CN104531707A - siRNA sequence for inhibiting survivin gene expression and use - Google Patents

Abstract

The invention discloses a siRNA sequence for inhibiting survivin gene expression. The siRNA sequence has a broad spectrum siRNA antitumor effect, and has a good inhibitory effect on breast cancer, lung cancer, colon cancer, liver cancer, leukemia, gastric carcinoma, cervical cancer, oral epithelial carcinoma in vitro, and particularly on breast cancer, lung cancer and cervical cancer cells. When entering an organism through a certain manner, the siRNA double strand sequence can efficiently and specially inhibit the expression of related proteins of diseases to silence related disease protein, and can effectively remove the expression of target gene to achieve the purpose of treatment.

Description

A kind of siRNA sequence and purposes suppressing survivin genetic expression purposes

Technical field

The present invention discloses a kind of Double-stranded siRNA molecules suppressing survivin genetic expression, additionally provides the medical application of siRNA molecule simultaneously, belongs to biomedicine technical field.

Background technology

Malignant tumour is one of primary killers of current threat human life health, although various methods for the treatment of constantly occurs, also do not find real effective radical cure method at present, the treatment of tumour is the global problem of long-standing problem medical circle always.Escape the feature that apoptosis is all tumours, it makes tumour cell survival under misgrowth stimulates, and resists chemotherapy and radiation.Human anti-apoptotic protein survivin is by becoming with apoptotic signal effect the growth that new the most ergastic therapeutic targets carrys out inhibition tumor cell.

RNA disturbs (RNA interference, RNAi) phenomenon to be a kind of defense mechanism resisting transgenosis or adventitious viruses infringement conservative on evolving, and is a kind of sequence-specific PTGS.The treatment being found to be disease treatment especially cancer of this technology opens brand-new approach, for researcher provides a brand-new gene therapy theory.Application RNA perturbation technique, with target to being that the siRNA of survivin gene is as targeted drug, disturb after the transcribing of gene, thus inducing apoptosis of tumour cell, the mitotic division of inhibition tumor cell, the tolerance of vascularization during Tumor suppression transfer and reduction tumour cell chemicotherapy, suppresses tumour, may become a kind of new tumor therapeuticing method in the near future.

summary of the invention:

The present invention discloses a kind of Double-stranded siRNA molecules and the medical application thereof that suppress 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 a kind of small molecules interference RNA (siRNA) of wide spectrum double-strand of efficient target survivin gene, the forming with antisense strand of positive-sense strand by following nucleotide sequence:

Positive-sense strand 5 ＇-GCAGGUUCCUUAUCUGUCA-3 ＇, (SEQ ID NO:1)

Antisense strand 5 ＇-UGACAGAUAAGGAACCUGC-3 ＇; (SEQ ID NO:2)

According to a further aspect in the invention, the invention allows for a kind of Double-stranded siRNA molecules, the forming with antisense strand of positive-sense strand by following nucleotide sequence:

Positive-sense strand 5 ＇-GCAGGUUCCUUAUCUGUCAX-3 ＇, (SEQ ID NO:3)

Antisense strand 5 ＇-UGACAGAUAAGGAACCUGCX-3 ＇; (SEQ ID NO:4)

Wherein X represents tt, TT or UU.T represents deoxythymidine.After preferably, in above-mentioned sequence, " X " of 3 ＇ ends is tt, i.e. two deoxythymidines.

The backbone sequences of Double-stranded siRNA molecules of the present invention is the nucleotide sequence shown in SEQ ID NO:1 and SEQ ID NO:2, and namely foregoing positive-sense strand and antisense strand have the duplex molecule of the nucleotide sequence shown in SEQ ID NO:1 and 2 respectively.

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, its advantage is that prepared siRNA is randomly distributed in survivin gene open reading frame section, length controllability is distributed in 19-23bp, can improve the hit rate of Effective target site.

The invention allows for a kind of plasmid, described plasmid comprises the DNA sequence of any one Double-stranded siRNA molecules foregoing.Thus, utilize plasmid transfection 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 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 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 mammary cancer, lung cancer, liver cancer, leukemia, cancer of the stomach, cervical cancer, bladder cancer, Skin Squamous Cell Carcinoma, oral epithelium cancer, colorectal cancer, carcinoma of the pancreas, multiple myeloma, prostate cancer, melanoma or osteosarcoma.

Thus, according to the another aspect that this law is bright, the invention allows for can the application in antitumor drug by any one double-strand siRNA foregoing or plasmid.

