CN102125696B - Oligomeric ribonucleic acid composite for inhibiting tumor growth and angiogenesis and application thereof - Google Patents
Oligomeric ribonucleic acid composite for inhibiting tumor growth and angiogenesis and application thereof Download PDFInfo
- Publication number
- CN102125696B CN102125696B CN2011100045827A CN201110004582A CN102125696B CN 102125696 B CN102125696 B CN 102125696B CN 2011100045827 A CN2011100045827 A CN 2011100045827A CN 201110004582 A CN201110004582 A CN 201110004582A CN 102125696 B CN102125696 B CN 102125696B
- Authority
- CN
- China
- Prior art keywords
- nucleic acid
- sirna
- sequence
- acid oligomer
- oligomeric
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Abstract
The invention discloses an oligomeric ribonucleic acid composite for inhibiting tumor growth and angiogenesis and application thereof. In the oligomeric ribonucleic acid composite, a main ingredient is double-stranded ribonucleic acid (RNA), wherein a nucleotide sequence in a first strand of the double-stranded RNA is shown by a sequence 1 in a sequence table, and a nucleotide sequence in a second strand of the double-stranded RNA and a reverse complementary strand of the first strand have homology of over 63.6 percent. In the oligomeric ribonucleic acid composite serving as a novel anti-tumor medicament of small interference RNA, all oligomeric ribonucleic acid ingredients in the composite are chemically-synthesized and modified oligomeric ribonucleic acid, so the composite has the characteristics that: the chemically-synthesized and modified oligomeric ribonucleic acid is difficult to degrade, has the long effect half-life period, and can be used for extracorporal experiments and intracorporal treatment. Compared with the medicaments with a single ingredient, the anti-tumor effect achieved by the combination of part of oligomeric ribonucleic acids is better.
Description
Technical field
The present invention relates to suppress tumor growth and vasculogenesis oligomeric ribonucleic acid composite and application thereof.
Background technology
Small nucleic acid (miRNA) is a class non-coding small nucleic acids, and wide expression, regulate important physiology and the pathologic process of body in vivo.The generation of small nucleic acid and tumour, development and prognosis are closely related.Research discovery in recent years, miRNA is at tumor tissues and obviously different from the expression between healthy tissues.MiRNA both can be used as the genesis that proto-oncogene promotes tumour, can suppress as cancer suppressor gene the genesis of tumour again, regulate the different mechanism of tumor development according to miRNA, they are formed different formulas or the antineoplastic siRNA of they and other (siRNA) is formed different formulas, will more can effectively treat neoplastic disease than single miRNA.
Summary of the invention
The object of the present invention is to provide a kind of nucleic acid oligomer.
Nucleic acid oligomer provided by the invention is a kind of double-stranded RNA, and wherein: the nucleotide sequence of article one chain of described double-stranded RNA is as shown in sequence in sequence table 1; The reverse complemental chain of the nucleotide sequence of second chain and described article one chain has the homology more than 63.6%.Sequence 1 direction from left to right is 5 '-3 '.
The nucleotide sequence of above-mentioned second chain can be as shown in sequence 2 or 3 in sequence table.Sequence 2 and 3 direction from left to right is 3 '-5 '.
Above-mentioned nucleic acid oligomer through as 1) or 2) modify the nucleic acid oligomer that obtains:
1) phosphodiester bond of the connection Nucleotide of described nucleic acid oligomer is modified, preferably the oxygen with described phosphodiester bond replaces with sulphur;
2) to the modification of 2 '-OH of the ribose of described nucleic acid oligomer, preferably described 2 '-OH is replaced with methoxyl group or fluorine or described 2 '-OH carried out deoxidation modify.
Within the DNA that gives expression to above-mentioned nucleic acid oligomer also belongs to protection scope of the present invention.
Another object of the present invention is to provide a kind of pharmaceutical composition that suppresses tumor growth.
The activeconstituents of the pharmaceutical composition of inhibition tumor growth provided by the invention is oligomeric ribonucleic acid composite, can be following a) or b) or c):
A) modify body by VEGF-siRNA or its and above-mentioned nucleic acid oligomer forms;
B) modify body by PTN-siRNA or its and above-mentioned nucleic acid oligomer forms;
C) modified by VEGF-siRNA or its that body, PTN-siRNA or its are modified body and above-mentioned nucleic acid oligomer forms;
Described VEGF-siRNA is double chain nucleotide, and the nucleotide sequence of article one chain of described VEGF-siRNA is as shown in sequence in sequence table 4, and the nucleotide sequence of the second chain of described VEGF-siRNA is as shown in sequence in sequence table 5; Sequence 4 direction from left to right is 5 '-3 ', and sequence 5 direction from left to right is 3 '-5 '.
Described PTN-siRNA is double chain nucleotide, and the nucleotide sequence of article one chain of described PTN-siRNA is as shown in sequence in sequence table 6, and the nucleotide sequence of the second chain of described PTN-siRNA is as shown in sequence in sequence table 7; Sequence 6 direction from left to right is 5 '-3 ', and sequence 7 direction from left to right is 3 '-5 '.
