CN104561004A - Small molecular RNA of interference TGM2 gene for inhibiting proliferation of glioma as well as preparation method and application of small molecular RNA - Google Patents
Small molecular RNA of interference TGM2 gene for inhibiting proliferation of glioma as well as preparation method and application of small molecular RNA Download PDFInfo
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- CN104561004A CN104561004A CN201510023923.3A CN201510023923A CN104561004A CN 104561004 A CN104561004 A CN 104561004A CN 201510023923 A CN201510023923 A CN 201510023923A CN 104561004 A CN104561004 A CN 104561004A
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Abstract
The invention discloses a small molecular RNA of an interference TGM2 gene for inhibiting proliferation of glioma as well as a preparation method and an application of the small molecular RNA. The small molecular RNA of the interference TGM2 gene is base sequences which are shown in SEQ ID NO.1 and SEQ ID NO.2. The invention designs the small molecular RNA of the interference TGM2 gene, having promoting function on proliferation of glioma, and the inhibitory effect of the small molecular RNA on proliferation of glioma is researched by using a glioma cell (U373 cell) model, so that a novel molecular drug is provided for gene therapy of glioma.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of small molecules interference RNA (i.e. shRNA), particularly relating to a kind of microRNA of the interference TGM2 gene for suppressing glioma to be bred.In addition, the invention still further relates to the preparation method and application of the microRNA of this interference TGM2 gene.
Background technology
TGM2 albumen is a kind of protein-crosslinking enzyme of wide expression, on cell proliferation, cell adhesion and apoptosis serve important regulating and controlling effect, high expression level in multiple cancer cells, and participate in drug resistance and metastases, and closely related with multiple nerve degenerative diseases.
Glioma betides neuroectodermal tumour, therefore also known as neuroectodermal tumors or neurotic triad.Tumor originates in neural mesenchymal cell, i.e. neuroglia, ependyma, choroid epithelium and neural parenchyma, i.e. neurone.Most of tumor originates in dissimilar neuroglia, but according to tissue generation source and biological property similar, to betiding neuroectodermal various check neoplastic disease, be generally all called neurospongioma.
The sorting technique of glioma is a lot, and what clinical workers often adopted is fairly simple Kernohan classification of classifying.In various glioma, maximum with astrocytoma, secondly be glioblastoma multiforme, be followed successively by thereafter medulloblastoma, ependymoma, oligodendroglioma, pinealoma, Mixed Gliomas, papilloma of choroid plexus, unfiled glioma and neuronal tumour.The predilection site of various glioma is different, and as astrocytoma, adult is more common in hemicerebrum, and children are then multiple at cerebellum; Glioblastoma multiforme almost all betides hemicerebrum; Medulloblastoma betides vermis of cerebellum; Ependymoma is more common in the 4th ventricles of the brain; Oligodendroglioma betides hemicerebrum mostly.
Glioma is more common with the male sex, and especially at glioblastoma multiforme, medulloblastoma, the male sex is obviously more than women.Various glioblastoma multiforme is more common in the middle age, and ependymoma is more common in children and youth, and medulloblastoma nearly all occurs in children.Also there are certain relation at the position of glioma and age, and as cerebral astrocytoma and glioblastoma multiforme are more common in adult, little cerebral glioma (astrocytoma, medulloblastoma, ependymoma) is more common in children.
Glioma is slowly fallen ill mostly, and certainly occur that symptom to consultation time is generally several weeks to the several months, minority can reach the several years.High shorter with PFT medical history of grade malignancy, tumour medical history that is more optimum or that be positioned at dead zone is longer.If tumour has hemorrhage or capsule change, symptom can increase the weight of suddenly, even has the pathogenic process of similar cerebro-vascular diseases.The clinical symptom of glioma can divide two aspects, and one is symptoms of intracranial hypertension, as headache, vomiting, visual deterioration, diplopia, mental symptom etc.; The focal symptom that another is oncothlipsis, infiltration, destruction cerebral tissue produce, can show as irritation in early days as localized epilepsy, and the later stage shows as neurological deficit symptom as paralysis.
The diagnosis of glioma, analyze according to its biological property, age, sex, predilection site and clinical course, in medical history and sign basis, adopt the auxiliary examinations such as electro physiology, ultrasonic wave, radionuclide, radiology and nucleus magnetic resonance, correct localization is almost 100%, and etiologic diagnosis accuracy can more than 90%.
RNA interference (RNA interference, RNAi) refers in the cell by double chain RNA mediate, with the mRNA of its sequence homology, selective degradation occurs, thus causes the phenomenon of silenced gene expression, and this is a kind of gene silencing mechanism of post-transcriptional level.The ultimate principle of RNA interference (RNAi) is the expression activity utilizing the double-stranded RNA (dsRNA) with homology to block rapidly sequence-specific target gene, is the instrument that in laboratory, a kind of effective blocking gene is expressed.
