CN104561004B - A kind of microRNA and its preparation method and application of the interference TGM2 gene for inhibiting glioma to be proliferated - Google Patents

Abstract

MicroRNA of interference TGM2 gene that the invention discloses a kind of for inhibiting glioma to be proliferated and preparation method thereof and its application.The microRNA of interference TGM2 gene is base sequence shown in SEQ ID NO.1 and SEQ ID NO.2.Present invention design has the microRNA of the interference TGM2 gene of facilitation to glioma proliferation, its inhibiting effect to glioma proliferation is studied using glioma cell (U373 cell) model, provides a kind of recruit's drug for the gene therapy of glioma.

Description

It is a kind of for inhibit glioma be proliferated interference TGM2 gene microRNA and its Preparation method and application

Technical field

The present invention relates to field of biotechnology, and in particular to a kind of small molecules interference RNA (i.e. shRNA) more particularly to one The microRNA of interference TGM2 gene of the kind for inhibiting glioma to be proliferated.Moreover, it relates to interference TGM2 gene MicroRNA preparation method and application.

Background technique

TGM2 albumen is a kind of protein-crosslinking enzyme of wide expression, and cell proliferation, cell adherence and Apoptosis play Important regulating and controlling effect, the high expression in a variety of cancer cells, and drug resistance and metastases are participated in, and move back with a variety of nerves Row disease is closely related.

Glioma is to betide the tumour of neuroderm, therefore also known as neuroectodermal tumors or neurotic triad.It is swollen Tumor originates from neural interstitial cell, i.e. neuroglia, endyma, choroid epithelium and neural parenchyma, i.e. neuron.Mostly Number tumours originate from different types of neuroglia, but similar according to tissue generation source and biological property, to betiding The various check neoplastic diseases of neuroderm, generally referred to as glioma.

There are many classification method of glioma, and clinical workers is often classified using the fairly simple Kernohan that classifies Method.It is most with astrocytoma in various glioma, secondly it is glioblastoma, is followed successively by medulloblastoma, room pipe thereafter Film tumor, oligodendroglioma, pinealoma, Mixed Gliomas, papilloma choroideum, unfiled glioma and neuronal are swollen Tumor.The predilection site of various glioma is different, and if astrocytoma adult is more common in cerebral hemisphere, children are then multiple in cerebellum； Glioblastoma almost betides cerebral hemisphere；Medulloblastoma betides vermis of cerebellum；Ependymoma is more common in the 4th brain Room；Oligodendroglioma mostly occurs in cerebral hemisphere.

Glioma is more common with male, and especially in glioblastoma multiforme, medulloblastoma, male is significantly more than female Property.Various glioblastoma is more common in the middle age, and ependymoma is more common in children and youth, and medulloblastoma nearly all occurs Children.Also there are certain relationship at the position of glioma and age, if cerebral astrocytoma and glioblastoma are more common in adult, Small glioma (astrocytoma, medulloblastoma, ependymoma) is more common in children.

Glioma is slowly fallen ill mostly, is occurred symptom to consultation time certainly and is generally several weeks to the several months, a small number of up to the several years. Grade malignancy it is high and posterior fossa tumor medical history it is shorter, more benign or tumour medical history positioned at dead zone is longer.If tumour has out Blood or capsule become, and symptom can aggravate suddenly, or even have the pathogenic process of similar cerebrovascular disease.The clinical symptoms of glioma can be divided to two sides Face, first is that symptoms of intracranial hypertension, such as headache, vomiting, hypopsia, diplopia, mental symptom；Another is oncothlipsis, leaching Profit destroys focal symptom caused by brain tissue, can behave as irritation such as localized epilepsy in early days, the later period shows as nerve Afunction symptom is as paralysed.

The diagnosis of glioma is analyzed according to its biological property, age, gender, predilection site and clinical process, On the basis of medical history and sign, using the auxiliary examinations such as electro physiology, ultrasonic wave, radionuclide, radiology and nuclear magnetic resonance, positioning Accuracy is almost 100%, and etiologic diagnosis accuracy can be 90% or more.

RNA interference (RNA interference, RNAi) refers to by intracellular and its sequence homology of double chain RNA mediate Selective degradation occurs for mRNA, and the phenomenon that so as to cause silenced gene expression, this is a kind of gene silencing machine of post-transcriptional level System.The basic principle of RNA interference (RNAi) is to block sequence specific rapidly using the double-stranded RNA (dsRNA) with homology The expression activity of target gene is a kind of tool for being effectively blocked gene expression in laboratory.