The modification body of described VEGF-siRNA is through following 1) or 2) modify the material obtain:
1) phosphodiester bond of the connection Nucleotide of described VEGF-siRNA is modified, preferably the oxygen with described phosphodiester bond replaces with sulphur;
2) to the modification of 2 '-OH of the ribose of described VEGF-siRNA, preferably described 2 '-OH is replaced with methoxyl group or fluorine or described 2 '-OH carried out deoxidation modify;
The modification body of described PTN-siRNA is through following 1) or 2) modify the material obtain:
1) phosphodiester bond of the connection Nucleotide of described PTN-siRNA is modified, preferably the oxygen with described phosphodiester bond replaces with sulphur;
2) to the modification of 2 '-OH of the ribose of described PTN-siRNA, preferably described 2 '-OH is replaced with methoxyl group or fluorine or described 2 '-OH carried out deoxidation modify.
Wherein, activeconstituents a) in, in claim 1-3, to modify the mass ratio of body be (0.5-2) for arbitrary described nucleic acid oligomer and described VEGF-siRNA or its: (0.5-2), preferably 1: 1;
Activeconstituents b) in, in claim 1-3, arbitrary described nucleic acid oligomer and described PTN-siRNA or its mass ratio of modifying body are (0.5-2): (0.5-2), and preferably 1: 1;
Activeconstituents c) in, in claim 1-3, arbitrary described nucleic acid oligomer and described VEGF-s iRNA or its mass ratio of modifying body, PTN-siRNA or its modification body are (0.5-2): (0.5-2): (0.5-2), and preferably 1: 1: 1.
Aforementioned pharmaceutical compositions comprises at least a and at least a pharmaceutically acceptable carrier in above-mentioned nucleic acid oligomer molecule." pharmaceutically acceptable carrier " used herein should with pharmaceutical composition of the present invention in double stranded rna molecule compatible.In a preferred embodiment, described " pharmaceutically acceptable carrier " refers to transfection reagent in body, as polymine (PEI), and jetPEI (L-PEI), liposome, Transferrins,iron complexes, folic acid etc.
Above-mentioned nucleic acid oligomer suppresses within the reagent of tumor growth or the application in the reagent of preparation inhibition tumor cell expression of cyclin D 1 also belong to protection scope of the present invention in preparation.
Further, mentioned reagent can be the pharmaceutical composition of above-mentioned inhibition tumor growth.
Specifically, above-mentioned tumour is intestinal cancer, nasopharyngeal carcinoma, cervical cancer, liver cancer, mammary cancer or lung cancer.
Described nucleic acid oligomer is chemosynthesis, can carry out 2 ' fluoro (2 '-F), 2 ' methoxyl group (2 '-OMe), sulfo-(PS) and 2 ' deoxidation (2 '-deoxy) chemically modified.
Experimental result with chemosynthesis and the above-mentioned nucleic acid oligomer molecule transfection human nasopharyngeal epithelioma 1 CNE that modifies, colon-cancer cell strain HCT116, breast carcinoma cell strain MCF7, the tumour cells such as cervical cancer cell strain Hela, hepatoma cell strain HepG2 shows that described nucleic acid oligomer miR-210 can effectively suppress the propagation of above-mentioned tumour cell.SiPTN also can suppress the propagation of above-mentioned tumour cell.SiVEGF and siPTN all can suppress tumor-blood-vessel growth.
Described nucleic acid oligomer miR-210 suppresses the expression of CCND1; SiVEGF suppresses the expression of VEGF, and siPTN suppresses the expression of PTN.By the combination of above-mentioned three kinds of different nucleic acid oligomer molecules, realize suppressing the growth of tumour and the effect of vasculogenesis.
Tumor cell inoculation in nude mice, is formed the described oligomeric nucleic acid combination that experimental results show that for the treatment of with described nucleic acid oligomer after tumour and can effectively suppress in vivo the growth of tumour.
As the antitumor drug of novel small nut acids, chemosynthesis and the described nucleic acid oligomer of modifying are difficult for being degraded, and the long effect transformation period is arranged, and can be used for experiment in vitro, more can be used for interior therapeutic.
Description of drawings
Figure 1A be mtt assay detect 2 '-OMe-miR-210 suppresses the column diagram of nasopharyngeal carcinoma CNE cell proliferation, Figure 1B be mtt assay detect 2 '-OMe-siPTN suppresses the column diagram of Hela cell proliferation.
Fig. 2 be RT-PCR detect 2 '-expression of OMe-miR-210** inhibition tumor cell CCND1.
Fig. 3 be RT-PCR detect 2 '-expression of OMe-siVEGF inhibition tumor cell VEGF.
Fig. 4 be RT-PCR detect 2 '-PTN of OMe-siPTN inhibition tumor cell expresses, swimming lane 1-4 is respectively the cell of untransfected, the negative control of transfection stochastic sequence (NC), transfection 2 '-OMe-siPTN24 hour, transfection 2 '-OMe-siPTN 48 hours.
