CN103961720B - MicroRNA is for the preparation of reducing glioma to the purposes of the medicine of TRAIL toleration - Google Patents
MicroRNA is for the preparation of reducing glioma to the purposes of the medicine of TRAIL toleration Download PDFInfo
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- CN103961720B CN103961720B CN201410165917.7A CN201410165917A CN103961720B CN 103961720 B CN103961720 B CN 103961720B CN 201410165917 A CN201410165917 A CN 201410165917A CN 103961720 B CN103961720 B CN 103961720B
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Abstract
The present invention is the divisional application of the application for a patent for invention of application number 201310089306.4.The present invention relates to a kind of microRNA for the preparation of reduction glioma to the purposes of the medicine of TRAIL toleration, does described pharmaceutical pack contain sequence as SEQ? ID? microRNA shown in NO:3; SEQ? ID? microRNA shown in NO:3 is not combined with death receptor DR5 gene 3 '-UTR, improves the expression of DR5 gene, reduces tumor to the toleration of TRAIL.Described microRNA, by the regulation and control to DR5 gene expression, reaches the object of modulate tumor cell to TRAIL toleration.
Description
The divisional application that the application is application number 201310089306.4, the applying date is on March 19th, 2013, denomination of invention is the application for a patent for invention of " a kind of modulate tumor is to the medicine of TRAIL toleration ".
Invention field
The present invention relates to biomedicine field, the modulate tumor especially containing microRNA is to the medicine of TRAIL toleration.
Background technology
Tumor necrosin relative death inducing ligand (TRAIL) is newfound TNF family newcomer in recent years, in vitro can induced various types of tumors apoptosis, but normal tissue is insensitive.Show the safe and effective of it in the clinical I phase experimental data of the U.S., carry out II/III phase clinical research at present.TRAIL is mainly by playing the function of cell death inducing with the receptors bind of cell surface, death receptor DR4 and DR5 is 2 specific receptors of TRAIL, because these two receptors exist only in tumor, so TRAIL is to not normal cells side effect.Although TRAIL provides new approach for oncotherapy, but find in clinical and laboratory research, the tumor cell of nearly 50% has certain toleration to TRAIL, and when namely going to process tumor cell with TRAIL, tumor cell does not have obvious apoptosis phenomenon to occur.
The non-coding tiny RNA of MicroRNA (miRNA) to be length be 20 ~ 24 nucleotide, can the expression of specific suppression target gene by the combination with target spot.Nearly ten years, researcher finds that microRNA plays extremely important function in the physiological process such as the propagation of fetal development and cell, differentiation and apoptosis.Up to the present, there is several microRNA energy modulate tumor to the toleration of TRAIL, such as, microRNA-221/222 weakens the therapeutic effect (GarofaloM of TRAIL to nonsmall-cell lung cancer by lowering PTEN and TIMP3 expression, DiLevaG, RomanoG, NuovoG, SuhSS, NgankeuA, TaccioliC, PichiorriF, AlderH, SecchieroP, GaspariniP, GonelliA, CostineanS, AcunzoM, CondorelliG, CroceCM.miR-221 & 222regulateTRAILresistanceandenhancetumorigenicitythroug hPTENandTIMP3downregulation.CancerCell2009, 16:498-509), and it is contrary, microRNA-130 and microRNA-212 improves tumor cell to the sensitivity (IncoronatoM of TRAIL by target PED, GarofaloM, UrsoL, RomanoG, QuintavalleC, ZancaC, IaboniM, NuovoG, CroceCM, CondorelliG.miR-212increasestumornecrosisfactor-relateda poptosis-inducingligandsensitivityinnon-smallcelllungcan cerbytargetingtheantiapoptoticproteinPED.CancerRes2010, 70:3638-3646.AcunzoM, VisoneR, RomanoG, VeroneseA, LovatF, PalmieriD, BottoniA, GarofaloM, GaspariniP, CondorelliG, ChiarielloM, CroceCM.miR-130atargetsMETandinducesTRAIL-sensitivityinN SCLCbydownregulatingmiR-221and222.Oncogene2012, 31:634-642).Recently, microRNA-25 is in the news by regulating and controlling the expression of DR4 and improves the toleration (RazumilavaN of cholangiocarcinoma cell to TRAIL, BronkSF, SmootRL, FingasCD, WerneburgNW, RobertsLR, MottJL.miR-25targetsTNF-relatedapoptosisinducingligand (TRAIL) deathreceptor-4andpromotesapoptosisresistanceincholangio carcinoma.Hepatology2012; 55:465-475).