At present, there is not yet the report of the identical sequence small molecules interference RNA about the TGM2 gene for suppressing glioma to be bred.
Summary of the invention
One of the technical problem to be solved in the present invention is to provide a kind of microRNA of the interference TGM2 gene for suppressing glioma to be bred.
Two of the technical problem to be solved in the present invention is to provide the preparation method of the microRNA of this interference TGM2 gene.
Three of the technical problem to be solved in the present invention is to provide the application of the microRNA of this interference TGM2 gene.
For solving the problems of the technologies described above, the present invention adopts following technical scheme:
In one aspect of the invention, provide a kind of microRNA of the interference TGM2 gene for suppressing glioma to be bred, the sequence of its correspondence is:
Sense (positive-sense strand): 5 '-CTAGACCACTTCATTTTGCTCTTCCTCGAGGAAGAGCAAAATGAAGTGGG-3 ', as shown in SEQ ID NO.1;
Antisense (antisense strand): 5 '-GATCCCCACTTCATTTTGCTCTTCCTCGAGGAAGAGCAAAATGAAGTGGT-3 ', as shown in SEQ ID NO.2.
In another aspect of this invention, provide a kind of method preparing the microRNA of the above-mentioned interference TGM2 gene for suppressing glioma to be bred, the concrete steps of the method are:
Step one, specific requirement according to PMT98 carrier design, design corresponds to TGM2 gene 499-517 position, that is: CCACTTCATTTTGCTCTTC shRNA oligonucleotide sequence and synthesize, the oligonucleotide sequence sequence of described shRNA is the base sequence as shown in SEQ ID NO.1 and SEQ ID NO.2;
The requirement of step 2, foundation PMT98 carrier design, being connected the shRNA sequence of design with PMT98 plasmid, obtaining a kind of microRNA of the interference TGM2 gene for suppressing glioma.
In another aspect of this invention, a kind of microRNA of above-mentioned interference TGM2 gene is provided to suppress the application in the medicine of glioma propagation in preparation.
The present invention's design is to the RNAi of the TGM2 gene of glioma propagation association, and apply glioma cell model (U373 cell model) and study its restraining effect to glioma proliferation resistant, for RNAi provides a kind of novel drugs molecule at the treatment use of glioma.Through experimental verification, the tumor growth in transfection TGM2RNA interference group is suppressed, and this experimental result shows, small molecules interference RNA of the present invention can lower the TGM2 genetic expression in glioma, obviously can suppress the growth of glioma.
Accompanying drawing explanation
Fig. 1 is the oligonucleotide sequence of shRNA in the embodiment of the present invention 1 and the connection diagram of PMT98 plasmid.
Fig. 2 is that in the embodiment of the present invention 1, TGM2RNAi is to the interference effect schematic diagram of TGM2 in brain glioblastoma cell, and wherein, blank represents ghost group, and empty vector represents transfection empty carrier group; TGM2RNAi represents transfection TGM2RNA interference group.
Fig. 3 be in the embodiment of the present invention 1 TGM2RNAi brain glioblastoma cell is bred affect schematic diagram, wherein, blank represents ghost group, and empty vector represents transfection empty carrier group; TGM2RNAi represents transfection TGM2RNA interference group.
Fig. 4 is that in the embodiment of the present invention 2, three microRNAs are to the interference effect contrast schematic diagram of TGM2 in brain glioblastoma cell, and wherein, blank represents ghost group, and empty vector represents transfection empty carrier group.
Embodiment
Following examples are only not used in for illustration of the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in embodiment, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.
Embodiment 1
One, the cultivation of glioma cell:
Glioma cell U373 clone (buying from school of life and health sciences cellular resources center, Chinese Academy of Sciences Shanghai) is incubated at 37 DEG C, 5%CO
2constant incubator.The cell trysinization of logarithmic phase takes off wall, adds appropriate nutrient solution afterwards and repeatedly blows and beats to mix cell; The cell suspension obtained is moved in 15ml centrifuge tube, the centrifugal 3min of 1500rpm; Remove supernatant; With 5ml aseptic 1 × PBS, cell is hanged, piping and druming mixing; Add about 1ml cell suspension in the new culturing bottle that 5ml fresh culture is housed, put into 37 DEG C, 5%CO
2cultivate in incubator, within about about 2 days, go down to posterity 1 time (mainly checking that cell is with or without confluent culture bottle).