Fig. 5 is 2 '-5 later knurl volume broken line graphs of OMe oligomeric nucleic acid combination treatment nasopharyngeal carcinoma transplanted tumor.
Fig. 6 is 2 '-the OMe oligomeric nucleic acid combination adds the heavy column diagrams of 6 later knurls for the treatment of nasopharyngeal carcinoma transplanted tumor.
Fig. 7 is 2 '-OMe oligomeric nucleic acid combination treatment nasopharyngeal carcinoma transplanted tumor 6 times after tumor effect figure.
Embodiment
The invention will be further described below in conjunction with specific embodiment, but the present invention is not limited to following examples.
In following embodiment, if no special instructions, be ordinary method.
The preparation of embodiment 1, nucleic acid oligomer
The sequence of nucleic acid oligomer described in the present invention positive-sense strand is: 5 '-CUGUGCGUGUGACAGCGGCUGA-3 ' (SEQID No.1); Antisense strand is 3 '-UUGACACGCACACUGUCGCCGA-5 ' sequence (SEQ ID No.2) or 3 '-CACACGCCACCCGUCCCCGACG-5 ' (SEQ ID No.3).In the present invention, the VEGF-siRNA positive-sense strand is 5 '-GGAGUACCCUGAUGAGAUCTT-3 ' (SEQ ID No.4); Antisense strand is 3 '-TTCCUCAUGGGACUACUCUAG-5 ' (SEQ ID No.5); The PTN-siRNA positive-sense strand is 5 '-GCCCAAACCUCAAGCAGAATT-3 ' (SEQ ID No.6); Antisense strand is 3 '-TTCGGGUUUGGAGUUCGUCUU-5 ' (SEQ ID No.7); Stochastic sequence described in the present invention is as negative control (NC), the positive-sense strand sequence of NC is: 5 '-UUCUCCGAACGUGUCACGUTT-3 ' (SEQ ID No.8), the antisense strand sequence is: 3 '-TTAAGAGGCUUGCACAGUGCA-5 ' (SEQ ID No.9).A, G, C, U in described nucleic acid oligomer and NC sequence represents Adenosine acid, guanosint sugar nucleotide, cytosine(Cyt) ribonucleotide and uracil ribonucleotide, and T represents thymidylic acid.
Described nucleic acid oligomer and NC entrust Shanghai lucky agate (GenePharma) Pharmaceutical Technology Inc. to synthesize and carry out following any one chemically modified: 2 ' methoxyl group (2 '-OMe), 2 ' fluoro (2 '-F), sulfo-and 2 '-deoxidation (2 '-deoxy), then obtain the nucleic acid oligomer powder through frozen dried, be used for the experiment of embodiment 2-4.
5 kinds of nucleic acid oligomers in embodiment 1 are respectively: the nucleic acid oligomer negative control (called after NC) that nucleic acid oligomer (called after PTN-siRNA), sequence 8 and the sequence 9 that nucleic acid oligomer (called after VEGF-siRNA), sequence 6 and the sequence 7 that nucleic acid oligomer (called after miR-210**), sequence 4 and the sequence 5 that the nucleic acid oligomer that sequence 1 and sequence 2 are paired into (called after miR-210), sequence 1 and sequence 3 are paired into is paired into is paired into is paired into.
Wherein: miR-210 after 2 ' methoxyl group is modified called after 2 '-OMe-miR-210; MiR-210** after above-mentioned 2 ' methoxyl group is modified called after 2 '-OMe-miR-210**; VEGF-siRNA after 2 ' methoxyl group is modified called after 2 '-OMe-siVEGF, PTN-siRNA after 2 ' methoxyl group is modified called after 2 '-OMe-siPTN.NC after 2 ' methoxyl group is modified called after 2 '-OMe-NC.
Above-mentioned synthetic method be derived from one piece nineteen ninety-five disclosed document: Wincott F, DiRenzo A, Shaffer C, GrimmS, Tracz D, Workman C, Sweedler D, Gonzalez C, Scaringe S and Usman N.Synthesis, deprotection, analysis and purification of RNA and ribozymes.Nucleic Acids Res.1995,23:2677-84.Whole chemosynthesis can roughly be divided into process (1) oligomerization Yeast Nucleic Acid synthetic of four; (2) deprotection; (3) purifies and separates; (4) the aseptic sterilization of desalination annealing.
(1) the oligomerization ribonucleotide is synthetic
Automated DNA/the RNA synthesizer (for example, Applied Biosystems EXPEDITE8909) the upper RNA that sets synthetic 1 mmole, the coupling time of setting simultaneously each circulation is 10-15 minute, initiator is that 5 '-O-of connecting of solid phase is to dimethoxy-thymidine upholder, first circulates in and connects a base on solid support, then in the n time (19 〉=n 〉=2) circulation, connect a base on the base that the n-1 time circulation connects, repeat this circulation until complete the synthetic of whole nucleotide sequences.