Summary of the invention
Therefore, for tumor cell, TRAIL is had to the problem of toleration, the invention provides modulate tumor containing microRNA to the medicine of TRAIL toleration.
To achieve these goals, the present invention adopts following technical scheme.
Modulate tumor, to a medicine for TRAIL toleration, is characterized in that, described medicine contains sequence for microRNA shown in SEQIDNO:1-4 and variant thereof.
Preferably, microRNA shown in SEQIDNO:1-2 and variant thereof are combined with death receptor DR5 gene 3 '-UTR, suppress the expression of DR5 gene, improve tumor to the toleration of TRAIL.
Preferably, microRNA shown in SEQIDNO:3-4 and variant thereof are not combined with death receptor DR5 gene 3 '-UTR, improve the expression of DR5 gene, reduce tumor to the toleration of TRAIL.
Preferably, the microRNA shown in SEQIDNO:1 is microRNA-133a; MicroRNA shown in SEQIDNO:2 is microRNA-133b.
Preferably, the microRNA shown in SEQIDNO:3 is the antisense sequences of microRNA-133a; MicroRNA shown in SEQIDNO:4 is the antisense sequences of microRNA-133b.
Preferably, described microRNA is obtained by chemosynthesis or from organism sample.
Further preferably, described organism sample is tissue or cell.
Preferably, described microRNA is diagnostic markers or the therapeutic targets of a kind of TRAIL treatment.
Preferably, described tumor comprises all solid tumors and leukemia.
Further preferably, described tumor is glioma.
Modulate tumor of the present invention is to the medicine of TRAIL toleration, and contained microRNA and antisense sequences thereof, by the regulation and control to DR5 gene expression, reach the object of modulate tumor cell to TRAIL toleration.Described microRNA can by reducing the expression of DR5 thus improving the toleration of TRAIL in neuroglial cytoma, and its antisense sequences then significantly improves the expression of DR5 and facilitates the apoptosis of TRAIL induction.So microRNA-133a/b is likely as a diagnostic markers or the molecular target of TRAIL oncotherapy.
Accompanying drawing explanation
Fig. 1 is the toleration figures of 5 kinds of neuroglial cytomas to TRAIL.Wherein, in Fig. 1, A is the proliferation results that mtt assay measures 5 kinds of neuroglial cytomas; In Fig. 1, B is the apoptosis result of PI Determination Staining 5 kinds of neuroglial cytomas.
Fig. 2 is the expression of results figure of microRNA in the neuroglial tumor of different TRAIL tolerance type.
Fig. 3 is the schematic diagram that bioinformatics software prediction microRNA and DR5 combines.
Fig. 4 is the data result figure that extracorporeal biology functional experiment checking microRNA and DR5 combines.
Fig. 5 is that microRNA-133a affects result figure to the toleration of the TRAIL of neuroglial cytoma.
In above-mentioned figure, U87, M059K, M059J, A172 and T98G are neuroglial cytoma strain in 5, purchased from American Type culture collection warehousing (Americantypeculturecollection, ATCC); MiR-133a represents microRNA-133a, and miR-133b represents microRNA-133b, and anti-133a represents the antisense sequences of microRNA-133a; Mock represents blank, and NC represents negative control.
Detailed description of the invention
Below in conjunction with specific embodiment, such scheme is described further.Should be understood that these embodiments do not limit the scope of the invention for illustration of the present invention.The implementation condition adopted in embodiment can do further adjustment as the case may be, and not marked implementation condition is generally the condition in normal experiment.
