CN109536498A - A kind of PABPC5 gene inhibitor and application thereof - Google Patents
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Abstract
本发明属于生物医药领域，具体涉及一种PABPC5基因抑制剂及其在抗胶质瘤中的用途。所述的PABPC5基因靶向抑制剂的基因序列为5’‑GGAACATTCTGTCCTGCAAAG‑3’（SEQ ID No.1），与SEQ ID No.1所示的DNA序列具有90%同源性且具有相同或相近功能的DNA序列。PABPC5基因抑制剂用于制备抗肿瘤药物中的用途，所述肿瘤为脑胶质瘤。通过其在脑胶质瘤中的应用，可以抑制胶质瘤细胞的迁移和侵袭，诱导胶质瘤细胞凋亡，具有针对性的预防和提高胶质瘤治疗效果的优点。
The invention belongs to the field of biomedicine, and in particular relates to a PABPC5 gene inhibitor and its use in anti-glioma. The gene sequence of the PABPC5 gene targeting inhibitor is 5'-GGAACATTCTGTCCTGCAAAG-3' (SEQ ID No.1), which has 90% homology and is identical or similar to the DNA sequence shown in SEQ ID No.1 functional DNA sequences. Use of a PABPC5 gene inhibitor for preparing an antitumor drug, wherein the tumor is glioma. Through its application in glioma, the migration and invasion of glioma cells can be inhibited, and the apoptosis of glioma cells can be induced, which has the advantages of targeted prevention and improvement of the therapeutic effect of glioma.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to the inhibitor of a kind of PABPC5 gene and its in anti-glioma
Purposes.
Background technique
Glioblastoma be cental system it is most common be also the highest malignant tumour of grade of malignancy.Treatment means are main at present
Based on operation, chemicotherapy is adjuvant treatment.But since glioblastoma has high invasive growth, angiogenesis prosperous
The biological properties such as Sheng are cut under iconography and microscope entirely even if operation reaches, and recurrence is still unavoidable.After complex treatment
Its median survival interval is still no more than 15 months, and survival rate is no more than 15% within 5 years.In recent years, not with the research of gene molecule level
Disconnected deeply domestic and international medical field has new understanding and understanding to the pathogenesis of glioma, more and more sends out with glioma
Hair tonic is opened up closely related specific gene and is found, and a large amount of clinical researches show expression and the chemotherapeutics of these specific genes
Validity it is closely related.Accordingly, it is determined that the specific gene of glioma, specifies its expression status, and be directed to the gene development
With exploitation pharmaceutical preparation, the therapeutic efficiency of glioma can be effectively improved, the toxic side effect of drug is reduced, had boundless
Application prospect.
PABPC5 (Poly (A) binding protein cytoplasmic 5) is that one kind can be with poly (A) caudal knot
The rna binding protein of conjunction.PABPC5 is located at Xq21.31, contains 2 exons.Studies have reported that being examined in the brain of adult
The expression of PABPC5 is measured, also some researches show that the formation of PABPC5 and sex chromosome is closely related.It not yet finds at present
Expression of the PABPC5 in malignant glioma and the regulation for participating in glioma cell biological behaviour.Early period, using real-
Time PCR method has detected the expression of PABPC5.The results show that the expression of PABPC5 is significant in Glial cells
Higher than normal star spongiocyte group, this result prompt PABPC5 may participate in the function tune of generation and development to glioma
Control.
The principle of RNA perturbation technique is that RNA molecule is cut using Dicer digestion, forms RNA silencing complex, and targeting combines
The process of target RNA molecule and then degradation of rna molecule.The present invention develops the inhibitor of TSNAX gene using RNA perturbation technique,
It plays a role in glioma field of gene.
Currently, effect and related mechanism of the PABPC5 in glioma occurrence and development have not been reported, about PABPC5
The current still blank out of application in terms of glioma gene therapy.Therefore, developing a kind of relevant drug of PABPC5 becomes
Urgent problem to be solved at present.Implementation of the invention can be realized the theoretical research of glioma pathogenesis and turn to clinical application
Change, solves the problems, such as that glioma recurrence and treatment are resisted, effectively improve the therapeutic effect of glioma.