(2) deprotection
The solid support that is connected with RNA is joined in test tube, and add the ethanol/ethamine (volume ratio is 1: 3) of 1 milliliter in this test tube, then sealing, be placed in 55-70 ℃ of incubator, hatched 2-30 hour, fech connection has the solid support of siRNA and with distilled water drip washing 2 times (each 1 milliliter), collects elutriant, and at room temperature dry 30 minutes.Then, add the tetrahydrofuran solution (1M) of 1 milliliter of tetrabutyl ammonium fluoride, room temperature was placed 4-12 hour, then added 2 milliliters of ethanol, and collecting precipitation namely obtains the crude product of little RNA.
(3) purifies and separates
It is in the ammonium acetate solution of 1 mole/milliliter that the crude product of the RNA that obtains is dissolved in 2 ml concns, then separates by the C18 high pressure liquid chromatography, obtains the RNA product of purifying.
(4) desalination annealing
Be RNA product 2-4 time (each 2 milliliters) of the aqueous ethanolic solution washing purifying of 75 % by weight with concentration, removing salt, and drying under room temperature.Then with oligomerization Yeast Nucleic Acid mixed dissolution (10mM Tris in the damping fluid of 1-2 milliliter of positive-sense strand and antisense strand, pH=7.5-8.0,50mM NaCl), this solution is heated to 95 ℃, then slowly this solution is cooled to room temperature, and kept room temperature 16-22 hour, obtain containing the solution of double-stranded Microrna.
Experimental procedure is: with embodiment 1 obtain 2 '-OMe-miR-210 (being called for short miR-210 in figure) and 2 '-OMe-siPTN (being called for short siPTN in figure) and negative control 2 '-OMe-NC (being called for short NC in figure) powder is dissolved in respectively in sterilized water without the RNA enzyme, the nucleic acid oligomer solution that to be made into 3 kinds of final concentrations be 20pmol/L.Collect logarithmic phase nasopharyngeal carcinoma CNE cell (available from the Shanghai cell bank, for 2 '-OMe-miR-210 and 2 '-OMe-NC transfection) or the Hela cell (available from the Shanghai cell bank, confession 2 '-OMe-siPTN and 2 '-OMe-NC transfection), adjust concentration of cell suspension, divide and plant in 96 orifice plates, every hole 150 μ l are totally 5000 cells; Be placed in 37 ℃, contain 5%CO
2Incubator in make cell attachment, cultivated 6-24 hour.during transfection, respectively with 0.40 μ l nucleic acid oligomer solution (above-mentioned 2 '-F nucleic acid oligomer or 2 '-F nucleic acid oligomer negative control) and the lipofectamine 2000 (Invitrogen) of 0.20 μ l be diluted in respectively in 20 μ l serum-free culture nutrient solutions (Opti-MEM), and at room temperature hatched 5 minutes, then above-mentioned nucleic acid oligomer solution is mixed with lipofectamine 2000 solution, mixture is in room temperature after standing 20 minutes, the 10%FBS-DMEM of 60ul is added in nucleic acid oligomer-liposome complex of 40 μ l, be added to again in Tissue Culture Plate (the Growth of Cells fusion rate is 50-70%) transfection 6-8 hour.For making cells Synchronous, with cell serum starvation 12 hours, continue to cultivate 72 hours with being changed to again the 2.5%FBS-DMEM substratum afterwards after transfection; Carefully suck supernatant 120ul, making in the hole fertility nutrient solution is 80 μ l, then adds 20 μ lMTT (5mg/ml), and namely 0.5% MTT continues to cultivate 4 hours; Suck the dimethyl sulfoxide (DMSO) DMSO that supernatant adds 100 μ l, the light absorption value in each hole of measurement, enzyme-linked immunosorbent assay instrument 492nm place; Arrange simultaneously and transfer 0 hole (substratum, MTT, dimethyl sulfoxide (DMSO)), every group arranges 3 multiple holes; Result as shown in Figure 1, Figure 1A show with transfection 2 '-OMe-NC relatively, transfection 2 '-the cell OD value of OMe-miR-210 (Figure 1A) or 2 '-OMe-siPTN (Figure 1B) obviously reduces, illustrate 2 '-propagation that OMe-miR-210 or 2 '-OMe-siPTN can obvious inhibition tumor cell.
In the present embodiment, miR-210 through 2 ' fluoro (2 '-F), sulfo-(PS) and 2 ' deoxidation modify after on the impact of tumor cell proliferation with through 2 ' methoxyl group (2 '-OMe) the effect there was no significant difference after modification.
The RT-PCR that embodiment 3, nucleic acid oligomer suppress its expression of target gene detects
Use the l ipofectamine of Invi trogen
TM2000 liposomes carry out transfection, the working specification that all operations all provides by Invitrogen.CNE or Hela cell are inoculated into (5X10 in 6 orifice plates
5Individual cells/well), support liquid (available from GIBCO) cultivation after 16-24 hour with the DMEM that contains 10% foetal calf serum, cell culture fluid is changed into the DMEM nutrient solution that contains antibiotic-free.With the nucleic acid oligomer powder (2 '-OMe-miR-210**, 2 '-OMe-siPTN, 2 '-OMe-siVEGF and 2 '-OMe-NC) is dissolved in respectively in sterilized water without the RNA enzyme, being made into final concentration is the nucleic acid oligomer solution of 20pmol/L.
