CN105617401A - Tumor radiation-sensitizing and radiation bystander effect reducing function of miRNA, implementation method, and uses - Google Patents
Tumor radiation-sensitizing and radiation bystander effect reducing function of miRNA, implementation method, and uses Download PDFInfo
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Abstract
The invention belongs to the technical field of biomedicine, and particularly relates to a radiation-sensitizing function of miR214, a radiation-sensitizing and radiation bystander effect damage reducing function of miR495, uses of the miR214 and the miR495 in improvement of a tumor radiotherapy treatment effect, and an application method. In the invention, miRNA can be used as an auxiliary medicine for improving a tumor clinical radiotherapy effect, particularly, the miR495 can improve the tumor radiation sensitivity and can reduce the damage to a liver caused by a bystander effect, and a new idea is provided for preparation of a clinical tumor radiotherapy auxiliary medicine.
Description
Technical field
The invention belongs to biotechnology and medical field, it is specifically related to the radiation sensitizing effect of a kind of RNA small molecules miR214, and the radiation sensitivity of another kind of RNA small molecules miR495, the effect that weakens radiation bystander cell, and both implementation methods and purposes.
Background technology
Microrna (micoRNA, it is called for short miRNA) it is a class by the non-coding single stranded RNA that the length of endogenous gene is 18-24 Nucleotide, the miRNA precursor (pre-miRNA) of length 70-80 the Nucleotide having hairpin ring structure by one section is by generation after Dicer enzyme (the RNA restriction endonuclease that double-stranded RNA is single-minded) shearing. They participate in posttranscriptional gene expression regulation in animals and plants, are combined in and transcribe rear level by being close to complete complementary with the 3'-UTR district of said target mrna and make it degrade, or not exclusively complementation is combined in the synthesis of translation skill arrestin with it. It is that Lee, Wightman etc. in 1993 have found miRNA and lin-4[LeeR.C etc. that adjustable nematode growth is grown in beautiful nematode (C.Elegans) body about miRNA report the earliest, TheC.elegansheterochronicgenelin-4encodessmallRNAswithan tisensecomplementarytolin-14.Cell.1993,75:843-854.]. Hereafter, more than 5000 miRNA from 58 different plant species is found out [MeltzerPS.Cancergenomics:smallRNAswithbigimpacts.Nature gradually, 2005,435 (7043): 745-46..], and each miRNA has corresponding gene target spot, thus regulate and control the expression of about 30% protein coding gene, miRNA is extensively present in biology, plays important biological regulation effect in fields such as tumour, infectious diseases, cardiovascular disorder, immunity, metabolism.
Tumour radiotherapy is a kind of classical way of clinical upper treatment tumour. the tumour patient of nearly 60% needs to accept radiotherapy. ionizing rays effect can cause DNA of tumor cell to damage, and then cell death inducing, reaches the effect for the treatment of tumour. but owing to tumour cell exists certain radiation resistance, have impact on the effect [JossonS of tumour radiotherapy to a great extent, SungSY, LaoK, etal.RadiationmodulationofmicroRNAinprostatecancercellli nes [J] .Prostate, 2008,68 (15): 1599-606.]. another defect that the radiotherapy of tumour is applied clinically is, the damage of biological cells and tissues is nonspecific by ionizing rays, radiological medicine is in tumour traditional therapy, not only tumour cell is caused damage, the normal cell that ionizing rays also can make tumor vicinity even remote organization's cell damage. this kind of damage derives from organizes the release cells factor (Organizationreleasingfactor by irradiating cell, ORF), TNF-a (tumornecrosisfactor-a (TNF-��) [MuthusamyV, PivaTJ..UVB-stimulatedTNF �� releasefromhumanmelanocyteandmelanomacellsismediatedbyp3 8MAPK.IntJMolSci..2013, 14 (8): 17029-17054], Interleukin-1�� (interleukin-1 ��, IL-1 ��) [PaquetteB, TherriaultH, WagnerJR.Roleofinterleukin-1 �� inradiation-enhancementofMDA-MB-231breastcancercellinvas ion.RadiatRes.2013, 180 (3): 292-298.], epithelical cell growth factor (epidermalgrowthfactor, EGF), transforming growth factor (transforminggrowthfactor �� 1, TGF-�� 1) [ShenL, LiX, ShanB, ZhangL, GongY, DongZetal.TherapeuticeffectofcompoundofWhitePeonyRootOra lLiquidsonradiation-inducedesophagealtoxicityviatheexpre ssionofEGFandTGF-�� 1.BiomedRep.2013, 1 (2): 308-314], active oxygen radical (ReactiveOxygenSpecies, ROS), nitrogen protoxide (nitricoxide, NO) [the .Nitricoxideisakeymoleculeservingasabridgebetweenradiati on-inducedbystanderandadaptiveresponses.CurrMolPharmacol .2011 such as MatsumotoH, 4 (2): 126-134.], radiation injury signal can be transmitted to the cell directly not radiated by these informational molecules, in tissue, then the damaging effect brought out. this kind of phenomenon is called radiation bystander cell phenomenon. such as, in the radiotherapy of liver cancer, liver injury is exactly a severe complication of radiation bystander cell, a lot of patient after carrying out radiotherapy all with radiation liver injury, for the radiotherapy of tumour brings great side effect [Maor, Y, al.LiverInjuryInducedbyAnticancerChemotherapyandRadiatio nTherapy.IntJHepatol.2013:815105.]. therefore, in the radiotherapy of clinical tumor, how to improve the radiosensitivity of tumour cell, reduce the key that the damage to non-irradiated healthy tissues becomes tumour radiotherapy simultaneously.
The relation of miRNA and tumour is the focus of research always, research shows that miRNA is in multiple cancer, such as [CalinGA such as lymphatic cancer, large bowel cancer, lung cancer, mammary cancer, liver cancer, colloid parent cell cancer and cancer of the stomach, CroceCM.MicroRNAsignaturesinhumancancers [J] .NatRevCancer, 2006,6 (11): 857-66.] abnormal and participate in regulation and control. recently, the regulating effect of miRNA in tumor radiation susceptibility starts to be paid close attention to. the radiosensitivity of cell reflects the efficiency that DNA repairs, simultaneously, in NHEJ and HRR path, the disappearance of core parts result in the cell phenotype [JeggoP of molecular cell biology, Cellularradiosensitivity:howmuchbetterdoweunderstandit? IntJRadiatBiol2009, 85 (12): 1061-1081.], the key gene that miRNA replys path except DNA damage after participation radiation carries out except radiosensitivity regulation and control, the externalitiess such as tumor microenvironment can also be regulated to affect radiosensitivity, reach the object making Cell irradiation sensitivity or radioresistance. as in neurocytoma, the change of S phase cell cycle checkpoint can be caused by the miR-421 unconventionality expression of N-myc mediation regulation and control ATM and strengthened cell to the susceptibility [HailiangHu of radiation, a, LiutaoDu, aGindyNagabayashi, aRobertCetal.ATMisdown-regulatedbyN-Myc regulatedmicroRNA-421 [J] .ProcNatlAcadSciUSA, 2010, 107 (4): 1,506 1511.]. during miR-101 is damaged to DNA double chain by target, important gene ATM and DNA-PKcs makes its down-regulated expression, thus increase the radiosensitivity [ChenS of tumour cell, WangH, NgWL, et.al.RadiosensitizingeffectsofectopicmiR-101onnon-small-celllungcancerdellsdependontheendogenousmiR-101level [J] .IntJRadiatOncolBiolPhys.2011:81 (5): 1524-9.]. miR-22 relies on mode with ATM and raises, and suppressing the expression of PTEN so that radiosensitivity increases [TanG, ShiY, WuZH.microRNA-22promotescellsurvivaluponUVradiationbyrep ressingPTEN [J] .BiochemBiophysResCommun, 2012, 417 (1): 546-51.]. these miRNA improving tumour cell radiosensitivity likely become the novel targets improving patient's radiotherapy effect. on the contrary, some miRNA can also Cell protection, reduce the caused DNA damage of radiation. have in the naked mouse tumor experiment of liver cancer cell as transplanted in irradiation, the downward of miR210 can reduce cell to the tolerance [YangW of radiation, WeiJ, SunT, LiuF.EffectsofknockdownofmiR-210incombinationwithionizin gradiationinhumanendothelialcells [J] .RadiatOncol.2013, 8:102.].
