CN104561003B

CN104561003B - A kind of microRNA and its preparation method and application of the interference MCM7 gene for inhibiting glioma to be proliferated

Info

Publication number: CN104561003B
Application number: CN201510022968.9A
Authority: CN
Inventors: 俞磊; 吴小江; 祁伟; 楼小燕; 吴雪松
Original assignee: SHANGHAI SHENGBO BIOMEDICAL TECHNOLOGY Co Ltd
Current assignee: SHANGHAI SHENGBO BIOMEDICAL TECHNOLOGY Co Ltd
Priority date: 2015-01-16
Filing date: 2015-01-16
Publication date: 2019-03-22
Anticipated expiration: 2035-01-16
Also published as: CN104561003A

Abstract

The microRNA for the interference MCM7 gene that the invention discloses a kind of for inhibiting glioma to be proliferated and its application.The microRNA of interference MCM7 gene is base sequence shown in SEQ ID NO.1 and SEQ ID NO.2.Present invention design has the microRNA of the interference MCM7 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 MCM7 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 MCM7 gene of the kind for inhibiting glioma to be proliferated；In addition, the invention further relates to interference MCM7 genes MicroRNA preparation method and application.

Background technique

MCM7 albumen is one of the constituent of MCM (minichromosome maintenance) compound, has DNA Helicase activity participates in DNA replication dna and originates, cell proliferation, DNA damage reparation and regulating and controlling effect important from the cell cycle, Have studies have shown that MCM7 high expression (including glioma) in kinds of tumors, can be used as Several Kinds of Malignancy diagnosis and Prognostic indicator.

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 MCM7 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 MCM7 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 MCM7 gene.

The third technical problem to be solved by the present invention is to provide the application of the microRNA of interference MCM7 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 MCM7 gene for inhibiting glioma to be proliferated is provided, Its corresponding sequence are as follows:

Positive-sense strand: 5 '-CTAGAGCATTGATGAGTTCGACAAGACTCGAGTCTTGTCGAACTCATCAATGCG-3 ', As shown in SEQ ID NO.1；

Antisense strand: 5 '-GATCCGCATTGATGAGTTCGACAAGACTCGAGTCTTGTCGAACTCATCAATGCT-3 ', As shown in SEQ ID NO.2.

RNA interference (RNA interference, RNAi) refer to it is being highly conserved during evolution, by double-strand The phenomenon that RNA (double-stranded RNA, dsRNA) induces, homologous mRNA efficient selective degradation.Host cell pair These dsRNA immediately generate reaction, and dsRNA is cut into multiple with specific length by the endonuclease Dicer in cytoplasm With the small fragment RNA (about 21~23bp) of structure, i.e. siRNA.SiRNA in the cell under the action of RNA helicase unwinding at Positive-sense strand and antisense strand are followed by combined with some enzymes (including restriction endonuclease, excision enzyme, unwindase etc.) in vivo by antisense siRNA again Form the silencing complex (RNA-induced silencing complex, RISC) of RNA induction.RISC and allogenic gene The homologous region of the mRNA of expression is specifically bound, and RISC has the function of nuclease, cuts mRNA, cutting in binding site Site is the both ends of combination complementary with antisense strand in siRNA.Fracture mRNA after being cut degrades immediately, to induce host Cell is directed to the degradation reaction of these mRNA.

In another aspect of this invention, it provides and a kind of prepares the above-mentioned interference MCM7 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 MCM7 gene 1974-1994 according to the specific requirement that PMT98 carrier designs, That is: the shRNA oligonucleotide sequence of GCATTGATGAGTTCGACAAGA is simultaneously synthesized, the oligonucleotides sequence of the shRNA Column sequence are as follows: 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 small molecules interference RNA of the MCM7 gene for inhibiting glioma to be proliferated.

In another aspect of this invention, the microRNA for providing a kind of above-mentioned interference MCM7 gene inhibits colloid in preparation Application in the drug of tumor proliferation.

The present invention designs the RNAi that associated MCM7 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 MCM7 RNA interference group in tumour growth be suppressed, should the experimental results showed that, this The small molecules interference RNA of invention can lower the MCM7 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 MCM7 RNAi to the interference effect schematic diagram of MCM7 in brain glioblastoma cell, In, blank indicates ghost group, and empty vector indicates transfection empty carrier group；MCM7 RNAi indicates that transfection MCM7 RNA is dry Disturb group.

Fig. 3 is the influence schematic diagram that MCM7 RNAi is proliferated brain glioblastoma cell in the embodiment of the present invention 1, wherein Blank indicates ghost group, and empty vector indicates transfection empty carrier group；MCM7 RNAi indicates transfection MCM7 RNA interference Group.