Summary of the invention
In view of the existing technical defect, the present invention provides one kind highly expressed specific gene suppression in glioma
Preparation can inhibit the migration and invasion of glioma cell by its application in glioma, and induction gum oncocyte withers
It dies, has the advantages that targetedly to prevent and improve therapeutic effect on glioma.
To achieve the goals above, the present invention uses following technical scheme.
A kind of targeted inhibition agent of PABPC5 gene, the gene order of the targeted inhibition agent are 5 '-
GGAACATTCTGTCCTGCAAAG-3 ' (SEQ ID No.1), or it is same with 90% with DNA sequence dna shown in SEQ ID No.1
Source property and the DNA sequence dna with same or similar function.
The targeted inhibition agent of the PABPC5 gene is able to suppress the shRNA sequence of PABPC5 gene expression, shRNA mould
Plate sequence includes positive-sense strand and antisense strand, and the positive-sense strand and antisense strand are respectively.
Positive-sense strand.
5’-CACCGGAACATTCTGTCCTGCAAAGTTCAAGAGACTTTGCAGGACAGAATGTTCCTTTTTTG-3’
(SEQ ID No.2).
Antisense strand.
5’-GATCCAAAAAAGGAACATTCTGTCCTGCAAAGTCTCTTGAAGGAACATTCTGTCCTGCAAAG-3’
(SEQ ID No.3).
A kind of transcription product for transcribing above-mentioned shRNA, sequence are.
5 '-GGAACATTCTGTCCTGCAAAGTTCAAGAGAGGAACATTCTGTCCTGCAAAG-3 ' (SEQ ID
No.4).
A kind of inhibitor of PABPC5 gene is used to prepare the purposes in anti-tumor drug, and the tumour is glioma.
The anti-tumor drug is the inhibitor and carrier or figuration pharmaceutically of the PABPC5 gene containing therapeutic dose
The pharmaceutical composition of agent composition.
The anti-tumor drug is pharmaceutically acceptable dosage form, optimizing injection.
Compared with prior art, beneficial effects of the present invention.
1) rna binding protein is able to maintain that the stemness of tumor stem cell, and the stability by enhancing tumour correlation RNA participates in
, there is drug resistance so as to cause chemotherapeutics in generation, development, transfer and the angiogenesis of tumour.Therefore rna binding protein can lead to
The effect of a variety of mechanisms play oncogenes is crossed, tumor development, suppression therapy effect are promoted.PABPC5 is that glioma is special
Property highly expressed rna binding protein, the occurrence and development of tumour can be promoted and induce drug resistance.Mainly for glioma specificity
Highly expressed PABPC5 gene reaches targeted therapy, reduces side effect.
2) feature of inhibitor of the present invention is dosage form acceptable in any pharmacotherapeutics, and there is great clinic to answer
With value.
3) research and development are the masters of current tumor therapeutic agent research and development for the inhibitor of tumor cell specific height expression molecule
Target is wanted, but due to medicament research and development period length and therapeutic effect difference etc., constrains the development of tumor-targeting drug.RNA is dry
Disturbing technology has the features such as safe and reliable, to deliver related neoplasms therapeutic agent by extracellular and intracellular number of ways, is swollen
The treatment of tumor provides relatively rapid and there is revolutionary tumour medicine to research and develop approach.
4) present invention prepares rna binding protein PABPC5 gene inhibitor using RNA perturbation technique, have targeted therapy,
Safely and effectively, relatively rapid and permanently effective feature is produced, the deficiency in anti-tumor drug research and development has been filled up.
Detailed description of the invention
Fig. 1 is Real-time PCR method detection PABPC5 in normal cerebral tissue's sample and samples of human glioma sample
Expression.Compared with normal cerebral tissue's sample, PABPC5 is expressed in samples of human glioma significantly to be increased, and difference has statistics
Meaning (n=5,** P< 0.01 vs. normal cerebral tissue)；Compared with Low grade glioma sample, PABPC5 is in glioblastoma group
It knits middle expression significantly to increase, difference has statistically significant.(n=5,## P< 0.01 vs. Low grade glioma).