At present, miR214 regulate the research of various oncobiology active function many, such as osteosarcoma [XuZ, WangT..miR-214promotestheproliferationandinvasionofosteo sarcomacellsthroughdirectsuppressionofLZTS1.BiochemBioph ysResCommun.2014:449 (2): 190-5.], hepatic metastasis of colonic carcinoma [ChenDL, WangZQ, ZengZL.et.al.IdentificationofMicroRNA-214asanegativeregu latorofcolorectalcancerlivermetastasisbywayofregulationo ffibroblastgrowthfactorreceptor1expression..Hepatology.2 014.doi:10.1002/hep.27118.], nasopharyngeal carcinoma [ZhangZC, LiYY, WangHY.KnockdownofmiR-214promotesapoptosisandinhibitscel lproliferationinnasopharyngealcarcinoma [J] .PLoSOne.2014:9 (1): e86149.], cancer of the stomach [YangTS, YangXH, WangXD, et.al.MiR-214regulategastriccancercellproliferation, migrationandinvasionbytargetingPTEN.CancerCellInt.2013:1 3 (1): 68.] etc. miR495 and tumour relation also have several sections of reports. miR495 and multiple cancer, such as nonsmall-cell lung cancer [ChuH1, ChenX, WangH, et.al.MiR-495regulatesproliferationandmigrationinNSCLCby targetingMTA3 [J] .TumourBiol.2014:35 (4): 3487-94.], metastatic prostate cancer [FormosaA, MarkertEK, LenaAM, et.al.MicroRNAs, miR-154, miR-299-5p, miR-376a, miR-376c, miR-377, miR-381, miR-487b, miR-485-3p, miR-495andmiR-654-3p, mappedtothe14q32.31locus, regulateproliferation, apoptosis, migrationandinvasioninmetastaticprostatecancercells [J] .Oncogene.doi:10.1038/onc.2013.451.], glioblastoma multiforme [ChenSM, ChenHC, ChenSJ, et.al.MicroRNA-495inhibitsproliferationofglioblastomamul tiformecellsbydownregulatingcyclin-dependentkinase6 [J] .WorldJSurgOncol.2013, 11:87.], acute myelogenous lymphoma [LiZ1, CaoY, JieZ, et.al.MiR-495isatumor-suppressormicroRNAdown-regulatedin MLL-rearrangedleukemia [J] .ProcNatlAcadSciUSA.2012:109 (47): 19397-402.], cancer of the stomach [LiZ, CaoY, JieZ, et.al.miR-495andmiR-551ainhibitthemigrationandinvasionof humangastriccancercellsbydirectlyinteractingwithPRL-3 [J] .CancerLett.2012:323 (1): 41-7.]. growth regulating be correlated with. in addition, multidrug resistance is the common situations of chemotherapy of tumors, relevant with the process LAN of P glycoprotein. therefore suppress the expression of P glycoprotein may with to overcome multidrug resistance relevant. [the XuY such as Xu, OhmsSJ, LiZ, the miRNA express spectra comparing wild-type cell K562 and mdr cell K562/ADM finds et.al.ChangesintheexpressionofmiR-381andmiR-495areinvers elyassociatedwiththeexpressionoftheMDR1geneanddevelopmen tofmulti-drugresistance [J] .PLoSOne.2013:8 (11): e82062.], in the latter, miR495 expresses and significantly lowers, research show miR495 can target to adjustment multidrug resistance gene (Multidrugresistance1, MDR1) 3'-UTR of gene.MiR495 is replied and expresses, it is possible to reduce the expression of P glycoprotein, increase cell to the uptake ratio of medicine. Therefore the chemotherapy resistance that miR495 and P albumen is relevant is correlated with.
At miR214 to, in the relevant research of radiation, only one section around radiation-induced skin injury Mechanism Study. Skin injury is one of complication of local radiotherapy, Zhang etc. [ProteinandmiRNAprofilingofradiation-inducedskininjuryinr ats:theprotectiveroleofperoxiredoxin-6againstionizingrad iation] irradiate skin with 45Gy electron beam in rat model, albumen and the change of miRNA express spectra of position and contiguous normal skin tissue is irradiated in research, determine miR214 abduction delivering after irradiation, and PRDX-6 may be suppressed to express, process LAN PRDX-6 can alleviate the degree of injury of skin. By miRNA chip of expression spectrum, the lung cancer patient that is responsive and tolerance of the radiotherapy on clinical is detected, compared with finding insensitive with radiotherapy group, the responsive group of radiotherapy there are 5 miRNA (miR126, let7a, miR495, miR451, miR12sb) express and significantly raise [the refreshing .MicroRNA of Cui Shuan expresses the research with nonsmall-cell lung cancer radiation sensitivity relation]. Except above-mentioned research, there is no miR214 and miR495 at present and increase research report that is quick and that weaken in radiation bystander cell damaging action at tumor radiotherapy.