The formulation of medicine of the present invention can be various ways, as long as be suitable for the administration of corresponding disease and keep the activity of siRNA molecule rightly.Such as, for injection drug delivery system, formulation can be lyophilized powder.

Optionally, the acceptable auxiliary of any pharmacy in said medicine formulation, can be comprised, as long as it is suitable for corresponding drug delivery system and keep the activity of siRNA molecule rightly.

positively effect of the present invention is:provide a kind of small molecules interference RNA (siRNA) of wide spectrum double-strand of efficient target survivin gene newly, also disclose its purposes in antitumor drug simultaneously.

Accompanying drawing explanation

Fig. 1 is that after siRNA transfection two kinds of cells of chemosynthesis, real-time quantitative PCR detects survivin mrna expression amount column diagram, and ordinate zou represents the mrna expression level of survivin relative to GAPDH; X-coordinate represents four process experimental group, is respectively the groups of cells of untransfected, and the negative siRNA control group of transfection, transfection experiment comprises two siRNA concentration, is respectively 10nM and 20nM.

Fig. 2 is the survivin expressing quantity after Western Blot detects siRNA transfection MDA-MB-231 cell, Hela cell and A549 cell.

embodiment:

For the ease of understanding the present invention, especially exemplified by following examples.Its effect is understood to be explaination of the present invention but not to any type of restriction of the present invention.

embodiment 1:the siRNA of chemosynthesis is to the test experience of survivin Gene silencing efficacy

1. key instrument, reagent and material.

1.1 instruments: nucleic acid synthesizer (GE company), PCR instrument (ABI company); Real-time quantitative PCR instrument (Bio-Rad); Cell culture incubator (Thermo) etc.

1.2 materials and reagent: RNAiMAXTM(invitrogen), DMEM substratum (Gibco),

TurboCapture mRNA kit(QIAGEN), SensiMix ^tMone-Step Kit(Quantace) etc.Other biochemical reagents are all purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.

1.3 PCR primers (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 '-GCAGGUUCCUUAUCUGUCA dTdT-3 '

Antisense strand: 5 '-UGACAGAUAAGGAACCUGC dTdT-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. the checking of silence efficiency

3.1 cell cultures: cervical cancer cell Hela and breast cancer cell MCF-7 containing 10 % FBS DMEM substratum in, 37 DEG C, 5 % CO2 incubators cultivate.

3.2 plating cells transfection: by cell by 1 × 10 ⁴/ hole is inoculated in 24 porocyte culture plates, at 37 DEG C, 5% CO ₂incubator cell cultures 24 hours, containing in the DMEM substratum of 10 % FBS without dual anti-, transfection is according to RNAiMAX ^tMspecification sheets transfection, siRNA adds by 20 nM/ holes.Use transfection reagent RNAiMAX simultaneously ^tMnegative control NT-siRNA is proceeded to Hela and MCF-7 cell.

3.3 real-time quantitative PCRs detect survivin gene mRNA levels: with mRNA extraction purification test kit TurboCapture mRNA kit extraction purification cell RNA, operation is undertaken by test kit specification sheets, dissolve RNA with 80 μ L without RNase water/hole, getting 4 μ L RNA is that template carries out real-time quantitative PCR reaction.

Detect survivin mrna 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 L 2 × SensiMix one-step, l μ L 5 ＇ forward primer (6 μMs), 0.5 μ L 50 × SYBR Green I, supplies system to 25 μ L with without RNase water.Reaction conditions: 40 DEG C of reverse transcription 30 min, 95 DEG C of denaturation 7 min, 95 DEG C of sex change 20 sec, 60 DEG C of annealing 30 sec, 72 DEG C extend 30 sec, circulate 45 times.

3.4 interpretations of result: use Real-time PCR Analysis experimental result, and make histogram, experiment is divided into four groups, wherein " untransfected " is the cell sample without any process, NT-siRNA is the negative control of concentration 20nM, and other two groups is siRNA of the present invention, and concentration is respectively 10nM, 20nM.As shown in Figure 1, the siRNA silencing efficiency of result display target survivin gene is remarkable, has certain concentration dependent.When siRNA is 20nM, the silence efficiency of survivin gene can reach more than 80%.

embodiment 2:survivin gene silencing affects tumor cell proliferation

By cell by 5 × 10 ³/ hole is inoculated in 96 porocyte culture plates, at 37 DEG C, 5% CO ₂incubator cell cultures 24 hours, containing in the DMEM substratum of 10 % FBS without dual anti-, according to the specification sheets transfection of RNAiMAXTM, siRNA adds by 10,20,40 nM/ holes, 37 DEG C hatch 48 hours after, every hole adds 20 μ L MTT(5mg/mL), 37 DEG C are continued to hatch 4h, absorb substratum, add 200 μ L DMSO and dissolve the crystallization of first a ceremonial jade-ladle, used in libation, measure absorbance in 540nm place.

Mtt assay detects siRNA to the suppression result of tumor cell proliferation, in table 1.

Table 1 siRNA acts on 48 hours inhibiting rates to growth of tumour cell

Because cell line A549, cell strain Hela and cell strain MCF-7 are respectively lung cancer cell line, cervical cancer cell lines and breast carcinoma cell strain, so siRNA of the present invention may be used for the treatment of lung cancer, cervical cancer and mammary cancer.

embodiment 3:western Blot detects the suppression situation that siRNA expresses survivin

By MDA-MB-231 cell, Hela cell and A549 cell by 1.5 × 10 ⁵/ hole is inoculated in 6 porocyte culture plates, at 37 DEG C, 5% CO ₂incubator cell cultures 24 hours, is containing in the DMEM substratum of 10 % FBS, according to the specification sheets transfection of RNAiMAXTM without dual anti-, siRNA adds by 20 nM/ holes, 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 primary antibodie and two process resistant, then carry out chemiluminescence reaction after closing, development.The results are shown in Figure 2, in three 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 mammary cancer, cervical cancer and lung cancer.

　SQE ID NO:1

<110> Jilin University

　　<120>

　　<140>

　　<160> 1

　　<210> 1

　　<211> 19

　　<212> RNA

<213> synthetic

　　<400> 1

gcagguuccuuaucuguca 19

　　SEQ ID NO:2

<110> Jilin University

　　<120>

　　<140>

　　<160> 4

　　<210> 2

　　<211> 19

　　<212> RNA

<213> artificial sequence

　　<400> 1

ugacagauaaggaaccugc 19

SEQ ID NO:3

<110> Jilin University

　　<120>

　　<140>

　　<160> 4

　　<210> 3

　　<211> 21

　　<212> RNA

<213> artificial sequence

　　<400> 1

gcagguuccuuaucugucadTdT 21

SEQ ID NO:4

<110> Jilin University

　　<120>

　　<140>

　　<160> 4

　　<210> 4

　　<211> 21

　　<212> RNA

<213> artificial sequence

　　<400> 1

ugacagauaaggaaccugcdTdT 21

 

 

 

Claims (6)

1. suppressing a Double-stranded siRNA molecules for survivin genetic expression, it is characterized in that the positive-sense strand and antisense strand by having following nucleotide sequence forms:

Positive-sense strand 5 ＇-GCAGGUUCCUUAUCUGUCA-3 ＇,

Antisense strand 5 ＇-UGACAGAUAAGGAACCUGC-3 ＇.

2. suppressing a Double-stranded siRNA molecules for survivin genetic expression, it is characterized in that the positive-sense strand and antisense strand by having following nucleotide sequence forms:

Positive-sense strand: 5 ＇-GCAGGUUCCUUAUCUGUCA X-3 ＇,

Antisense strand: 5 ＇-UGACAGAUAAGGAACCUGC X-3 ＇;

Wherein, X represents tt, TT or UU, and t represents deoxythymidine.

3. a siRNA expression plasmid, is characterized in that, described plasmid is the DNA sequence comprising Double-stranded siRNA molecules described in any one of claim 1 ~ 2.

4. a Survivin gene silencing agent box, is characterized in that, described test kit comprises Double-stranded siRNA molecules described in any one of claim 1 ~ 2 or siRNA expression plasmid according to claim 3.

5. Double-stranded siRNA molecules or the siRNA expression plasmid according to claim 3 of the suppression survivin genetic expression according to any one of claim 1 ~ 2 are preparing the application in antitumor drug.

6. in claim 5 the application that describes, wherein tumour is at least one in mammary cancer, lung cancer, liver cancer, leukemia, cancer of the stomach, cervical cancer, bladder cancer, Skin Squamous Cell Carcinoma, oral epithelium cancer, colorectal cancer, carcinoma of the pancreas, multiple myeloma, prostate cancer, melanoma or osteosarcoma.