CN105063048A - SiRNA (small interfering ribonucleic acid) capable of inhibiting expression of Survivin genes and application of siRNA - Google Patents
SiRNA (small interfering ribonucleic acid) capable of inhibiting expression of Survivin genes and application of siRNA Download PDFInfo
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- CN105063048A CN105063048A CN201510497040.6A CN201510497040A CN105063048A CN 105063048 A CN105063048 A CN 105063048A CN 201510497040 A CN201510497040 A CN 201510497040A CN 105063048 A CN105063048 A CN 105063048A
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Abstract
The invention discloses siRNA (small interfering ribonucleic acid) capable of inhibiting expression of Survivin genes and an application of siRNA and particularly relates to an application of the efficient siRNA in preparation of medicines for controlling the tumor cell cycle. Efficient siRNA double-strand sequence can specifically inhibit expression of protein related to a disease after entering an organism in a certain way, so that related disease genes are silenced, and the purpose of treatment is achieved. SiRNA molecules have spectral anti-tumor activity, have a remarkable inhibition effect on diseases such as leukemia, gastric cancer, prostate, bladder cancer, cervical cancer, breast cancer, lung cancer, liver cancer and the like and can be used for preparing medicines for inhibiting proliferation and transfer of tumor cells.
Description
Technical field
The invention discloses a kind of survivin of suppression genetic expression siRNA molecule and preparing the application of antitumor drug, belong to biomedicine technical field.
Background technology
At present, malignant tumour clinical progress is fast, lifetime is short and the medium-long term curative effect of traditional clinical treatment is all undesirable, and chemotherapeutics plays therapeutic action on the one hand, and on the other hand, the health of toxic side effect to patient of chemotherapeutics plays destruction simultaneously.Therefore, thus finding new ideas of cancer therapy is the significant problem that the clinical and researcher in the whole world faces.Along with molecular biological development, carry out for oncogene the direction that targeted therapy becomes cancer therapy research.Drug effect, in targeting moieties such as tumour molecule, certain genes, improves curative effect on the one hand, decreases the consumption of medicine on the other hand.The target that searching can become oncotherapy has become one of prerequisite for the treatment of tumour.Pharmaceutically-active target may be become comprise: the various molecules in the special antigen that cancer cells has, special growth factor receptors, tumor vascular growth correlation factor, cancer cells specific information pipeline, cancer cells inner cell cycle and apoptotic regulatory molecule.
Mankind survivin gene is that the Ambrosini etc. of Yale in 1997 utilizes effector cell's proteolytic enzyme acceptor-1 (effectorcellproteasereceptor-1, EPR-1) cDNA to be separated and to clone out in the screening by hybridization of human genome, karyomit(e) 17q25 is located on EPR-1, total length 14.7kb, be made up of 3 introns and 4 exons, its mRNA is about 1.9kb, and the albumen that its coding produces is made up of 142 amino acid, and relative molecular weight is 16.5 × 10
3the IAP (inhibitorofapoptosisprotein of discovered in recent years, IAP) newcomer in family, has the dual-use function of inhibited apoptosis and maintenance cell mitogen, expresses hardly in normal Zhong Mo differentiated tissue.Under physiological status, survivin is only expressed in adult's secretory endometrium, placenta, testis, thymus gland and embryo, but under pathological state, survivin wide expression in the various malignant tumor tissue of the mankind, as cancer of the stomach, the esophageal carcinoma, colorectal cancer, liver cancer, mammary cancer, cervical cancer, lung cancer, leukemia, melanoma, lymphoma, III ~ IV phase neuroblastoma etc.Survivin expresses that increase can inhibition tumor cell apoptosis and promote it to breed, and its expression is obviously relevant to course advancement.Therefore, survivin as a kind of new tumor markers, may become the index detecting malignant tumour.
RNA disturbs survivin expression technology, RNA disturbs (RNAinterference, RNAi) refer in biological cell, exogenous or endogenic double-stranded RNA (double-strandedRNA) causes degraded specific with the mRNA of its homology, thus suppress the transcript and expression of survivin gene, there is the feature of high degree of specificity, high efficiency.Sum up achievement in research in recent years, it has more wide prospect in clinical application.