Fig. 2 is the detection of Real-time PCR method using after PABPC5 gene inhibitor, and PABPC5 is in human glioma
Expression in U87 and U251 cell.Compared with blank control group, in U87 and U251 cell, PABPC5 is in inhibitor group
Expression is significant to lower, and difference is statistically significant.(n=3,** P< 0.01 vs. blank control group).
Fig. 3 is human glioma U87, U251 cell migration energy after PABPC5 gene inhibitor is applied in Transwell detection
The variation of power.(n=3,** P< 0.01 vs. blank control group).
Fig. 4 is human glioma U87, U251 cell invasion after PABPC5 gene inhibitor is applied in Transwell detection
The variation of ability.(n=3,** P< 0.01 vs. blank control group).
After Fig. 5 is flow cytomery application PABPC5 gene inhibitor, human glioma U87 Apoptosis ability
Variation.
Specific embodiment
The preparation method of the present invention is described in detail antitumorigenic substance with reference to the accompanying drawings and examples.
1. the expression of real-time quantitative PCR detection PABPC5.
1.1 Trizol methods extract the total serum IgE in tissue and cell.
1.1.1 with cold PBS wash collect cell (liquid nitrogen cryopreservation organizations tissue refiner is with 5000rpm at 0 DEG C
Crush 30 seconds), the piping and druming of 1 ml Trizol reagent is added for several times, microscopic observation cell is rear to move at oil droplet shape (sufficiently cracking)
In 1.5 ml EP pipes, 5 minutes are stood, cracks it sufficiently.
1.1.2 0.2 ml chloroform is added into sample, acutely concussion stands 3 minutes at room temperature manually.
1.1.3 being centrifuged 15 minutes in 4 DEG C of 12000g, takes upper strata aqueous phase into new EP pipe, add 0.5 ml isopropyl
Alcohol, mixing of turning upside down stand 10 minutes at room temperature.
1.1.4 4 DEG C of 12000g abandon supernatant after being centrifuged 15 minutes, and 1 ml, 75% ethyl alcohol is added.
1.1.5 4 DEG C of 7500g are centrifuged 5 minutes, and 40 μ l DEPC water are added in drying at room temperature after 15 minutes, sample can freeze
In -80 DEG C of refrigerators.
The expression of 1.2 1 step dye method qRT-PCR detection PABPC5.
Total serum IgE is extracted using Trizol: Trizol is applied directly to blow and beat on cell.It is placed at room temperature for after five minutes, Xiang Guan
0.2 ml chloroform of middle addition, acutely concussion 15 seconds, are placed 2-3 minutes at room temperature.4 DEG C, 12000g is centrifuged 15 minutes, takes upper layer
Water phase is into another new pipe.The concussion of 0.5 ml isopropanol is added, places 5 minutes under room temperature.4 DEG C, 12000g is centrifuged 10 points
Clock abandons supernatant, and tube bottom white precipitate is with 1 ml, 75% ethanol washing, vortex device oscillation.4 DEG C, 7500g is centrifuged 5 minutes, abandons supernatant
Liquid is inverted EP pipe on clean filter paper, until visible ethyl alcohol volatilizees completely.Appropriate DEPC water is added, slightly piping and druming to RNA
It is completely dissolved.Total serum IgE Nanodrop spectrophotometric determination RNA concentration and OD260/OD280 ratio verify purity
(RNA purity validation criteria: when OD260/OD280 is in 1.7-2.0, illustrating that RNA purity is very high, as OD260/OD280 < 1.7,
Show there is protein or phenol pollution；As OD260/OD280 > 2.0, show there may be isothiocyanic acid remaining).
Using One Step SYBR® PrimeScript®PLUS RT-PCR Kit (TaKaRa, China), according to saying
The operation of bright book is expanded.Key step includes, by synthesized target gene and GAPDH upstream and downstream primer with 0.1%
After the dissolution of DEPC water, configure RT-PCR reaction system (20 μ l): One Step SYBR buffer, 10 μ l；TaKaRa
Ex Taq HS Mis, 1.2 μ l；PrimeScript PLUS TRase Mix, 0.4 μ l；PCR upstream primer, 0.8 μ l；
PCR downstream primer, 0.8 μ l；ROX Refference Dye, 0.4 μ l；Total serum IgE, 2 μ l；RNase Free dH2O, 4.4 μ l.