Currently, there is not yet in relation to the identical sequence small molecules interference RNA for inhibiting the TGM2 gene of glioma proliferation Report.

Summary of the invention

One of the technical problem to be solved in the present invention is to provide a kind of interference TGM2 gene for inhibiting glioma to be proliferated MicroRNA.

The second technical problem to be solved by the present invention is to provide the preparation method of the microRNA of interference TGM2 gene.

The third technical problem to be solved by the present invention is to provide the application of the microRNA of interference TGM2 gene.

In order to solve the above technical problems, the present invention adopts the following technical scheme:

In one aspect of the invention, a kind of microRNA of interference TGM2 gene for inhibiting glioma to be proliferated is provided, Its corresponding sequence are as follows:

Sense (positive-sense strand): 5 '-CTAGACCACTTCATTTTGCTCTTCCTCGAGGAAGAGCAAAATGAAGTGGG- 3 ', as shown in SEQ ID NO.1；

Antisense (antisense strand): 5 '-GATCCCCACTTCATTTTGCTCTTCCTCGAGGAAGAGCAAAATGAAGT GGT-3 ', as shown in SEQ ID NO.2.

In another aspect of this invention, it provides and a kind of prepares the above-mentioned interference TGM2 gene for inhibiting glioma to be proliferated The method of microRNA, the specific steps of this method are as follows:

Step 1: design corresponds to TGM2 gene 499-517 according to the specific requirement that PMT98 carrier designs, it may be assumed that The shRNA oligonucleotide sequence of CCACTTCATTTTGCTCTTC is simultaneously synthesized, the oligonucleotide sequence sequence of the shRNA It is classified as: the base sequence as shown in SEQ ID NO.1 and SEQ ID NO.2；

Step 2: the requirement according to the design of PMT98 carrier, the shRNA sequence of design and PMT98 plasmid are attached, Obtain a kind of microRNA for inhibiting the interference TGM2 gene of glioma.

In another aspect of this invention, the microRNA for providing a kind of above-mentioned interference TGM2 gene inhibits colloid in preparation Application in the drug of tumor proliferation.

The present invention designs the RNAi that associated TGM2 gene is proliferated to glioma, and applies glioma cell model (U373 Cell model) its inhibiting effect to glioma proliferation resistant is studied, one kind is provided newly in the treatment use of glioma for RNAi Drug molecule.Experiments verify that transfection TGM2RNA interference group in tumour growth be suppressed, should the experimental results showed that, this hair Bright small molecules interference RNA can lower the TGM2 gene expression in glioma, can obviously inhibit the growth of glioma.

Detailed description of the invention

Fig. 1 is the connection schematic diagram of the oligonucleotide sequence of shRNA and PMT98 plasmid in the embodiment of the present invention 1.

Fig. 2 be in the embodiment of the present invention 1 TGM2RNAi to the interference effect schematic diagram of TGM2 in brain glioblastoma cell, In, blank indicates ghost group, and empty vector indicates transfection empty carrier group；TGM2RNAi indicates transfection TGM2RNA interference Group.

Fig. 3 is the influence schematic diagram that TGM2RNAi is proliferated brain glioblastoma cell in the embodiment of the present invention 1, wherein blank Indicate ghost group, empty vector indicates transfection empty carrier group；TGM2RNAi indicates transfection TGM2RNA interference group.

Fig. 4 is that three microRNAs show the interference effect comparison of TGM2 in brain glioblastoma cell in the embodiment of the present invention 2 It is intended to, wherein blank indicates ghost group, and empty vector indicates transfection empty carrier group.

Specific embodiment

Following embodiment is only illustrative of the invention and is not intended to limit the scope of the invention.It is not specified in embodiment specific The experimental method of condition, usually according to normal condition, for example (,) Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or built according to manufacturer The condition of view.