Summary of the invention
The present invention discloses a kind of its application at antitumor drug of Double-stranded siRNA molecules suppressing survivin genetic expression, is a kind of new siRNA sequence, can the propagation of inhibition tumor cell and transfer.
Double-stranded siRNA molecules of the present invention, is characterized in that, this Double-stranded siRNA molecules is a kind of small molecules interference RNA (siRNA) of wide spectrum double-strand of efficient target survivin gene, has following sequential structure:
Positive-sense strand 5 ＇-GAGACAGAAUAGAGUGAUA-3 ＇ SEQIDNO:1
Antisense strand 5 ＇-UAUCACUCUAUUCUGUCUC-3 ＇ SEQIDNO:2
The backbone sequences of Double-stranded siRNA molecules of the present invention is the nucleotide sequence shown in SEQIDNO:1 and SEQIDNO:2, and namely positive-sense strand and antisense strand have the duplex molecule of the nucleotide sequence shown in SEQIDNO:1 and 2 respectively.
Preferably, described Double-stranded siRNA molecules, has following sequential structure:
Positive-sense strand: 5 ＇-GAGACAGAAUAGAGUGAUAtt-3 ＇ SEQIDNO:3
Antisense strand: 5 ＇-UAUCACUCUAUUCUGUCUCtt-3 ＇ SEQIDNO:4.
The siRNA molecule storehouse that siRNA molecule of the present invention derives from the function conserved regions for survivin gene open reading frame and prepares, the positive-sense strand of this sequence is combined with the mRNA of survivin gene, the translation of interference survivin gene mRNA, thus the division of inhibition tumor cell, make tumour cell be arrested in the G2/M phase.
The advantage of siRNA molecule of the present invention is, prepared siRNA is randomly distributed in survivin gene open reading frame section, and length controllability is distributed in 19-23bp, can improve the hit rate of Effective target site.
The invention allows for a kind of siRNA recombinant plasmid, described siRNA recombinant plasmid comprises the sequence of any one Double-stranded siRNA molecules foregoing.Thus, utilize siRNA Transfected Recombinant Plasmid zooblast, can effective reticent surivivin gene, and then cell death inducing, inhibition tumor cell division, Tumor suppression blood vessel generation, thus effectively can treat tumour.Further, this siRNA molecule and siRNA recombinant plasmid can effectively for the preparation of antitumor drug, thus utilize the antitumor drug of preparation to carry out administration to patient, effectively treat tumour.
According to the invention allows for a kind of survivin gene silencing agent box, described test kit comprises any one Double-stranded siRNA molecules foregoing or siRNA recombinant plasmid.Utilize test kit of the present invention effectively the cell of tumour patient can be carried out survivin gene silencing, thus effectively can treat tumour.
SiRNA molecule of the present invention can as the effective constituent of antitumor drug, and tumor disease comprises as leukemia, cancer of the stomach, prostate cancer, bladder cancer, cervical cancer, mammary cancer, lung cancer, liver cancer etc.
Thus, the invention allows for any one Double-stranded siRNA molecules foregoing or siRNA recombinant plasmid is preparing the application in antitumor drug.
The present invention proposes a kind of antitumor drug, this medicine contains siRNA molecule or the siRNA recombinant plasmid of significant quantity, this antitumor drug can adopt ordinary method to be prepared into corresponding preparations, as injection form can be made into, such as, be prepared by ordinary method with physiological saline or the aqueous solution containing glucose and other assistant agents.Injection, solution etc. should aseptically manufacture.
Positively effect of the present invention is: the small molecules interference RNA (siRNA) providing a kind of wide spectrum double-strand of efficient target survivin gene newly, also discloses it simultaneously and is preparing the application in antitumor drug.
Accompanying drawing explanation
Fig. 1 is that after siRNA transfection two kinds of cells of chemosynthesis, real-time quantitative PCR detects survivinmRNA expression amount column diagram, and ordinate zou represents the mrna expression level of survivin relative to GAPDH; X-coordinate represents four process experimental group, and be respectively the groups of cells of untransfected, transfection experiment comprises three siRNA concentration, is respectively 10nM, 20nM and 30nM.