It is put into gene-amplificative instrament after above-mentioned each ingredient is mixed mark number.The condition and parameter of amplification are as follows: 42 DEG C, 5 min, 95
DEG C, 10s；95 DEG C of 5s, 60 DEG C of 34s are recycled for 40 totally.CT value is measured, using GAPDH as internal reference, with 2-△△Ct is indicated
The relative expression quantity of PABPC5.
Expression of the PABPC5 gene in normal astroglia and glioma cell is detected, as shown in Figure 1, with
Normal cerebral tissue's sample is compared, and PABPC5 is expressed in glioblastoma tissue significantly to be increased, and expression is with tumour
The raising of rank and increase.
2. the preparation and application of PABPC5 gene inhibitor.
The interference sequence of PABPC5 gene is designed, targeting people PABPC5 gene is selected and specificity inhibits PABPC5 gene
The target-gene sequence of expression is as follows.
5 '-GGAACATTCTGTCCTGCAAAG-3 ' (SEQ ID No.1).
GGAACATTCTGTCCTGCAAAG is inputted in the same original sequence alignment analysis nucleotide blast of NCBI
Sequence is compared, the results showed that other mRNA genes of the sequence and people do not have high homology, and it is dry can to make specificity
Disturb the specific sequence of PABPC5 gene.
Targeting people PABPC5 gene is designed for the above target sequence and inhibits the shRNA sequence of PABPC5 gene expression such as
Under, including positive-sense strand and antisense strand, this shRNA sequence be.
Positive-sense strand.
5’-CACCGGAACATTCTGTCCTGCAAAGTTCAAGAGACTTTGCAGGACAGAATGTTCCTTTTTTG-3’
(SEQ ID No.2).
Antisense strand.
5’-GATCCAAAAAAGGAACATTCTGTCCTGCAAAGTCTCTTGAAGGAACATTCTGTCCTGCAAAG-3’
(SEQ ID No.3).
The transcription product of above-mentioned shRNA is transcribed, sequence is.
5 '-GGAACATTCTGTCCTGCAAAGTTCAAGAGAGGAACATTCTGTCCTGCAAAG-3 ' (SEQ ID
No.4).
The above sequence information is designed and synthesized into corresponding plasmid, as PABPC5 gene inhibitor.PABPC5 gene inhibits
Agent transfection: the plasmid U6/GFP/Neo of sh-NC, sh-PABPC5 make PABPC5 expression silencing, without containing PABPC5 sequence or
The empty plasmid of shRNA is experiment negative control；Using 24 well culture plate culture glioma cells, when cell growth reaches 80% left side
It is transfected when right；Plasmid, the Opti-MEM that configuration transfection needs®I and LTX and Plus reagent (Life
Technologies) transfection reagent.A pipe: a hole is dissolved in 50 μ l Opti-MEM, I+1 μ l according to 1 μ g Plasmid DNA
P3000 places 5 min, and B pipe: hole is dissolved in 50 μ l Opti-MEM according to 1 μ l LTX and Plus®In I；A, B two is managed
5 min are stood after mixing；Culture solution is sucked out, every hole is added 100 μ L of transfection cocktail, adds 400 μ L of EBM-2 culture solution；
It is screened after 48 h with containing the culture medium that concentration is 0.4 mg/mL antibiotic G418, is continuously increased the concentration of G418, about
Obtain to stablize the cell line of silencing PABPC5 after 4 weeks.It is divided into 3 groups in subsequent experimental, is respectively: normal group, transfection
The blank control group of PABPC5 silencing empty plasmid transfects the inhibitor group of PABPC5 silencing plasmid.
After PABPC5 gene inhibitor is applied in detection, the expression of PABPC5 in U87 and U251 glioma cell.As a result
As shown in Fig. 2, PABPC5 expresses significant downward in inhibitor group compared with blank control group.
3. cell Transwell migration and Matrigel.
1) the sugared culture solution of DMEM high that 500 μ l contain 10% serum is added in every hole in 24 porocyte culture plates, puts in hole
Set the cell transwell that aperture is 8 μm.