Wherein, 2 '-OMe-miR-210** and 2 '-OMe-NC transfection CNE cell; 2 '-OMe-siPTN and 2 '-OMe-NC transfection Hela cell; 2 '-OMe-siVEGF and 2 '-OMe-NC transfection CNE cell.
during transfection, getting respectively the above-mentioned oligonucleotide solution of 10 μ l and the lipofectamine 2000 of 5 μ l is diluted in respectively in 250 μ l serum-free culture nutrient solutions (Opti-MEM), and at room temperature hatched 5 minutes, then above-mentioned nucleic acid oligomer solution is mixed with lipofectamine 2000 solution, mixture is in room temperature after standing 20 minutes, nucleic acid oligomer-liposome complex of 500 μ l with after 1.5ml serum-free DMEM nutrient solution mixes, be added in Tissue Culture Plate, be changed to the 10%FBS-DMEM substratum after 6 hours, respectively at 24, 48 collecting cells carry out RT-PCR and detect.
The step that RT-PCR detects is: use CNE or the Hela cell of 1ml TRIzol (invitrogen) the described nucleic acid oligomer of cracking transfection or nucleic acid oligomer negative control, and press the total RNA of working specification extraction of the TRIzol description of product.Because the effect that RNA disturbs may be by potential a small amount of genomic dna impact, experiment adopts DNase I (RNase-free) (TaKaRa) further to digest residual DNA in total RNA that degrades.With 37 ℃ of digestion of DNase I after 30 minutes, according to again purify total RNA and RNA is carried out quantitatively and quality evalution of the DNase I description of product.Reverse transcription reaction is the M-MLV reversed transcriptive enzyme system of using TaKaRa company, and total RNA of 1ug is mixed with the Oligo dT of 0.5ug, heats 5 minutes, is cooled to rapidly 0 ℃.Add damping fluid by the description of product, hatched 1 hour for 42 ℃.CDNA after reverse transcription reaction, template detection target gene as the PCR reaction, transfection 2 '-expression of the pattern detection CCND1 of OMe-miR-210**, upstream primer is 5 '-GCTGGAGCCCGTGAAAAAGA-3 ', downstream primer is 5 '-CTCCGCCTCTGGCATTTTG-3 '.Transfection 2 '-the pattern detection vegf expression upstream primer of OMe-siVEGF is 5 '-GAGGGCAGAATCATCACGAA-3 '; Downstream primer is 5 '-GGGAACGCTCCAGGACTTAT-3 '.Transfection 2 '-the pattern detection PTN of OMe-siPTN expresses, and upstream primer is 5 '-CACGGGAGGGCACTCGGACT-3 ', and downstream primer is 5 '-GTCTTCTGGCATTCGGCATTG-3 '.The PCR reaction system is that 94 spend 5 minutes, 94 degree 1 minute, 54 degree 30 seconds, 72 degree 20 seconds, totally 28 circulations.The PCR product is carried out agarose gel electrophoresis.
Result is as shown in Fig. 2-4, with the negative control group of transfection nucleic acid oligomer (2 '-OMe-NC, be called for short NC in figure) relatively, transfection 2 '-the obviously CCND1 expression (Fig. 2) of inhibition tumor cell of OMe-miR-210** (being called for short miR-210** in figure), transfection 2 '-the obviously expression (Fig. 3) of inhibition tumor cell VEGF of OMe-siVEGF (being called for short siVEGF in figure), and transfection 2 '-PTN of the obvious inhibition tumor cell of OMe-siPTN expresses (Fig. 4).In Fig. 2 and Fig. 3, upper figure is electrophorogram, and figure below is that gel image analysis software changes into electrophoresis band the histogram of relative expression quantity.
In the present embodiment, miR-210** through 2 ' fluoro (2 '-F), sulfo-(PS) and 2 ' deoxidation modify after on the impact of the transformation period test result of tumor cells expression CCND1 with through 2 ' methoxyl group (2 '-OMe) affect there was no significant difference after modification.
The restraining effect of embodiment 4,2 '-OMe nucleic acid oligomer to tumor growth
Cultivate nasopharyngeal carcinoma CNE cell strain with the DMEM nutrient solution that contains 10% foetal calf serum, the cell in the vegetative period of taking the logarithm is made single cell suspension, regulates cell concn 5 * 10
7Individual cell/ml.At the dorsal part alar part subcutaneous injection 0.1ml tumour cell suspension of male BALB/C nude mice in 5 ages in week, make it form transplanted tumor.nude mice is divided into eight groups, be respectively 2 '-OMe-NC (being called for short the NC group), 2 '-OMe-miR-210 (being called for short the miR-210 group), 2 '-OMe-miR-210 adds 2 '-OMe-siVEGFA (being called for short the miR-210+siVEGF group), 2 '-OMe-siVEGF (being called for short the siVEGF group), 2 '-OMe-siVEGF+2 '-OMe-siPTN (being called for short the siVEGF+siPTN group), 2 '-OMe-miR-210+2 '-OMe-siPTR (being called for short the miR-210+siPTN group), 2 '-OMe-miR-210+2 '-OMe-siVEGF+2 '-OMe-siPTN (being called for short the miR-210+siVEGF+siPTN group), 5 fluoro-uracil (being called for short 5 ' FU group).The first day that the inoculated tumour cell is later and the 3rd day are carried out respectively tail vein injection 1 time.Reach 50mm to gross tumor volume
3After change intratumor injection into.