Two, the design of TGM2-RNAi plasmid and transfection:
1.TGM2-RNAi the design of plasmid:
According to the specific requirement of PMT98 carrier design, design correspond to TGM2 gene 499-517 position, that is: CCACTTCATTTTGCTCTTC shRNA oligonucleotide sequence and synthesize, send Shanghai Jierui Biology Engineering Co., Ltd to synthesize.
The sequence that TGM2-RNAi is corresponding is:
Sense (positive-sense strand): 5 '-CTAGACCACTTCATTTTGCTCTTCCTCGAGGAAGAGCAAAATGAAGTGGG-3 ', as shown in SEQ ID NO.1;
Antisense (antisense strand): 5 '-GATCCCCACTTCATTTTGCTCTTCCTCGAGGAAGAGCAAAATGAAGTGGT-3 ', as shown in SEQ ID NO.2.
According to the specific requirement of PMT98 carrier design, be connected with PMT98 plasmid by the interference fragment of the TGM2-RNAi of design, see Fig. 1, then glioma cell is entered in transfection.Fig. 1 is the diagram of PMT98 plasmid construction principle, and it uses based on the CMV polymerase Il promoters of RNA and the loop-stem structure of optimization, thus accurately can form small molecules interference RNA (shRNA) in target cell.PMT98 is a carrier cut in advance, by the selection markers that carrier carries, carries out the cotransfection of mammalian cell, sets up the stable cell lines that shRNA expresses.PMT98 carrier (element orders: pLVX-PGK-PURO-CMV-EGFP-mir30arm-polyA) transforms on pLVX-PURO carrier (element orders: the pLVX-CMV-MSC-PGK-PURO-polyA) basis of clontech company.PMT98 carrier complete sequence is as shown in SEQ ID NO.7.
2.TGM2-RNAi the transfection of plasmid:
Day before transfection, plants upper 2 × 10 in the 60mm culture dish of the DMEM growth medium (DMEM growth medium a kind ofly to develop on the basis of MEM substratum, the cell culture medium containing each seed amino acid and glucose) containing 5ml antibiotic-free
6individual glioma cell (glioma cell by above-mentioned one, cultivate obtain).Within after bed board second day, prepare transfection: first use the substratum (DMEM) of 500 μ l serum-frees to dissolve the TGM2-RNAi expression vector of 8.0 μ g, dissolve cationic-liposome Lipofectamine with 500 μ l substratum (DMEM or RPMI1640)
tM2000 (20 μ l), mix, incubated at room 5 minutes lightly.After hatching, for impelling shRNA expression vector and Lipofectamine
tM2000 fully combine, and they are mixed gently, and incubated at room 20 minutes, to form DNA-Lipofectamine
tMthe mixture of 2000.By DNA-Lipofectamine
tM2000 mixtures join 60mm culture dish, afterwards will containing DNA-Lipofectamine
tMthe Tissue Culture Dish of 2000 mixtures puts into 37 DEG C, 5%CO
2cultivate 6 hours in incubator, change normal cell nutrient solution.
Three, experiment grouping and Testing index
Blank group (ghost group): glioma cell, nutrient solution is glioma cell nutrient solution;
Empty vector group (transfection empty carrier group); The glioma cell of empty vector is crossed in transfection, and nutrient solution is glioma cell nutrient solution;
TGM2RNAi group (transfection TGM2RNA interference group): the glioma cell of TGM2-RNAi is crossed in transfection, and nutrient solution is glioma cell nutrient solution.
Fig. 2 represents the interference effect of TGM2RNAi to TGM2 in brain glioblastoma cell, and Fig. 2 is that in U373 cell, TGM2 interference effect QPCR detects.In Fig. 2, TGM2RNAi group is compared with empty vector group, and TGM2 gene is obviously suppressed (* * p<0.01).
Fig. 3 represents the impact that TGM2-RNAi breeds brain glioblastoma cell.As shown in Figure 3, TGM2RNAi group is compared with emptyvector group, and the cell proliferation of transfection TGM2RNAi interference group (TGM2RNAi group) is significantly lower than the cell proliferation rate (* * p<0.01) of transfection empty carrier group (empty vector group).
Four, experimental result:
Above-mentioned experiment shows, the TGM2 gene in transfection TGM2RNAi interference group is significantly struck to be subtracted, and tumor growth is also subject to obvious suppression.This experimental result shows, small molecules interference RNA of the present invention can lower the TGM2 genetic expression in glioma, obviously can suppress the growth of glioma.
Embodiment 2 contrast experiment
Select the microRNA that 3 score values are higher, carrier construction also disturbs sieve target, and experimental procedure, with embodiment 1, is below relevant comparative's experimental data.