" microRNA " in the present invention can be chemosynthesis, also can be to extract from the cell that there is this gene order, tissue.Described tissue can select neoplastic lesion tissue, and with paraffin mass, paraffin section that these tissues are made.
" sample " in the present invention refers to potential all in vitro samples that may contain sequence shown in SEQIDNO:1-4, preferably from the sample of human body.More preferably from the sample of tumor tissues.The purification of samples of the blood total serum IgE certainly extracted also can as sample of the present invention.
" microRNA-133a " in the present invention refers to the microRNA comprising the sequence shown in SEQIDNO:1 or its homologous sequence.The microR-133a in known various source, such as people, chimpanzee, Mus etc. in this area, these homologous sequences are all included in term of the present invention " microRNA-133a ".In the present invention, microRNA-133a variant comprises through replacing, lacking or add one or several nucleotide in above-mentioned naturally occurring microRNA-133a sequence, or through biochemical modification, and still there is the derivative RNA of biologic activity.The meaning of the antisense sequences of MicroRNA-133b, microRNA-133a and the antisense sequences of microRNA-133b and their variant and scope are with reference to above-mentioned explanation.
" oncotherapy " in the present invention refers to various solid tumor and leukemia, preferably glioma.
" medicine " of " modulate tumor is to the medicine of TRAIL toleration " in the present invention can also comprise other active component or the auxiliary element for the treatment of or prophylaxis of tumours.
The toleration of embodiment one, neuroglial cytoma TRAIL
With 5 kinds of common neuroglial cytomas for model, with the TRAIL (50ng/ml of variable concentrations; 100ng/ml) process respectively, adopt cell proliferation (mtt assay) and PI Determination Staining 5 kinds of neuroglial cytomas to the toleration of TRAIL.Statistical analysis finds, with U87 cell for standard, M059K and M059J belongs to TRAIL tolerance type cell strain, and A172 and T98G belongs to the strain of TRAIL sensitive cell.The results are shown in Figure 1.
Embodiment two, the microRNA-133a/b expression in the neuroglial cytoma of different TRAIL tolerance type
Real-Time Fluorescent Quantitative PCR Technique is adopted to determine the expression of microRNA-133a and microRNA-133b in the neuroglial cytoma of different TRAIL tolerance type.Statistical analysis finds, microRNA-133a and microRNA-133b expresses and raised in TRAIL tolerance type cell strain; And express suppressed in the cell strain of TRAIL responsive type.The results are shown in Figure 2.
Embodiment three, Bioinformatics Prediction microRNA-133a may be combined with DR5 gene 3 '-UTR
Adopt bioinformatics software TargetScan prediction to find, hsa-microRNA-133a may be combined with DR5 gene 3 '-UTR, the results are shown in Figure shown in 3.
The experiment of embodiment four, extracorporeal biology proves that microRNA-133a suppresses the expression of DR5
MicroRNA-133a is transfected into U87 respectively, in M059J and A172 cell after 72 hours, collecting cell, extraction RNA and albumen carry out the expression that Q-PCR and Westernblotting detects DR5 respectively, result shows no matter at mRNA level in-site or protein level, microRNA-133a significantly lowers the expression of DR5, and the antisense sequences of microRNA-133a (anti-microRNA-133a) but raises its expression.Therefore, above-mentioned extracorporeal biology functional experiment confirms, microRNA-133a can be combined with DR5 gene 3 '-UTR, thus suppresses the expression of DR5, the results are shown in Figure 4.
Embodiment five, microRNA-133a are on the impact of the TRAIL toleration of neuroglial cytoma
Adopt the impact of microRNA-133a on the TRAIL toleration of neuroglial cytoma of cell proliferation (mtt assay) and PI Determination Staining.Statistical analysis, microRNA-133a adds the toleration of the TRAIL of neuroglial cytoma, and its antisense sequences reduces the toleration of the TRAIL of neuroglial cytoma, facilitates the apoptosis of TRAIL induction.The results are shown in Figure 5.