To sum up, although the mechanism that miRNA participates in the adjustment of tumor radiation susceptibility is still in the primary research stage, but the research that deepens continuously along with miRNA function, the impact that tumor radiation susceptibility is regulated by miRNA is unquestionable, and miRNA becomes tumour cell susceptibility and regulates novel targets. In this research field, urgent need to develop can effectively strengthen tumour cell self radiosensitivity, the miRNA medicine that simultaneously weakens radiation bystander cell to improve radiotherapeutic effect while reduce radiotherapy to the damaging action of non-irradiated tissue.
MiRNA is participated in the work in radiosensitivity in early stage by the present inventor, finding that miR495 and miR214 all can improve the radiosensitivity of tumour cell, miR-495 obviously alleviates radiation bystander cell to the damaging action of liver in can also testing in vivo and in vitro in addition.
Summary of the invention
Previous work of the present invention determines and strengthens radiosensitivity, weakens relevant miRNA miR214 and miR495 of radiation bystander cell, and thus the present invention provides the novelty teabag that miR214 and miR495 improves tumor radiotherapy effect, weakens normal tissue injury.
On the one hand, the present invention provides Microrna miR214 radiation sensitivity in for the preparation of tumour radiotherapy and improves the purposes of the medicine of radiotherapeutic effect.
On the other hand, the present invention provides Microrna miR495 radiation sensitivity in for the preparation of tumour radiotherapy and improves the purposes of the medicine of radiotherapeutic effect.
On the other hand, the present invention provides the pharmaceutical composition improving radiotherapeutic effect for radiation sensitivity in tumour radiotherapy, wherein comprises: the Microrna miR214 of (a) significant quantity; And (b) pharmaceutically acceptable carrier.
On the other hand, the present invention provides the pharmaceutical composition improving radiotherapeutic effect for radiation sensitivity in tumour radiotherapy, wherein comprises: the Microrna miR495 of (a) significant quantity; And (b) pharmaceutically acceptable carrier.
On the other hand, the present invention provides the pharmaceutical composition improving radiotherapeutic effect for radiation sensitivity in tumour radiotherapy, wherein comprises: the Microrna miR214 and Microrna miR495 of (a) significant quantity; And (b) pharmaceutically acceptable carrier.
On the other hand, the present invention provides Microrna miR495 for the preparation of the purposes weakening the medicine radiating bystander cell in tumour radiotherapy.
Tumour radiotherapy described in the present invention radiates bystander cell and includes but not limited to liver injury.
On the other hand, the present invention provides the pharmaceutical composition radiating bystander cell in tumour radiotherapy for weakening, and described composition comprises: the Microrna miR495 of (a) significant quantity; And (b) pharmaceutically acceptable carrier
Tumour of the present invention can be but be not limited to liver cancer, mammary cancer, cervical cancer or lung cancer.
In an embodiment of the invention, Microrna miR214 and Microrna miR495 is from people.
In an embodiment of the invention, described Microrna miR214 has the nucleotide sequence as shown in SEQIDNO.2.
In an embodiment of the invention, described Microrna miR495 has the nucleotide sequence as shown in SEQIDNO.4.
In the present invention, the form of described composition is suitable for (but being not limited to): direct naked RNA injection, liposome RNA direct injection, Jin Bao are bombarded method, breeding defect bacterium or replication defective adenoviral by rna gene rifle and carry plasmid DNA method.