Fig. 2 is the survivin expressing quantity after WesternBlot detects siRNA transfected with human prostate cancer cell PC-3, hepatocellular carcinoma H22, breast cancer cell MCF-7 and cervical cancer cell Hela, corresponding to NC is negative control group, and corresponding to EG is the siRNA group adding 20nM/ hole.
Embodiment
For making the present invention easier to understand, below in conjunction with specific embodiment, set forth the present invention further.These embodiments are only not used in for illustration of the present invention and limit the scope of the invention.
Embodiment 1:siRNA silence efficiency is verified
1. key instrument, reagent and material.
1.1 instruments: nucleic acid synthesizer (GE company), PCR instrument (ABI company); Real-time quantitative PCR (Bio-Rad); Cell culture incubator (Thermo) etc.
1.2 materials and reagent: RNAiMAX
tM(invitrogen), DMEM substratum (Gibco),
TurboCapturemRNAkit (QIAGEN), SensiMix
tMone-StepKit (Quantace) etc.Other biochemical reagents are all purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
1.3PCR primer (Biomics Bioisystech Co., Ltd's synthesis):
Survivin forward primer: 5 ＇-AGAACTGGCCCTTCTTGGAGG-3 ＇
Survivin reverse primer: 5 ＇-CTTTTTATGTTCCTCTATGGGGTC-3 ＇
GAPDH forward primer; 5 ＇-GAAGGTGAAGGTCGGAGTC-3 ＇
GAPDH reverse primer: 5 ＇-GAAGATGGTGATGGGATTTC-3 ＇
2. chemosynthesis siRNA
According to the nucleotide sequence of survivin gene, design small molecules interference siRNA:
Positive-sense strand: 5 '-GAGACAGAAUAGAGUGAUAtt-3 '
Antisense strand: 5 '-UAUCACUCUAUUCUGUCUCtt-3 '
By the comparison in human EST database of this sequence, determine other not identical with it gene order.SiRNA is synthesized by Rui Bo bio tech ltd, Guangzhou.
3.RT-PCR detects survivin gene mRNA levels
3.1 cell cultures: cervical cancer cell Hela and human lung cancer cell A549 respectively containing 10%FBS DMEM substratum in, 37 DEG C, 5%CO
2incubator is cultivated.
3.2 plating cells transfection: by cervical cancer cell Hela and human lung cancer cell A549 by 1 × 10
5individual/hole is inoculated in 96 porocyte culture plates, and two kinds of cells arrange untransfected group, siRNA10nM group, siRNA20nM group and siRNA30nM group respectively, totally 8 groups.At 37 DEG C, 5%CO
2incubator cell cultures is spent the night, and containing in the DMEM substratum of 10%FBS without dual anti-, transfection is according to RNAiMAX
tMspecification sheets transfection, by embodiment 1 step 2 synthesis siRNA add grouping corresponding to two groups of cells by 10nM/ hole, 20nM/ hole and 30nM/ hole respectively.
Detect survivinmRNA expression level in sample with gene-specific primer, the house-keeping gene GAPDH that simultaneously increases contrasts as internal reference.Each reaction do 3 parallel.Set up following 25 μ L reaction systems: 4 μ L template ribonucleic acids, 12.5 μ L2 × SensiMixone-step, l μ L5 ＇ forward primer (6 μMs), l μ L3 ＇ reverse primer (6 μMs) 0.5 μ L50 × SYBRGreenI, supplies system to 25 μ L with without RNase water.Reaction conditions: 40 DEG C of reverse transcription 30min, 95 DEG C of denaturation 7min, 95 DEG C of sex change 20sec, 60 DEG C of annealing 30sec, 72 DEG C extend 30sec, circulate 45 times.
3.4 interpretations of result: use Real-time PCR Analysis experimental result, and make histogram, as shown in Figure 1, experiment is divided into four groups, wherein " untransfected " is the cell sample without any process, and other three groups is siRNA of the present invention, and concentration is respectively 10nM, 20nM, 30nM.The siRNA silencing efficiency of result display target survivin gene is remarkable, has certain concentration dependent.When siRNA is 30nM, the silence efficiency of survivin gene can reach more than 80%.