Above-mentioned example, only for technical conceive of the present invention and feature are described, its object is to person skilled in the art can be understood content of the present invention and implement according to this, can not limit the scope of the invention with this.All equivalent transformations of doing according to spirit of the present invention or modification, all should be encompassed within protection scope of the present invention.
Claims (5)
- MicroRNA shown in 1.SEQIDNO:3 is for the preparation of reducing glioma to the purposes of the medicine of TRAIL toleration.
- 2. purposes according to claim 1, is characterized in that, microRNA shown in SEQIDNO:3 is not combined with death receptor DR5 gene 3 '-UTR, improves the expression of DR5 gene, reduces tumor to the toleration of TRAIL.
- 3. purposes according to claim 1, is characterized in that, microRNA shown in SEQIDNO:3 is the antisense sequences of microRNA-133a.
- 4. purposes according to claim 1, is characterized in that, described microRNA is obtained by chemosynthesis or from organism sample.
- 5. purposes according to claim 4, is characterized in that, described organism sample is tissue or cell.
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| CN201410165917.7A CN103961720B (en) | 2013-03-19 | 2013-03-19 | MicroRNA is for the preparation of reducing glioma to the purposes of the medicine of TRAIL toleration |
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| CN201410165917.7A CN103961720B (en) | 2013-03-19 | 2013-03-19 | MicroRNA is for the preparation of reducing glioma to the purposes of the medicine of TRAIL toleration |
| CN201310089306.4A CN103157117B (en) | 2013-03-19 | 2013-03-19 | Medicine for regulating and controlling TRAIL (Tnf (Tumor necrosis factor) Related Apoptosis Inducing Ligand) tolerance of tumour |
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Citations (4)
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| WO2010041140A2 (en) * | 2008-10-10 | 2010-04-15 | National University Of Ireland | Treatment of proliferative disorders with a death receptor agonist |
| CN102080086A (en) * | 2009-12-01 | 2011-06-01 | 中国科学院上海药物研究所 | Human miR-133a antisense nucleic acid and application thereof |
| CN102573856A (en) * | 2009-09-10 | 2012-07-11 | 弗莱明·韦林 | Method for the preparation of micro-RNAand its therapeutic application |
| CN102803511A (en) * | 2009-11-23 | 2012-11-28 | 俄亥俄州立大学 | Materials and methods useful for affecting tumor cell growth, migration and invasion |
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2013
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010041140A2 (en) * | 2008-10-10 | 2010-04-15 | National University Of Ireland | Treatment of proliferative disorders with a death receptor agonist |
| CN102573856A (en) * | 2009-09-10 | 2012-07-11 | 弗莱明·韦林 | Method for the preparation of micro-RNAand its therapeutic application |
| CN102803511A (en) * | 2009-11-23 | 2012-11-28 | 俄亥俄州立大学 | Materials and methods useful for affecting tumor cell growth, migration and invasion |
| CN102080086A (en) * | 2009-12-01 | 2011-06-01 | 中国科学院上海药物研究所 | Human miR-133a antisense nucleic acid and application thereof |
Non-Patent Citations (4)
| Title |
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| Curcumin sensitizes tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis through reactive oxygen species-mediated upregulation of death receptor 5 (DR5);Eun Mi Jung等;《Carcinogenesis》;20050629;1905-1913 * |
| MicroRNA signatures of TRAIL resistance in human non-small cell lung cancer;M Garofalo等;《Oncogene》;20080204;第27卷;3845-3855 * |
| MiR-133b Targets Antiapoptotic Genes and Enhances Death Receptor-Induced Apoptosis;Juan P. Patron等;《PlosOne》;20120420;参见全文 * |
| miR-25 targets TNF-related apoptosis inducing ligand (TRAIL) death receptor-4 and promotes apoptosis resistance in cholangiocarcinoma;Nataliya Razumilava等;《HEPATOLOGY》;20121231;465-475 * |
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