The present inventor is through testing discovery in a large number: imports miR214 or miR495 in tumour cell or tumor tissues and all can effectively improve radiosensitivity, thus improves radiotherapeutic effect; In addition, miR495 simultaneously can by lowering Sp1, then the expression of eNOS is lowered, lower NO growing amount, reduce the content of TGF-��, and NO and TGF-�� are the important medium of mediation radiation bystander cell, therefore miR495 is as the ancillary drug of tumor radiotherapy, it is possible not only to improve the susceptibility of tumour self for radiotherapy, weaken the liver equivalent damage problem that radiation bystander cell brings simultaneously, thus, contriver has found that miR214 and miR495 increases, at tumor radiotherapy, the novelty teabag that quick and miR495 weakens radiation bystander cell damage, thus completes the present invention on this basis.
On the other hand, present invention also offers the present invention and provide Microrna miR495 for the preparation of the purposes in the medicine lowering eNOS expression in tumor in radiotherapy patient's body.
On the other hand, present invention also offers the present invention and provide the purposes of Microrna miR495 in the content reducing TGF-��.
On the other hand, present invention also offers the purposes of Microrna miR495 in the medicine for the preparation of the control non-irradiating cell DNA damage of tumor in radiotherapy patient.
As used herein, term " miR214 " refers to and comprises the sequence of miR214-5p/3p shown in SEQIDNO.1 or its homologous sequence, " miR495 " refers to and comprises the sequence of miR495-5p/3p shown in SEQIDNO.3 or its homologous sequence, comprise in addition through biology chemically modified in above-mentioned naturally occurring miR214 or miR495 sequence, and still there is the derivative RNA of radiation sensitivity.
In this area, the initial transcription product of known miR214 and miR495 encoding gene is after a series of processing maturations, forms ripe miR214 and miR495. MiR214 precursor and miR495 precursor only just have corresponding biologic activity at the miRNA being processed into maturation. Therefore, the technician of this area the mature sequence of Appropriate application miR214 or miR495 and the various other forms of nucleic acid substances (precursor of miR214 and miR495 as described in the present invention) that can produce or be processed into miR214 or miR495 all can obtain the effect of acquisition required for the present invention, namely radiation sensitizing effect is played, or radiation bystander cell can be weakened again radiation sensitivity, thus for improving the medicine of radiotherapeutic effect.
Expression vector and composition
The present invention is a kind of carrier also, and it contains the nucleotide sequence of miR214 or miR495. Described expression vector is usually also containing promotor, replication orgin and/or marker gene etc. Method well-known to those having ordinary skill in the art can be used for building the construction needed for thing of the present invention or expression vector. These methods include but not limited to: gene recombination technology, external synthetic technology etc. Composition should contain miR214 or miR495 of the present invention of significant quantity and pharmaceutically acceptable carrier.
Described " pharmaceutically acceptable " composition is applicable to people and/or animal and without the bad side reaction of transition (such as toxicity, stimulation and transformation reactions), namely has the material of rational benefit/risk ratio. Described " significant quantity " refers to amount that is that people and/or animal can produce function or activity and that can be accepted by people and/or animal.
Route of administration can adopt such as (but being not limited to): (1) direct naked RNA injection; (2) liposome RNA direct injection; (3) Jin Bao is bombarded method by rna gene rifle; (4) breeding defect bacterium or replication defective adenoviral carry plasmid DNA method; (5) electroporation, etc.