TGM2-si499:CCACTTCATTTTGCTCTTC (corresponding to TGM2 gene 499-517 position, the ordered sequence namely in the embodiment of the present invention 1); The sequence that TGM2-si499 is corresponding is:
Positive-sense strand: 5 '-CTAGACCACTTCATTTTGCTCTTCCTCGAGGAAGAGCAAAATGAAGTGGG-3 ', as shown in SEQ IDNO.1;
Antisense strand: 5 '-GATCCCCACTTCATTTTGCTCTTCCTCGAGGAAGAGCAAAATGAAGTGGT-3 ', as shown in SEQ IDNO.2.
TGM2-si666:CCTAGACATCTGCCTGATC (corresponding to TGM2 gene 666-684 position); The sequence that TGM2-si666 is corresponding is:
Positive-sense strand: 5 '-CTAGACCTAGACATCTGCCTGATCCTCGAGGATCAGGCAGATGTCTAGGG-3 ', as shown in SEQ IDNO.3;
Antisense strand: 5 '-GATCCCCTAGACATCTGCCTGATCCTCGAGGATCAGGCAGATGTCTAGGT-3 ', as shown in SEQ IDNO.4.
TGM2-si 1549:GAACATGGGCAGTGACTTT (corresponding to TGM2 gene 1549-1567 position).The sequence of TGM2-si 1549 correspondence is:
Positive-sense strand: 5 '-CTAGA GAACATGGGCAGTGACTTTCTCGAGAAAGTCACTGCCCATGTTCG-3 ', as shown in SEQ IDNO.5;
Antisense strand: 5 '-GATCC GAACATGGGCAGTGACTTTCTCGAGAAAGTCACTGCCCATGTTCT-3 ', as shown in SEQ IDNO.6.
As shown in Figure 4, as can be seen from Figure 4, TGM2-si499 (i.e. the microRNA of the embodiment of the present invention 1) is compared with TGM2-si666, TGM2-si 1549, and in U373 cell, TGM2 interference effect is the most remarkable for contrast and experiment.
Claims (3)
1., for a microRNA for the interference TGM2 gene that suppresses glioma to be bred, the sequence of its correspondence is:
Sense (positive-sense strand): 5 '-CTAGACCACTTCATTTTGCTCTTCCTCGAGGAAGAGCAAAATGAAGTGGG-3 ', as shown in SEQ ID NO.1;
Antisense (antisense strand): 5 '-GATCCCCACTTCATTTTGCTCTTCCTCGAGGAAGAGCAAAATGAAGTGGT-3 ', as shown in SEQ ID NO.2.
2. a preparation method for the microRNA of interference TGM2 gene according to claim 1, it is characterized in that, the concrete steps of the method are:
Step one, specific requirement according to PMT98 carrier design, design corresponds to TGM2 gene 499-517 position, that is: CCACTTCATTTTGCTCTTC shRNA oligonucleotide sequence and synthesize, described shRNA oligonucleotide sequence sequence is the base sequence as shown in SEQ ID NO.1 and SEQ ID NO.2;
The requirement of step 2, foundation PMT98 carrier design, being connected the shRNA sequence of design with PMT98 plasmid, obtaining the rna interference vector for disturbing TGM2 genetic expression.
3. the microRNA of interference TGM2 gene according to claim 1 suppresses the application in the medicine of glioma propagation in preparation.
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Citations (3)
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| CN102433336A (en) * | 2010-09-29 | 2012-05-02 | 上海生博生物医药科技有限公司 | Small interfering RNA for inhibiting glioma proliferation and promoting glioma apoptosis and preparation method and application thereof |
| CN102453715A (en) * | 2010-10-21 | 2012-05-16 | 上海生博生物医药科技有限公司 | Small interfering RNA (ribonucleic acid) for efficiently resisting glioma angiogenesis, preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102433336A (en) * | 2010-09-29 | 2012-05-02 | 上海生博生物医药科技有限公司 | Small interfering RNA for inhibiting glioma proliferation and promoting glioma apoptosis and preparation method and application thereof |
| CN102453715A (en) * | 2010-10-21 | 2012-05-16 | 上海生博生物医药科技有限公司 | Small interfering RNA (ribonucleic acid) for efficiently resisting glioma angiogenesis, preparation method and application thereof |
| CN102181445A (en) * | 2011-03-21 | 2011-09-14 | 上海生博生物医药科技有限公司 | Interference small molecule RNA of targeted LIN28 gene and glioma proliferation resistant application thereof |
Non-Patent Citations (1)
| Title |
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| JUN FU ET AL.: "TGM2 inhibition attenuates ID1 expression in CD44-high glioma-initiating cells", 《NEURO-ONCOLOGY》 * |
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