2) after cell count, different groups of cells is blown out into cell with the sugared culture solution of the DMEM high without containing serum and are hanged
Liquid is equably layered on respectively inside upper chamber, and 100 μ l cell suspensions containing about 10000 cells greatly are added in every hole cell.It is put into 37
It is cultivated 24 hours in DEG C constant incubator.
3) cell is taken out after 24 hours, upper indoor surface is not migrated into past cell with swab stick and is cleaned, according to methanol:
Glacial acetic acid=3: 1 proportional arrangement fixer.
4) cell is put into fixer, the cell of cell bottom surface is made to fix 30 minutes.
5) it is dried after cleaning cell with PBS.Prepare Giemsa stain, dye liquor: working solution 1:9.By Giemsa stain drop with
Cell bottom surface is dyed 1 hour.
6) it is cleaned twice with PBS, in the migration situation for being inverted 400 × microscopically observation cell, takes every group of cell at random
5 visuals field carry out number of cells statistics, the transfer ability of cell is represented with this.
4. cell Transwell Matrigel.
1) the matrigel Matrigel that 50 μ l concentration are 500ng/ μ l is uniformly spread in small indoor surface, is put into 37 DEG C of constant temperature
Make its solidification within 4 hours in case.
2) it is then then covered with cell suspension, method is same to migrate experiment.
Experimental result is as shown in Figure 3 and Figure 4, after PABPC5 gene inhibitor, compared with blank control group, and inhibitor
The cell migration of glioma U87, U251 and invasive ability of group significantly reduce.
5. cell apoptosis assay.
1) cell is cleaned twice with the PBS of pre-cooling.It is digested with the pancreatin without EDTA, cell is gently blown and beaten into cell
It is transferred to after suspension in 1.5ml centrifuge tube, 1000rpm is centrifuged 3 minutes collection cells.
2) continue to clean with PBS, 1000rpm is centrifuged 3 minutes, and supernatant is outwelled after centrifugation, is repeated twice.
3) 10 × binding of working solution buffer is diluted ten times.100 μ l Binding Buffer are added in every pipe, hang
Floating cell.
4) every pipe is sequentially added 5 μ l Annexin V-PI and 5 μ l FITC and mixes, and room temperature is protected from light 15 minutes.
5) 400 μ l1 × binding buffer are added to every pipe before machine on, after piping and druming uniformly, flow cytometer
The variation of (FACScan, BD company, the U.S.) detection Apoptosis.
For experimental result as shown in figure 5, compared with blank control group, PABPC5 gene inhibitor being capable of significant induction gum tumor
The apoptosis of U87 cell.
SEQUENCE LISTING
<110>Chinese Medical Sciences University
<120>a kind of PABPC5 gene inhibitor and application thereof
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>artificial sequence
<400> 1
ggaacattct gtcctgcaaa g 21
<210> 2
<211> 62
<212> DNA
<213>artificial sequence
<400> 2
caccggaaca ttctgtcctg caaagttcaa gagactttgc aggacagaat gttccttttt 60
tg 62
<210> 3
<211> 62
<212> DNA
<213>artificial sequence
<400> 3
gatccaaaaa aggaacattc tgtcctgcaa agtctcttga aggaacattc tgtcctgcaa 60
ag 62
<210> 4
<211> 53
<212> DNA
<213>artificial sequence
<400> 4
ggaacattct gtcctgcaaa gttcaagaga ggaacattct gtcctgcaaa g 51
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Non-Patent Citations (4)
| Title |
|---|
| FANGKUN JING等: "The PABPC5/HCG15/ZNF331 Feedback Loop Regulates Vasculogenic Mimicry of Glioma via STAU1-Mediated mRNA Decay", 《MOL THER ONCOLYTICS》 * |
| LYONS PA等: "Homo sapiens poly(A) binding protein cytoplasmic 5 (PABPC5), mRNA，NM_080832.3", 《NCBI GENBANK》 * |
| P BLANCO等: "A novel poly(A)-binding protein gene (PABPC5) maps to an X-specific subinterval in the Xq21.3/Yp11.2 homology block of the human sex chromosomes", 《GENOMICS》 * |
| 赵彦艳: "《医学生物学实验教程》", 31 March 2010, 人民军医出版社 * |
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