Accompanying drawing illustrates:
Fig. 1 verifies that miR214 and miR495 is to the colony formation result (experiment in vitro) of tumour cell radiation sensitizing effect. MiR214 and miR495 precursor polynucleotide sequence clone is utilized to build slow virus expression vector (pCDH-CMV-MCS-EF1-Puro), then infection HepG2 clone structure miR214 and miR495 stablizes overexpressing cell system respectively; Empty carrier is control group (control). The external radiation sensitization of two kinds of miRNA is verified, irradiation dose 6Gy by measuring the Clone formation of the rear miR214 group of radiation and miR495 group60Co-��. (result is shown as mean+SD (n=3); * P < 0.05 (miR214v.s.control); * P < 0.01 (miR214v.s.control);#P < 0.05 (miR495v.s.control);##P<0.01(miR495v.s.control)��
Fig. 2 is illustrated is that miR214 and miR495 is to the experimental result (experiment in vivo) of in-vivo tumour radiation sensitizing effect. In utilizing Fig. 1 to describe, miR214 and miR495 of preparation stablizes overexpressing cell system and compared with control cells (control), is inoculated in respectively in naked mouse and prepares tumour. Verify the radiation sensitizing effect of miR214 and miR495 after tumor by local radiation by measuring this gross tumor volume of three groups, radiation dose is 4Gy60Co-��, altogether partial radiation three times the 12nd day, the 15th day, the 18th day of inoculated tumour cell (time point be respectively). (result is shown as mean+SD (n=8); * P < 0.01 (miR214v.s.control); * * P < 0.001 (miR214v.s.control);##P < 0.01 (miR495v.s.control);###P<0.001(miR495v.s.control))
Fig. 3 is illustrated is the impact that radiation bystander cell signaling molecule eNOS and product NO thereof is generated by miR495 process LAN. Fig. 3 A is that (result is shown as mean+SD (n=3) for relative expression's situation of eNOS in the blood mRNA level in-site of periphery in miR495 and control group nude mouse; * P < 0.01 (miR495v.s.control)); Fig. 3 B is In vitro cell experiment, miR495 and control group measures the output of NO in 15 minutes after 6Gy radiates through flow cytometry, and result display miR495 can effectively reduce the output of bystander cell signaling molecule-NO, thus weakens radiation bystander cell.
Fig. 4 is illustrated is that miR495 process LAN is on the impact of radiation bystander cell signaling molecule TGF-�� content. Figure is radiation (radiation dose 6Gy60Co-��) afterwards miR495 group and control group get cell culture supernatant in different time points, by ELISA method measure TGF-�� relative expression's content (result is shown as mean+SD (n=3); * P < 0.05 (miR495v.s.control)).
The illustrated miR495 of the being process LAN of Fig. 5 is to the experiment in vitro weakening radiation bystander cell. MiR495 process LAN and control cell are at radiation (radiation dose 6Gy60Co-��) after, utilize media transfer the effects to normal liver cell LO2The impact of micronucleus quantity of formation, checking miR495 weakens the effect of radiation bystander cell. It is illustrated that in order to count measured micronucleus formation result under fluorescent microscope, (result is shown as mean+SD (n=3); * P < 0.05 (miR495v.s.control)). IR+: radiation; IR-: do not radiate.
The advantage of the present invention
The advantage of the present invention is mainly:
A () present invention is disclosed miR214 and miR495 at the novelty teabag strengthening tumor radio-sensitivity and weaken in the damage of radiotherapy bystander cell, for the research and development utilization of miRNA provides new thinking and approach;
B miR214 and miR495 or its precursor of () the present invention can be effective to tumour radiotherapy, for the tumour in tumor radiotherapy field increases the therapeutical agent that damage that is quick and that alleviate radiotherapy side effect normal tissue provides a kind of novelty.
Embodiment
In order to more clearly understand the technology contents of the present invention, being described with reference to the accompanying drawings especially exemplified by following instance, these examples are only not used in for illustration of the present invention and limit the scope of the invention simultaneously.
Embodiment 1 builds and identifies the HepG2 stable cell lines of process LAN miR214 and miR495
Slow virus packaging system (the SBI company purchased from the U.S.) is utilized to build process LAN miR214 and miR495 stable cell lines respectively. With MegaTran1.0 transfection reagent by packaging virus each component transfection 293T cell, preparation containing the viral supernatant of miR214 or miR495 precursor sequence (SEQIDNO:1 or SEQIDNO:2), then with virus Supernatant infection HepG2 cell thus obtain the stable cell lines being integrated into miR214 or miR495 precursor sequence. These stable cell lines can utilize host cell to be transcribed into RNA precursor, and is cut into ripe miR214 or miR495, therefore can continue process LAN miR214 or miR495. Meanwhile, empty carrier (control) is packed as cell line controls. Stable cell lines adopts TRIzol (Invitrogen company) extracting cell total rna, SYBRQPCR reagent (Exiqon company) is used to identify after RT-QPCR (Exiqon company), completing qualification on Chromo4Opticon2 real-time PCR (BioRad company), qualification result shows successfully to have prepared the HepG2 stable cell lines of process LAN miR214 and the HepG2 stable cell lines of process LAN miR495. Then carry out in body with the stable cell lines prepared and external functional experiment.