Embodiment 2:survivin gene silencing affects tumor cell proliferation
Cervical cancer cell Hela, liver cancer HepG2 and human lung cancer cell A549 are pressed 5 × 10
3individual/hole is inoculated in 96 porocyte culture plates respectively, and three kinds of cells are cultivated respectively, test respectively.At 37 DEG C, 5%CO
2incubator cell cultures 24 hours, is containing in the DMEM substratum of 10%FBS, according to RNAiMAX without dual anti-
tMspecification sheets transfection, in embodiment 1 step 2 siRNA of synthesis by 10,20,40nM/ hole adds, 37 DEG C hatch 48 hours after, every hole adds 20 μ LMTT (5mg/mL), 37 DEG C are continued to hatch 4h, absorb substratum, add 200 μ LDMSO and dissolve the crystallization of first a ceremonial jade-ladle, used in libation, measure absorbance in 490nm place.
Mtt assay detects siRNA to the suppression result of tumor cell proliferation, and siRNA strengthens the restraining effect of tumour lung cancer A549, liver cancer HepG2, cervical cancer Hela cells and mammary cancer MCF-7 gradually at 20nM to 50nM, and inhibiting rate the results are shown in Table 1.By the drug effect control drug of antineoplastic cis-platinum conventional clinically as siRNA, cis-platinum strengthens gradually 10 μMs to 40 μMs restraining effect to tumour lung cancer A549, liver cancer HepG2, cervical cancer Hela cells and mammary cancer MCF-7, act on after 48 hours, the inhibiting rate result to tumour cell that mtt assay detects, in table 2.
Table 1siRNA acts on 48 hours inhibiting rates to growth of tumour cell (%)
| Cell | 20nM | 30nM | 50nM |
| A549 | 20.4±2.2 | 49.8±3.6 | 61.3±2.4 |
| HepG2 | 22.6±1.4 | 50.3±2.8 | 64.9±4.3 |
| Hela | 25.1±0.6 | 55.6±2.4 | 68.7±3.6 |
| MCF-7 | 18.3±2.5 | 47.2±3.1 | 60.3±2.7 |
Table 2 cisplatin effect 48 hours inhibiting rates to growth of tumour cell (%)
| Cell | 10μM | 20μM | 40μM |
| A549 | 8.3±2.5 | 28.9±5.3 | 58.7±1.7 |
| HepG2 | 12.1±3.1. | 30.5±4.5 | 62.3±5.1 |
| Hela | 15.7±3.8 | 35.7±3.3 | 60.4±2.7 |
| MCF-7 | 12.7±1.2 | 37.8±3.7 | 57.6±1.5 |
Because cell line A549, Cell Line HepG2, cell strain Hela and cell strain MCF-7 are respectively lung cancer cell line, hepatoma cell strain, cervical cancer cell lines and breast carcinoma cell strain, so siRNA of the present invention may be used for the treatment of liver cancer, cervical cancer and mammary cancer.
Embodiment 3:WesternBlot detects the suppression situation that siRNA expresses survivin
By Human Prostate Cancer Cells PC-3, hepatocellular carcinoma H22, breast cancer cell MCF-7 cell and cervical cancer cell Hela respectively by 1.5 × 10
5individual/hole is inoculated in 6 porocyte culture plates, and four kinds of cells are cultivated respectively, test respectively, at 37 DEG C, 5%CO
2incubator cell cultures 24 hours, is containing in the DMEM substratum of 10%FBS, according to RNAiMAX without dual anti-
tMspecification sheets transfection, siRNA adds by 20nM/ hole, 37 DEG C hatch 48 hours after, absorb substratum, wash twice with PBS, add 100 μ L cell pyrolysis liquids, cracking 10min on ice bath, scrapes with cell and is scraped by cell, is transferred in 1.5ml centrifuge tube, after the centrifugal 5min of 10000rpm, get supernatant liquor and measure protein concentration.Often group is got 30 μ g albumen and is carried out SDS-PAGE, goes on pvdf membrane after electrophoresis completes, and carries out survivin primary antibodie and goat-anti rabbit two process resistant, then carry out chemiluminescence reaction after closing, development.The results are shown in Figure 2, after adding siRNA, the suivivin expressing quantity of cell is starkly lower than the negative control group without siRNA process.