The compound method of the nucleic acid oligomer preparation that the tail vein injection of each group is used is: single nucleic acid oligomer group (NC group, miR-210 group and siVEGF group) is to dissolve respectively every group of corresponding nucleic acid oligomer of 100 μ g with the sterilized water 165 μ l that do not contain the RNA enzyme; Duoupoly polynucleic acid group (miR-210+siVEGF group, siVEGF+siPTN group and miR-210+siPTN group) is mixing (cubic capacity 165ul) after two kind of 50 μ g nucleic acid oligomer in dissolving respectively every group with two parts of sterilized water 87.5 μ l that do not contain the RNA enzyme; Three nucleic acid oligomer groups (miR-210+siVEGF+siPTN group) are to mix (cubic capacity 165ul) after dissolving respectively the nucleic acid oligomer of three kind of 33.3333 μ g with three parts of sterilized water 55 μ l that do not contain the RNA enzyme.
Then each group adds transfection agents jetPEI 10 μ l (available from Polyplus company) again, and incubated at room mixed with equal-volume 10% glucose solution after 15 minutes, and cumulative volume 350ul is in incubated at room laggard end of line intravenous injection in 15 minutes.The oligonucleotide consumption of a shot is 100 μ g/.5 fluoro-uracil usages are: 25ng/kg/ time, and abdominal injection.
The compound method of the nucleic acid oligomer preparation that intratumor injection is used is with 2 of the sterilized water 11.3 μ l dissolving 20 μ g that do not contain the RNA enzyme '-OMe-nucleic acid oligomer.Single nucleic acid oligomer group is got the nucleic acid oligomer that 11.3 μ l do not contain the sterilized water dissolving 20 μ g of RNA enzyme, mix (cubic capacity 11.3ul) after two kind of 10 μ g nucleic acid oligomer in duoupoly polynucleic acid group is dissolved respectively every group with two parts of sterilized water 5.65 μ l that do not contain the RNA enzyme, three nucleic acid oligomer groups are dissolved respectively mixing (cubic capacity 11.3 μ l) after three kind of 6.667 μ g nucleic acid oligomer with three parts of sterilized water 3.77 μ l that do not contain the RNA enzyme.The consumption of transfection reagent is 1.2 μ l, adds equal-volume 10% glucose solution to mix, cumulative volume 25ul intratumor injection.The oligonucleotide consumption of a shot is 20 μ g/ points.Every injection in 2 days once, intratumor injection totally 5 times.5 fluoro-uracil usages are: 25ng/kg/ time, and abdominal injection.Inoculate rear 3 days and use every three days the major diameter (L) of vernier caliper measurement tumour and transverse diameter (S), calculate the approximate volumes of tumour.Calculation formula is: V (mm
3)=0.5 * L * S
2Intratumor injection finishes experiment 5 times afterwards, puts to death nude mice, takes out tumour and weighs.
Figure 5 shows that the broken line graph of the gross tumor volume contrast of the treatment group of different oligomeric nucleic acid combinations and control group after treatment, the increase for the treatment of group gross tumor volume is obviously little than control group, and the control group gross tumor volume is obvious ascendant trend.The tumor killing effect of oligomeric nucleic acid combination miR-210+s iVEGF is best.
Fig. 6 shows after treatment on nasopharynx transplantation tumor nude mice model, the knurl for the treatment of group nude mice is heavy obviously organize than contrast NC little, the inhibitory rate to 62.41% of oligomeric nucleic acid combination miR-210+siVEGF.
Fig. 7 is after oncotherapy, the oncotherapy design sketch for the treatment of group and control group.
In the present embodiment, miR-210 through 2 ' fluoro (2 '-F), sulfo-(PS) and 2 ' deoxidation modify after on the restraining effect of tumor growth with through 2 ' methoxyl group (2 '-OMe) affect there was no significant difference after modification.
Mice serum AST detects data to table 1 for experiment finishes later on.Detect nude mice aspartate amino transferase (AST) according to aspartate amino transferase test kit (available from the middle north control Biological Co., Ltd. of giving birth to) specification sheets operation.10ml/ bottle R2 (liquid) in test kit is joined in R1 (powder) bottle, be working fluid after dissolving, be placed in room temperature standing.Get fresh nude mice without haemolysis serum 20ul/ hole, join in 96 orifice plates, the working fluid 200ul/ hole for preparing is also joined in 96 orifice plates, mix.Set simultaneously 4 blank holes.Temperature of reaction insulation 1 minute, read initial absorbance under the 340nm wavelength, begin simultaneously timing, accurate 1, in the time of 2,3 minutes, read respectively absorbancy, determine that the per minute mean light absorbency changes, the formula that provides to specifications calculates, and obtains the numerical value of AST.The result demonstration raises without significance with the ALT numerical value that nonuser PBS group compares treatment group, and significantly hepatotoxicity of described nucleic acid oligomer treatment nothing is described.