With the radiation sensitizing effect of external checking miR214 and miR495 in embodiment 2 body
2.1miR214 with the external radiation of miR495 increases quick experiment
Utilizing the HepG2 stabilized cell of process LAN miRNA214, miR495 and control group to carry out colony formation, three kinds of cells are inoculated in six orifice plates with equal amts respectively, often the multiple hole of group, after cell pastes wall completely,60Co-gamma-rays single fraction irradiation cell (dosage 6Gy, dose rate 130cGy/min), then puts back to incubator by plate and hatches, every two days replaced medium. After cell grows population of cells in ware, take out flat board and it is fixed and dyes, counting of finally taking pictures. Shown by colony formation result: process LAN miR214 group is significantly lower than control group (see Fig. 1) with miR495 group Clone formation number under identical radiation dose. Experimental result shows the external radiosensitivity that all can improve tumour cell of miR214 and miR495.
2.2miR214 and radiation sensitivity experiment in the body of miR495.
Utilize the HepG2 stabilized cell of process LAN miRNA214, miR495 and control group in the big leg outer side in the both sides subcutaneous vaccination injection 10 respectively of naked mouse in 4 week age7Cell, partial radiation condition (60Co-��-ray, dosage 4Gy, dose rate 85cGy/min), radiated time is latter 12nd day, 15 days and 18 days of inoculation, totally 3 times. Different time points measures three groups of gross tumor volume changing conditions. Result shows: when radiating, the gross tumor volume of process LAN miR214, miR495 group is significantly lower than control group tumour (see Fig. 2). Experiment shows all can significantly improve in miRNA495 and miRNA214 body radiosensitivity.
Embodiment 3miR495 weakens the experiment in vivo of radiation bystander cell
At the end of in miR495 body, radiation sensitivity is tested, miR495 treated animal action enlivens, in good condition, and control animals is skinny, movable slow, is not in good state. Two groups of mouse are dissected, observe internal organs metamorphosis to dye in conjunction with HE, result shows: multiple necrosis region occurs in control group group liver naked-eye observation, and this phenomenon is supported in HE dyeing, and the liver of miR495 group dyes all close to normal liver from macroscopic appearance to HE. Experiment in vivo shows, process LAN miR495 can effectively weaken radiation bystander cell to the damage of liver.
Embodiment 4miR495 weakens the molecular mechanism of radiation bystander cell
Process LAN miR495, by lowering Sp1 transcription factor, lowers eNOS gene indirectly, thus reduces the signaling molecule NO of radiation bystander cell and the growing amount of TGF-��, finally weakens radiation bystander cell.
4.1miR495 on the impact of eNOS and product NO thereof
4.1.1 after measuring radiation by RT-QPCR, miR495 is on the impact of the mRNA expression level of periphery blood eNOS
The peripheral blood lymphocyte of control group and miR495 group mouse in Example 3, TRIReagentBD reagent (MolecularResearchCenter company) extracts its total serum IgE, then reverse transcription (Fermentas company) obtains cDNA and carries out the mRNA expression level that SYBRGreenI real-time quantitative PCR (Fermentas company) measures its eNOS. ENOS quantification PCR primer is: 5 ' AGGCAATCTTCGTTCA3 ' (eNOS upstream, SEQID:5) and 5 ' CCGCATAGCGTATCA3 ' (eNOS downstream, SEQID:6). Reference gene is GAPDH, and quantification PCR primer is: 5 ' AAGGCTGTGGGCAAGG3 ' (GAPDH upstream, SEQID:7) and 5 ' GGTGGAAGAGTGGGAGTT3 ' (GAPDH downstream, SEQID:8). The results are shown in Figure 3A. As can be seen from Figure 3A, miR495 group eNOS expression level is significantly lower than control group.
4.1.2 after measuring the checking radiation of NO growing amount by flow cytometry, process LAN miR495 is on the impact of NO expression level
The HepG2 stabilized cell of miR495 process LAN group and control group loads DAF-FM probe (purchased from green skies biotechnology research institute, specifically operate according to described in specification sheets) under tying up to equal amts, 6Gy's60Co-�� irradiates (dose rate is 130cGy/min) and carries out Flow cytometry NO growing amount immediately in latter 15 minutes, the results are shown in Figure 3B. From Fig. 3 B it will be seen that through radiation after miR495 group NO content relatively control group significantly reduce.