Embodiment 1

One, the culture of glioma cell:

Glioma cell U373 cell line (buys from Chinese Academy of Sciences's Shanghai school of life and health sciences cell resource center) culture In 37 DEG C, 5%CO₂Constant incubator.The cell of logarithmic growth phase digests de- wall with pancreatin, and appropriate culture solution and anti-is added later Multiple piping and druming is to mix cell；Obtained cell suspension is moved into 15ml centrifuge tube, 1500rpm is centrifuged 3min；Remove supernatant；With Sterile 1 × the PBS of 5ml hangs cell, and piping and druming mixes；Add about 1ml cell suspension in the new culture bottle that 5ml fresh culture is housed In, 37 DEG C are put into, 5%CO₂It is cultivated in incubator, 1 time (mainly checking cell, whether there is or not confluent cultures bottles) of passage in about 2 days or so.

Two, the design and transfection of TGM2-RNAi plasmid:

1.TGM2-RNAi the design of plasmid:

According to the specific requirement that PMT98 carrier designs, design corresponds to TGM2 gene 499-517, it may be assumed that The shRNA oligonucleotide sequence of CCACTTCATTTTGCTCTTC is simultaneously synthesized, send Shanghai Jierui Biology Engineering Co., Ltd into Row synthesis.

The corresponding sequence of TGM2-RNAi are as follows:

Sense (positive-sense strand): 5 '-CTAGACCACTTCATTTTGCTCTTCCTCGAGGAAGAGCAAAATGAAGTGGG- 3 ', as shown in SEQ ID NO.1；

Antisense (antisense strand): 5 '-GATCCCCACTTCATTTTGCTCTTCCTCGAGGAAGAGCAAAATGAAGT GGT-3 ', as shown in SEQ ID NO.2.

According to the specific requirement that PMT98 carrier designs, the interference fragment of the TGM2-RNAi of design and PMT98 plasmid are connected It connects, referring to Fig. 1, then transfects into glioma cell.Fig. 1 is the diagram of PMT98 plasmid construction principle, it is using based on RNA's The loop-stem structure of CMV polymerase Il promoters and optimization, so that small molecules interference RNA can be formed accurately in target cell (shRNA).PMT98 is that the carrier cut in advance by the selection markers carried on carrier carries out being total to for mammalian cell The stable cell lines of shRNA expression are established in transfection.PMT98 carrier (element orders: pLVX-PGK-PURO-CMV-EGFP- Mir30arm-polyA) it is pLVX-PURO carrier (element orders: pLVX-CMV-MSC-PGK- in clontech company PURO-polyA it is transformed on the basis of).PMT98 carrier complete sequence is as shown in SEQ ID NO.7.

2.TGM2-RNAi the transfection of plasmid:

The day before transfection, in the DMEM growth medium containing 5ml antibiotic-free, (DMEM growth medium is that one kind exists Developed on the basis of MEM culture medium, the cell culture medium containing various amino acid and glucose) 60mm culture dish in plant upper 2 ×10⁶A glioma cell (glioma cell is obtained by above-mentioned one, culture).Preparation in second day transfects after bed board: first with 500 The culture medium (DMEM) of μ l serum-free dissolves the TGM2-RNAi expression vector of 8.0 μ g, with 500 μ l culture mediums (DMEM or RPMI1640 cationic-liposome Lipofectamine) is dissolved^TM2000 (20 μ l), lightly mix, and are incubated at room temperature 5 minutes.It incubates After educating, to promote shRNA expression vector and Lipofectamine^TM2000 sufficiently combine, they are mixed gently, and are incubated at room temperature 20 minutes, to form DNA-Lipofectamine^TM2000 compound.By DNA-Lipofectamine^TM2000 compounds add Enter to 60mm culture dish, DNA-Lipofectamine will be contained later^TMThe Tissue Culture Dish of 2000 compounds is put into 37 DEG C, and 5% CO₂Culture 6 hours, change normal cell culture fluid in incubator.

Three, experimental group and Testing index

Blank group (ghost group): glioma cell, culture solution are glioma cell culture solution；

Empty vector group (transfection empty carrier group)；The glioma cell of empty vector was transfected, culture solution is Glioma cell culture solution；

TGM2RNAi group (transfection TGM2RNA interference group): the glioma cell of TGM2-RNAi was transfected, culture solution is glue Matter oncocyte culture solution.

Fig. 2 indicates TGM2RNAi to the interference effect of TGM2 in brain glioblastoma cell, and Fig. 2 is that TGM2 is interfered in U373 cell Effect QPCR detection.In Fig. 2, TGM2RNAi group compared with empty vector group, TGM2 gene be obviously suppressed (* * p < 0.01)。

Fig. 3 indicates the influence that TGM2-RNAi is proliferated brain glioblastoma cell.From the figure 3, it may be seen that TGM2RNAi group and empty Vector group is compared, and the cell Proliferation of transfection TGM2RNAi interference group (TGM2RNAi group), which is substantially less than, transfects empty carrier group The cell proliferation rate (p < 0.01 * *) of (empty vector group).