In four kinds of clone, siRNA can reduce the expression of survivin albumen significantly, so siRNA of the present invention may be used for the treatment of prostate cancer, liver cancer, mammary cancer and cervical cancer.
Claims (7)
1. suppress a Double-stranded siRNA molecules for survivin genetic expression, it is characterized in that, there is following sequential structure:
Positive-sense strand 5 ＇-GAGACAGAAUAGAGUGAUA-3 ＇ SEQIDNO:1
Antisense strand 5 ＇-UAUCACUCUAUUCUGUCUC-3 ＇ SEQIDNO:2.
2. suppress a Double-stranded siRNA molecules for survivin genetic expression, it is characterized in that, there is following sequential structure:
Positive-sense strand: 5 ＇-GAGACAGAAUAGAGUGAUAtt-3 ＇ SEQIDNO:3
Antisense strand: 5 ＇-UAUCACUCUAUUCUGUCUCtt-3 ＇ SEQIDNO:4.
3. a siRNA recombinant plasmid, is characterized in that, described siRNA recombinant plasmid comprises the Double-stranded siRNA molecules described in claim 1 or 2.
4. a Survivin gene silencing agent box, is characterized in that, described test kit comprises the Double-stranded siRNA molecules described in claim 1 or 2 or siRNA recombinant plasmid according to claim 2.
5. the Double-stranded siRNA molecules in claim 1 or described in 2 or the siRNA recombinant plasmid described in right 3 are preparing the application in antitumor drug or antineoplastic pharmaceutical compositions.
6. an antitumor drug, is characterized in that, the Double-stranded siRNA molecules as described in claim 1 or 2 containing significant quantity or siRNA recombinant plasmid according to claim 3.
7., according to the antitumor drug described in right 6, wherein tumour is leukemia, cancer of the stomach, prostate cancer, bladder cancer, cervical cancer, mammary cancer, lung cancer, at least one in liver cancer.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070072823A1 (en) * | 2002-11-14 | 2007-03-29 | Dharmacon Inc. | siRNA targeting survivin |
| CN101120099A (en) * | 2004-12-08 | 2008-02-06 | 株式会社百奥尼 | Method of inhibiting expression of target mrna using sirna consisting of nucleotide sequence complementary to said target mrna |
| US20100093085A1 (en) * | 2006-09-22 | 2010-04-15 | Dharmacon, Inc | Tripartite oligonucleotide complexes and methods for gene silencing by rna interference |
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2015
- 2015-08-13 CN CN201510497040.6A patent/CN105063048A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070072823A1 (en) * | 2002-11-14 | 2007-03-29 | Dharmacon Inc. | siRNA targeting survivin |
| CN101120099A (en) * | 2004-12-08 | 2008-02-06 | 株式会社百奥尼 | Method of inhibiting expression of target mrna using sirna consisting of nucleotide sequence complementary to said target mrna |
| US20100093085A1 (en) * | 2006-09-22 | 2010-04-15 | Dharmacon, Inc | Tripartite oligonucleotide complexes and methods for gene silencing by rna interference |
Non-Patent Citations (4)
| Title |
|---|
| LEIMING LI 等: "Developing Lipid Nanoparticle-Based siRNA Therapeutics for Hepatocellular Carcinoma Using an Integrated Approach", 《MOLECULAR CANCER THERAPEUTICS》 * |
| MGROSS: "603352- BACULOVIRAL IAP REPEAT-CONTAINING PROTEIN 5;BIRC5(SURVIVIN)", 《OMIM》 * |
| 夏荣耀 等: "SURVIVIN基因与肿瘤相关研究进展", 《现代肿瘤医学》 * |
| 文贤慧 等: "靶向Survivin基因与肿瘤", 《生命科学研究》 * |
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Application publication date: 20151118 |