Two experimental results of Fig. 3 A and Fig. 3 B all show that process LAN miR495 can significantly reduce the content of eNOS and product NO thereof, and the latter reduces finally weaken radiation bystander cell as one of signaling molecule radiating bystander cell, its growing amount.
4.2miR495 is on the content impact of radiation bystander cell signaling molecule TGF-��.
The HepG2 cell of the miR495 group of equal amts and control group is seeded to 6 orifice plates respectively, after pasting wall completely, carries out 6Gy's60Co-�� irradiates (dose rate is 130cGy/min), and pre-irradiation changes fresh culture, has irradiated 37 DEG C, 5%CO2Continuing to cultivate, the cell conditioned medium cultivating 0h, 24h, 48h after getting radiation respectively carries out enzyme linked immunosorbent assay (ELISA) and measures the TGF-�� (content of (SinoBiological company). The results are shown in Figure 4, as can be seen from Figure 4, the output of TGF-�� prolongation in time after irradiation and rise, wherein relatively control group level is all low for miR495 group, especially at 48h, there is significant difference, illustrate that process LAN miR495 can reduce the output of TGF-��, and TGF-�� radiates the long-acting signaling molecule of bystander cell as one, its content reduces then bystander cell signal weakening, then weakens radiation bystander cell.
Embodiment 5miR495 weakens the experiment in vitro of radiation bystander cell liver injury
For research different treatment group (miR495 group and control group) irradiates rear cell conditioned medium to the radiation bystander cell damage mechanisms of normal liver cell, by normal liver cell LO2Being inoculated in 6 orifice plates, miR495 group and the cellular control unit supernatant of cultivating 24h after getting radiation after pasting wall completely hatch LO respectively2Cell, processes LO with the cytochalasin B (Sigma company) of 1 �� g/mL simultaneously2, hatch supernatant discarded after 24h, with PBS 1 time, again to its fixation in situ cell, finally with 100 �� g/mL acridine orange (Sigma company) dyeing, use laser co-focusing technology to observe micronucleus microtexture, calculate its micronuclcus formation at fluorescence microscopy Microscopic observation simultaneously. The results are shown in Figure 5, as can be seen from Figure 5, after radiation, the micronuclcus formation of process LAN miR495 group is significantly lower than control group, has significant difference. This media transfer and micronucleus test illustrate that miR495 can protect the DNA damage effect of non-irradiating cell.
Claims (10)
1.miR214 or miR495 radiation sensitivity in for the preparation of tumour radiotherapy improves the purposes of the medicine of radiotherapeutic effect.
2.miR495 is for the preparation of the purposes weakening the medicine radiating bystander cell in tumour radiotherapy.
3. purposes according to claim 2, wherein said radiation bystander cell is liver injury.
4. purposes described in the arbitrary item of claims 1 to 3, wherein said miR214 or miR495 is from people.
5. purposes described in the arbitrary item of Claims 1-4, wherein said tumour is selected from liver cancer, mammary cancer, cervical cancer or lung cancer.
6. application described in the arbitrary item of claim 1 to 5, wherein said miR214 has the nucleotide sequence as shown in SEQIDNO:1.
7. application described in the arbitrary item of claim 1 to 6, wherein said miR495 has the nucleotide sequence as shown in SEQIDNO:2.
8. improve a pharmaceutical composition for radiotherapeutic effect for radiation sensitivity in tumour radiotherapy, described composition comprises: miR214 and/or miR495 of (a) significant quantity; And (b) pharmaceutically acceptable carrier.
9. the pharmaceutical composition radiating bystander cell in tumour radiotherapy for weakening, described composition comprises: the miR495 of (a) significant quantity; And (b) pharmaceutically acceptable carrier.
10. composition as claimed in claim 7 or 8, it is characterized in that, the form of described composition be suitable for direct naked RNA injection, liposome RNA injection, Jin Bao by rna gene rifle bombard method, breeding defect bacterium or replication defective adenoviral carry plasmid DNA method.
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| WO2010056737A2 (en) * | 2008-11-11 | 2010-05-20 | Mirna Therapeutics, Inc. | Methods and compositions involving mirnas in cancer stem cells |
| CN103691066A (en) * | 2013-12-27 | 2014-04-02 | 成都军区昆明总医院 | Gold labeling implant and manufacture method thereof |
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