migrates glioma cell, the scratch of A172 and U251 cell line has been carried out Experiment.Several interesting phenomenons are observed in an experiment.Firstly, the transfer ability of cell is bright under hypoxemia condition of culture as the result is shown Aobvious enhancing, this is consistent with expected results.Secondly, it can be observed that miR-588 obviously inhibits the transfer ability of glioma cell, together When also obvious antagonism hypoxia inducible rush migration effect.The glioma cell for knocking out miR-588 then shows the bright of transfer ability It is aobvious to be promoted, it is cultivated under hypoxia, more promotes the migration of cell.Oxygen water in miR-588 expression and incubator It is flat to regulate and control glioma cell transfer ability, and this regulating effect can interact.

Further to study influence of the miR-588 to glioma cell migration and invasive ability, using U87 and A172 cell System carries out Transwell experiment.The ability that U87 cell passes through polycarbonate membrane is obviously strong compared with A172 cell, and test is repeated several times It was found that U87 cell is cultivated in the cell Transwell is fixed dyeing for 6 hours, A172 cell was then fixed at 20 hours Dyeing.The experimental result of both cell lines is consistent.Compared with the control group, miR-588 significantly inhibits glioma cell Film transfer ability is worn, miR-588 inhibitor then significantly enhances the transfer ability of cell.Meanwhile hypoxemia promotes glioma Cell wears film migratory movement.Then, the observation that invasion of glioma cells ability is carried out in cell is layered on using Matrigel glue, As a result similar to the result of migration experiment.This illustrates that miR-588 can significantly inhibit the invasion transfer ability of glioma cell, and energy The rush of antagonism hypoxia inducible invades transfer ability simultaneously, and after having knocked out miR-588, the invasion transfer ability of glioma cell Significantly enhanced.

It is cultivated in Matrigel glue using U87 and A172 cell line and carries out VM formation Germicidal efficacy.In experiment, glioma Protrusion connection is assembled first and stretched out to cell, subsequently forms tubular structure i.e. VM, tubular structure is disintegrated after a period of time.Experiment knot Fruit shows that VM forms speed faster under hypoxia, and the tubular structure of formation is more.Compared with the control group, it is overexpressed miR-588 The speed and tubular structure quantity for obviously inhibiting VM to be formed are formed with antagonism to rush VM caused by hypoxemia.Knock out miR- The VM Forming ability of 588 cells significantly improves.These are the results show that VM is formed with the invasion transfer ability of cell with consistent Property, miR-588 can obviously inhibit the VM Forming ability of glioma cell.

3, Robo1 be miR-588 inhibit invasion of glioma cells migration and VM Forming ability in terms of downstream key gene.

To probe into, miR-588 regulates and controls the invasion migration of glioma cell and the mechanism of VM Forming ability, searching are closed downstream Key target gene.Robo1 is filtered out, relevant prediction result shows the 3 ' area-UTR the binding directly there are miR-588 of Robo1 Target spot.Robo1 is a kind of conservative transmembrane receptor protein, is mainly expressed in nervous system.Analyze TCGA database the results show that The expression of Robo1 is significantly raised compared with normal cerebral tissue in glioma, and Robo1 expression quantity is high in patients with gliomas Person is obviously shortened life cycle.These explanations Robo1 in glioma plays tumor promotion.RT-qPCR experiment display transfection miR-588 After mimics, Robo1 expression is obvious to be lowered, and knocking out miR-588 can induce Robo1 expression.For the miR-588 for examining protein level Inhibitory effect, 48 hours extractions albumen carries out Western Blot reality after mimics and the inhibitor transfection of miR-588 It tests, experimental result display, which is overexpressed miR-588, can reduce the expression of Robo1, conversely, knocking out miR-588 then increases Robo1's Expression, this illustrates that miR-588 can regulate and control the expression of Robo1, and negatively correlated therewith.For the straight of clear miR-588 and Robo1 It connects target spot relationship, carries out luciferase reporter gene experiment, it is glimmering after cell transfecting miR-588 as the result is shown compared to control group Light element enzymatic activity is reduced to about 61.3%, illustrates that Robo1 is by miR-588 direct regulation and control in glioma cell.