Fig. 4 is that three microRNAs show the interference effect comparison of MCM7 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 MCM7-RNAi plasmid:

1.MCM7-RNAi the design of plasmid:

According to the specific requirement that PMT98 carrier designs, design corresponds to MCM7 gene 1974-1994, it may be assumed that

The shRNA oligonucleotide sequence of GCATTGATGAGTTCGACAAGA is simultaneously synthesized, and extra large JaRa bioengineering is served Co., Ltd is synthesized.

The corresponding sequence of MCM7-RNAi are as follows:

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

Antisense (antisense strand): 5 '-GATCCGCATTGATGAGTTCGACAAGACTCGAG TCTTGTCGAACTCAT CAATGCT-3 ', as shown in SEQ ID NO.2.

According to the specific requirement that PMT98 carrier designs, the interference fragment of the MCM7-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- Mir30 arm-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.MCM7-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 MCM7-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；

MCM7 RNAi group (transfection MCM7 RNA interference group): the glioma cell of MCM7-RNAi was transfected, culture solution is Glioma cell culture solution.

Fig. 2 indicates MCM7 RNAi to the interference effect of MCM7 in brain glioblastoma cell, and Fig. 2 is that MCM7 is dry in U373 cell Disturb effect QPCR detection.In Fig. 2, for MCM7 RNAi group compared with empty vector group, MCM7 gene is obviously suppressed (* * p <0.01)。

Fig. 3 indicates the influence that MCM7-RNAi is proliferated brain glioblastoma cell.From the figure 3, it may be seen that MCM7 RNAi group and empty Vector group is compared, and the cell Proliferation of transfection MCM7 RNAi interference group (MCM7 RNAi 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 MCM7 gene in MCM7 RNAi 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 MCM7 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.

MCM7-si1456:CGTCAGCGTCACTGGTATTTT (corresponds to MCM7 gene 1456-1476)；MCM7- The corresponding sequence of si1456 are as follows:

Positive-sense strand: 5 '-CTAGACGTCAGCGTCACTGGTATTTTCTCGAGAAAATACCAGTGACGCTGACGG-3 ', As shown in SEQ ID NO.3；

Antisense strand: 5 '-GATCCCGTCAGCGTCACTGGTATTTTCTCGAGAAAATACCAGTGACGCTGACGT-3 ', As shown in SEQ ID NO.4.

MCM7-si1644:CGAAAAGCTGGCAGCTTCA (corresponds to MCM7 gene 1644-1664)；MCM7- The corresponding sequence of si1644 are as follows:

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

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

MCM7-si1974:GCATTGATGAGTTCGACAAGA (corresponds to MCM7 gene 1974-1994, that is, implements Ordered sequence in example 1), the corresponding sequence of MCM7-si1974 are as follows:

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

Antisense (antisense strand): 5 '-GATCCGCATTGATGAGTTCGACAAGACTCGAGTCTTGTCGAACTCAT CAATG CT-3 ', as shown in SEQ ID NO.2.

Contrast and experiment is as shown in figure 4, as can be seen from Figure 4, MCM7-si1974 (the i.e. small molecule of the embodiment of the present invention 1 RNA) compared with MCM7-si1456, MCM7-si1644, MCM7 interference effect is most significant in U373 cell.

Claims

1. a kind of small molecule shRNA of the interference MCM7 gene for inhibiting glioma to be proliferated, corresponding sequence are as follows:

Positive-sense strand:

5 '-CTAGAGCATTGATGAGTTCGACAAGACTCGAGTCTTGTCGAACTCATCAATGCG- 3 ', such as SEQ ID Shown in NO.1；

Antisense strand:

5 '-GATCCGCATTGATGAGTTCGACAAGACTCGAGTCTTGTCGAACTCATCAATGCT- 3 ', such as SEQ ID Shown in NO.2.

2. a kind of preparation method of the small molecule shRNA of interference MCM7 gene according to claim 1, which is characterized in that The specific steps of this method are as follows:

Step 1: design corresponds to MCM7 gene 1974-1994 according to the specific requirement that PMT98 carrier designs, it may be assumed that The shRNA oligonucleotide sequence of GCATTGATGAGTTCGACAAGA is simultaneously synthesized, the shRNA oligonucleotide sequence are as follows: Positive-sense strand 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 MCM7 gene expression.

3. the drug that the small molecule shRNA of interference MCM7 gene according to claim 1 inhibits glioma proliferation in preparation In application.