Table 1 detects data sheet to clear AST
| Group | Number of cases (N) | AST (U/L) | Standard error | The |
| PBS | ||||
| 6 | 38.5 | 3.2 | >0.05 | |
| |
6 | 32.6 | 7.2 | >0.05 |
| 5 ' |
6 | 38.2 | 5.5 | >0.05 |
| MiR-210+siPTN+ |
6 | 32.7 | 4.7 | >0.05 |
| Si PTN+ |
6 | 39.3 | 2.7 | >0.05 |
| MiR-210 |
6 | 38.9 | 9.3 | >0.05 |
| SiVEGF | 6 | 34.2 | 4.9 | >0.05 |
| MiR-210 | 6 | 31.0 | 7.3 | >0.05 |
| MiR-210 |
6 | 42.4 | 4.6 | >0.05 |
Claims (4)
1. composition that suppresses tumor growth, its activeconstituents be following a) or b) or c):
A) modify body by VEGF-siRNA or its and nucleic acid oligomer forms; Described VEGF-siRNA or its mass ratio of modifying body and described nucleic acid oligomer are 1:1;
B) modify body by PTN-siRNA or its and nucleic acid oligomer forms; Described PTN-siRNA or its mass ratio of modifying body and described nucleic acid oligomer are 1:1;
C) modify body, PTN-siRNA by VEGF-siRNA or its or it modifies body and nucleic acid oligomer forms; Described VEGF-siRNA or its mass ratio of modifying body, described PTN-siRNA or its modification body and described nucleic acid oligomer are 1:1:1;
Described VEGF-siRNA is double chain nucleotide, and the nucleotide sequence of its article one chain is as shown in sequence in sequence table 4, and the nucleotide sequence of second chain is as shown in sequence in sequence table 5;
Described PTN-siRNA is double chain nucleotide, and the nucleotide sequence of its article one chain is as shown in sequence in sequence table 6, and the nucleotide sequence of second chain is as shown in sequence in sequence table 7;
The modification body of described VEGF-siRNA is through following 1) or 2) modify the material obtain:
1) phosphodiester bond of the connection Nucleotide of described VEGF-siRNA is modified;
2) to the modification of 2 '-OH of the ribose of described VEGF-siRNA;
The modification body of described PTN-siRNA is through following 1) or 2) modify the material obtain:
1) phosphodiester bond of the connection Nucleotide of described PTN-siRNA is modified;
2) to the modification of 2 '-OH of the ribose of described PTN-siRNA;
Described nucleic acid oligomer is double-stranded RNA, and wherein, the nucleotide sequence of article one chain is as shown in sequence in sequence table 1, and the nucleotide sequence of second chain is as shown in sequence in sequence table 2.
2. composition as claimed in claim 1 is characterized in that: described nucleic acid oligomer is through following 1) or 2) modify the nucleic acid oligomer that obtains:
1) phosphodiester bond of the connection Nucleotide of described nucleic acid oligomer is modified;
2) to the modification of 2 '-OH of the ribose of described nucleic acid oligomer.
3. composition as claimed in claim 1 or 2, it is characterized in that: the described oxygen that phosphodiester bond is modified to phosphodiester bond replaces with sulphur;
Described being modified to of 2 ' of ribose-OH replaced described 2 '-OH or described 2 '-OH carried out deoxidation modify with methoxyl group or fluorine.