Four, experimental result:

Above-mentioned experiment shows to transfect the TGM2 gene in TGM2RNAi interference group and is significantly struck and subtracted, tumour growth also by It is obvious to inhibit.Should the experimental results showed that, small molecules interference RNA of the invention can lower the TGM2 gene expression in glioma, energy Enough obvious growths for inhibiting glioma.

2 comparative experiments of embodiment

3 higher microRNAs of score value are selected, carrier construction simultaneously interferes sieve target, and experimental procedure is with embodiment 1, below It is relevant comparative's experimental data.

TGM2-si499:CCACTTCATTTTGCTCTTC (corresponds to TGM2 gene 499-517, i.e., the present invention is implemented Ordered sequence in example 1)；The corresponding sequence of TGM2-si499 are as follows:

Positive-sense strand: 5 '-CTAGACCACTTCATTTTGCTCTTCCTCGAGGAAGAGCAAAATGAAGTGGG-3 ', such as SEQ Shown in ID NO.1；

Antisense strand: 5 '-GATCCCCACTTCATTTTGCTCTTCCTCGAGGAAGAGCAAAATGAAGTGGT-3 ', such as SEQ Shown in ID NO.2.

TGM2-si666:CCTAGACATCTGCCTGATC (corresponds to TGM2 gene 666-684)；TGM2-si666 Corresponding sequence are as follows:

Positive-sense strand: 5 '-CTAGACCTAGACATCTGCCTGATCCTCGAGGATCAGGCAGATGTCTAGGG-3 ', such as SEQ Shown in ID NO.3；

Antisense strand: 5 '-GATCCCCTAGACATCTGCCTGATCCTCGAGGATCAGGCAGATGTCTAGGT-3 ', such as SEQ Shown in ID NO.4.

TGM2-si 1549:GAACATGGGCAGTGACTTT (corresponds to TGM2 gene 1549-1567).TGM2-si 1549 corresponding sequences are as follows:

Positive-sense strand: 5 '-CTAGA GAACATGGGCAGTGACTTTCTCGAGAAAGTCACTGCCCATGTTCG-3 ', such as Shown in SEQ ID NO.5；

Antisense strand: 5 '-GATCC GAACATGGGCAGTGACTTTCTCGAGAAAGTCACTGCCCATGTTCT-3 ', such as Shown in SEQ ID NO.6.

Contrast and experiment is as shown in figure 4, as can be seen from Figure 4, TGM2-si499 (the i.e. small molecule of the embodiment of the present invention 1 RNA) compared with TGM2-si666, TGM2-si 1549, TGM2 interference effect is most significant in U373 cell.

Claims (3)

1. a kind of small molecule shRNA of the interference TGM2 gene for inhibiting glioma to be proliferated, corresponding sequence are as follows:

Positive-sense strand:

5 '-CTAGACCACTTCATTTTGCTCTTCCTCGAGGAAGAGCAAAATGAAGTGGG-3 ', such as SEQ ID NO.1 institute Show；

Antisense strand: 5 '-GATCCCCACTTCATTTTGCTCTTCCTCGAGGAAGAGCAAAATGAAGTG GT-3 ', such as SEQ Shown in ID NO.2.

2. a kind of preparation method of the small molecule shRNA of interference TGM2 gene according to claim 1, which is characterized in that The specific steps of this method are as follows:

Step 1: design corresponds to TGM2 gene 499-517 according to the specific requirement that PMT98 carrier designs, it may be assumed that The shRNA oligonucleotide sequence of CCACTTCATTTTGCTCTTC is simultaneously synthesized, the shRNA oligonucleotide sequence are as follows: just Adopted chain is as shown in SEQ ID NO.1, and antisense strand is as shown in SEQ ID NO.2；

Step 2: the requirement according to the design of PMT98 carrier, the shRNA sequence of design and PMT98 plasmid are attached, obtained For interfering the rna interference vector of TGM2 gene expression.

3. the drug that the small molecule shRNA of interference TGM2 gene according to claim 1 inhibits glioma proliferation in preparation In application.