In Western Blot experiment, it can also be observed that being overexpressed miR-588 suppression in U87 and A172 cell line The invasion transfer ability of the expression of MMP2 and MMP9 processed, MMP2 and glioma cell is positively correlated, and the invasion of MMP9 and cell are moved Shifting formed to VM it is related, this part illustrate miR-588 inhibit invasion of glioma cells migrate and VM Forming ability mechanism.For Further research knocks out the variation of MMP2 and MMP9 after Robo1, NC, miR-588mimics, the miR- loaded with slow virus 588inhibitor stable transfection U87 cell.U87 cell after steady turn of siRNA transfection of Robo1, extracts albumen and control group It compares, the expression of Robo1 is substantially reduced, and illustrates that the knockout efficiency of Robo1-siRNA is satisfied.It is tested simultaneously in Western Blot As a result confirm knock out Robo1 after, the expression of MMP2 and MMP9 reduce simultaneously, illustrate Robo1 expression decline inhibit MMP2 with The expression of MMP9 partially illustrates that miR-588 regulates and controls colloid to influence invasion migration and the VM Forming ability of glioma cell The mechanism that tumor invasion migration and VM are formed.

For effect of the verifying Robo1 in invasion of glioma cells migrates and VM is formed, is transfected and stablized with Robo1-siRNA The U87 glioma cell of NC, miR-588mimics, miR-588inhibitor are transfected, and scratch, Transwell are carried out with this And VM forms experiment, and not knock out the cell of Robo1 as a control group.Scratch experiment compared with the control group, strikes as the result is shown Except glioma cell transfer ability is decreased obviously after Robo1, surely turn the cell of miR-588 mimics after knocking out Robo1, carefully The ability of born of the same parents' migration minimizes.Surely turn the cell of miR-588 inhibitor after knocking out Robo1, cell migration ability and NC is similar, and the cellular control unit for not knocking out Robo1 is decreased obviously.

In subsequent Transwell experiment, same conclusion is obtained, Robo1 is knocked out and obviously inhibits glioma cell Transfer ability.Matrigel is carried out simultaneously, and experimental result and migration experiment are consistent, knock out Robo1 and equally also inhibit colloid The invasive ability of oncocyte.Experimental result illustrates that Robo1 is obviously promoted the invasion migration of glioma cell, and knocking out Robo1 can Enhance the ability for inhibiting invasion of glioma cells migration of miR-588.Downstream targets of the Robo1 as miR-588, miR-588 By inhibiting the expression of Robo1 to play cancer suppressing action.

For effect of the clear Robo1 in glioma cell VM is formed, the glioma cell of transfection Robo1 siRNA is used It carries out VM and forms experiment.The tubular structure that the VM that experimental result display knocks out Robo1 cell is formed is significantly reduced compared with control group, shape At time delay, illustrate that VM Forming ability is remarkably decreased, it is important that this illustrates that Robo1 is also functioned in the VM of glioma cell is formed Effect, this is consistent with the result of invasion migration.

To sum up, this example illustrates a kind of targeting Robo1 to inhibit the microRNA that glioma invasion migrate and VM is formed, MiR-588, and show that miR-588 can be used as a kind of biomarker of glioma, while being also that a glioblastoma is potential Therapy target.

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.

Claims

1. detecting the reagent of miR-588 expression or the reagent of detection Robo1 gene expression dose and its expression product level Application in preparation glioma testing product.

2. application as described in claim 1, which is characterized in that the glioma testing product is for diagnosing, detecting, monitoring or in advance Survey the progress of glioma.

3. application as claimed in claim 2, which is characterized in that the progress of the glioma includes the migration of glioma cell, invades It attacks and/or vascular mimicry is formed.

4. application as described in claim 1, which is characterized in that miR-588, Robo1 gene and its expression product are people Source.

5. promoting application of the substance of miR-588 expression in following (I)-(III) is at least one:

(I) product for inhibiting glioma cell to migrate is prepared；

(II) product for inhibiting invasion of glioma cells is prepared；

(III) product for inhibiting growth of glioma cells is prepared.

6. inhibiting application of the substance of Robo1 gene and its expression product in following (I)-(III) is at least one:

(I) product for inhibiting glioma cell to migrate is prepared；

(II) product for inhibiting invasion of glioma cells is prepared；

(III) product for inhibiting growth of glioma cells is prepared.