4. composition as claimed in claim 1 or 2, it is characterized in that: described tumour is intestinal cancer, nasopharyngeal carcinoma, cervical cancer, liver cancer, mammary cancer or lung cancer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100045827A CN102125696B (en) | 2011-01-11 | 2011-01-11 | Oligomeric ribonucleic acid composite for inhibiting tumor growth and angiogenesis and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100045827A CN102125696B (en) | 2011-01-11 | 2011-01-11 | Oligomeric ribonucleic acid composite for inhibiting tumor growth and angiogenesis and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102125696A CN102125696A (en) | 2011-07-20 |
| CN102125696B true CN102125696B (en) | 2013-06-12 |
Family
ID=44264127
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011100045827A Active CN102125696B (en) | 2011-01-11 | 2011-01-11 | Oligomeric ribonucleic acid composite for inhibiting tumor growth and angiogenesis and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102125696B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102178959B (en) * | 2011-03-15 | 2013-10-23 | 清华大学深圳研究生院 | siRNA for inhibiting growth of pulmonary metastasis tumor and oligonucleotide composition and application thereof |
| US9328348B2 (en) * | 2011-10-19 | 2016-05-03 | Suzhou Ribo Life Science Co., Ltd. | Small interference RNAs, uses thereof and method for inhibiting the expression of plk1 gene |
| CN102895672B (en) * | 2012-02-24 | 2013-11-20 | 中国科学院北京基因组研究所 | Composition for treating cancers and use thereof |
| CN114369130B (en) * | 2021-12-28 | 2023-10-03 | 杭州天龙药业有限公司 | Modified thio-oligonucleotides and their use |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1800387A (en) * | 2005-12-19 | 2006-07-12 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Small interfering RNA for suppressing multiple effect growth factor expression and its uses |
| CN1804038A (en) * | 2005-12-12 | 2006-07-19 | 清华大学深圳研究生院 | VEGF expression-inhibiting siRNA and application thereof |
| CN101831427A (en) * | 2010-04-01 | 2010-09-15 | 清华大学深圳研究生院 | Oligodeoxyribonudeotide for preventing recurrence of tumor and inhibiting tumor growth as well as application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009058907A2 (en) * | 2007-10-29 | 2009-05-07 | Isis Pharmaceuticals, Inc. | Targeting micrornas for the treatment of liver cancer |
-
2011
- 2011-01-11 CN CN2011100045827A patent/CN102125696B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1804038A (en) * | 2005-12-12 | 2006-07-19 | 清华大学深圳研究生院 | VEGF expression-inhibiting siRNA and application thereof |
| CN1800387A (en) * | 2005-12-19 | 2006-07-12 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Small interfering RNA for suppressing multiple effect growth factor expression and its uses |
| CN101831427A (en) * | 2010-04-01 | 2010-09-15 | 清华大学深圳研究生院 | Oligodeoxyribonudeotide for preventing recurrence of tumor and inhibiting tumor growth as well as application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 胡迎春等.siRNA沉默多效生长因子表达及其生物学意义.《癌症》.2006,第25卷(第10期),第1210-1215页. * |
| 陈善义等.VEGF特异的2′-脱氧修饰siRNA能够有效的抑制VEGF的表达和小鼠鼻咽癌异体移植瘤的生长.《中国科学(C辑:生命科学)》.2008,第38卷(第01期),第16-22页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102125696A (en) | 2011-07-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102575254B (en) | Methods and compositions for the specific inhibition of KRAS by asymmetric double-stranded RNA | |
| CN102325534B (en) | The DICER enzyme substrates extended and the method for specific inhibition of gene expression | |
| Ho et al. | Bioengineering of noncoding RNAs for research agents and therapeutics | |
| US10287582B2 (en) | Organic compositions to treat HSF1-related diseases | |
| JP2020188771A (en) | METHODS AND COMPOSITIONS FOR SPECIFIC INHIBITION OF α-1 ANTITRYPSIN BY DOUBLE-STRANDED RNA | |
| CN101980712A (en) | Targeting microRNAs for the treatment of liver cancer | |
| CN102125696B (en) | Oligomeric ribonucleic acid composite for inhibiting tumor growth and angiogenesis and application thereof | |
| CN113249380B (en) | Antisense oligonucleotide targeting COVID-19 novel coronavirus, NATAC chimeric molecule and application thereof | |
| CN102145181B (en) | siRNA for inhibiting tumor growth and oligomeric nucleic acid combination and application of siRNA | |
| CN106591308A (en) | Human pulmonary carcinoma erlotinib drug tolerance improving shRNA | |
| CN105861551A (en) | Vector for jointly expressing MicroRNAs to inhibit breast cancer cell proliferation and construction method and application thereof | |
| CN101831427B (en) | Oligodeoxyribonudeotide for preventing recurrence of tumor and inhibiting tumor growth as well as application thereof | |
| CN102140471B (en) | Oligo-nucleic acid for suppressing tumor growth and application thereof | |
| CN102178959B (en) | siRNA for inhibiting growth of pulmonary metastasis tumor and oligonucleotide composition and application thereof | |
| CN109097363B (en) | Biological recombinant miR34a-5p capable of effectively inhibiting growth of osteosarcoma | |
| CN105586391A (en) | Application of human GTPBP4 gene and related drugs of human GTPBP4 gene | |
| CN104147616B (en) | The application of double-stranded RNA or its trim in tumor inhibitor is prepared | |
| CN114796525B (en) | Use of inhibitors of cyclin-mediated proteins in tumor therapy | |
| CN100371444C (en) | VEGF expression-inhibiting siRNA and application thereof | |
| CN110179991A (en) | A kind of pro-drug with anti-tumor activity and preparation and application | |
| CN109022442B (en) | Biological recombinant miR124-3p capable of effectively inhibiting growth of osteosarcoma | |
| CN107709561A (en) | The siRNA of modification and siRNA containing modification pharmaceutical composition | |
| CN101270358A (en) | MiRNA sequence and uses thereof | |
| CN111996193A (en) | siRNA sequence for effectively inhibiting expression of epidermal growth factor receptor | |
| CN104611333A (en) | SiRNA for reducing human POKEMON gene expression and related application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |