CN104561017A - MM (multiple myeloma)-resistant small-interference RNA (ribonucleic acid) and application thereof - Google Patents
MM (multiple myeloma)-resistant small-interference RNA (ribonucleic acid) and application thereof Download PDFInfo
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Abstract
The invention relates to the field of biotechnology and medicine, and discloses an MM (multiple myeloma)-resistant small-interference RNA (ribonucleic acid) and an application thereof. The nucleotide sequence of a target polynucleotide is shown in any one of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 in a sequence table. Excessive SUMO (small ubiquitin-related modifier) modification is related to poor prognosis of MM, deep research on effects caused by SUMO modification Wnt/beta-catenin on MM cell proliferation and apoptosis as well as molecular mechanisms is beneficial to discovery of the source of the progress of the myeloma, and prognosis and regulatory mechanisms of the MM are further clarified; new therapeutic targets are developed for the key of recurrence of the MM. The provided target DNA has very important practical significance in realizing safer and more effective anti-myeloma treatment and curing the MM finally.
Description
Technical field
The present invention relates to biotechnology and medical field, be specifically related to a kind of small interference ribonucleic acid and application thereof of anti-multiple myeloma.
Background technology
Multiple myeloma (Multiple myeloma, MM) is that sickness rate is in the deputy malignant tumour of blood system, still cannot cure at present, and the life of serious threat its people is with healthy.Conventional chemotherapy and even new drug, while reduction tumor load, produce little effect to the myelomatosis stem cell maintaining self, are considered to MM and are finally in progress the key of recurrence.Therefore, study further in the urgent need to the source of recurring for MM, develop new safer and more effective targeted therapy, really improve existence and the prognosis of MM patient.
Wnt gene belongs to proto-oncogene, and being the key that kinds of tumors occurs, is also the important conditioning signal in tumor stem cell growth course.Our early stage research display: 1) Wnt signal transduction pathway high level activation in myeloma cell is relevant to the propagation of myeloma cell and resistance; 2) the crucial effect protein β-catenin in classical Wnt approach is degraded by ubiquitin-proteasome pathway, reducing β-catenin level can suppress myeloma cell to breed, promote its apoptosis, the activation of prompting Wnt/ β-catenin approach is very important to the progress of MM, further investigate further for this approach, be expected to find the key avoiding MM to recur.
In theory, the ubiquitination degraded of β-catenin, also should be subject to pining down and adjustment of other effects; In fact, research also has been found that: as the another large protein posttranslational modification system after phosphorylation, ubiquitination, intracellular small ubiquitin-related modifier proteins SUMO (small ubiquitin-relatedmodifier) is by competing the ubiquitination binding site of some substrate proteins, stop its proteasome pathway to be degraded, affect downstream signal transduction.A large amount of albumen is proved the adjustment being simultaneously subject to ubiquitination and SUMOization modification, comprises some signal of interest pathway protein, as IKB, Smad4, Huntingtin etc. avoid ubiquitination to degrade by SUMOization modification, keeps homeostasis, participates in tumour and occurs.The research that SUMOization is modified in myelomatosis is few, only there is one section of Blood latest report display: the myeloma cell line of all detections and primary cell all high expression level SUMOization modify necessary key enzyme, its high expression level degree is relevant to prognosis mala, prompting SUMOization modifies the progress that excessive activation promotes MM, but mechanism is unclear.
At present, modify whether can affect myeloma cell's biologic activity by the SUMOization of interference Wnt/ β-catenin approach, and then affect clinical efficacy and the prognosis of MM, do not have report both at home and abroad.That another large protein modifies system after ubiquitination, can the ubiquitination degraded of antagonism substrate protein.Early stage research shows: Wnt pathway activation promotes multiple myeloma (MM) cell proliferation resistance; Its crucial effect protein β-catenin is degraded by Ubiquitin-proteasome systerm.Preliminary experiment confirms again further: SUMO directly can modify and regulate β-catenin protein level.It is relevant to MM prognosis mala that current known SUMOization modifies excessive activation, but concrete mechanism is unclear.
SUMO-1 gene, No. GENBANK 7341 (see network address: http://www.ncbi.nlm.nih.gov/gene/7341), does not still have SUMO-1 gene high expression at present or suppresses the bibliographical information that the expression of SUMO-1 gene is relevant to multiple myeloma.
Summary of the invention
The object of the present invention is to provide a kind of small interference ribonucleic acid of anti-multiple myeloma, another object of the present invention is to provide the application of this small interference ribonucleic acid in preparation prevention or treatment multiple myeloma medicine.
The technical problem to be solved in the present invention is: for the current problem lacking effectively treatment molecular target in the treatment of multiple myeloma, a kind of effective solution is provided, by a kind of target polynucleotide of small interference ribonucleic acid of anti-multiple myeloma, by described target polynucleotide in the application prepared or screen in anti-multiple myeloma medicine.
The present inventor finds that SUMOization modification is degraded from ubiquitination by protection β-catenin, promotes Wnt pathway activation, finally causes the key signal path that multiple myeloma is in progress.Contriver in vitro with MM clone and primary cell for research object, disturbed by siRNA, Wnt pathway activities and the change of MM cell proliferation apoptosis ratio under more different SUMO level; Then regulate β-catenin protein expression, observe its impact on SUMOization modification, specify that SUMOization is modified affects myeloma cell's Proliferation and apoptosis activity by Wnt/ β-catenin approach.This invention exploits the therapeutic targets of new myelomatosis, significantly can improve the prognosis of MM patient.Contriver, by studying for following several respects, finally completes the present invention:
(1) disturb multiple myeloma cells SUMO-1 to express by siRNA, detect Wnt approach downstream molecules and express and the change of myeloma cell's Proliferation and apoptosis ratio; (2) specify that the expression of SUMO-1 gene in interference multiple myeloma cells is to the interact relation of β-catenin expression level; (3) build also transfection β-catenin plasmid, transfection SUMO-1 interference group cell, under observing the suppressed situation of SUMO-1, improve β-catenin expression level to the impact of multiple myeloma cells Proliferation and apoptosis; (4) have detected the inherence effect in multiple myeloma cell line between SUMO-1 gene and β-catenin albumen.
By analyzing experimental results, contriver surprisingly finds: it is the target spot promoting that Wnt activates that the SUMOization of (1) β-catenin albumen is modified; (2) modify the propagation that can reduce multiple myeloma cells by the SUMOization of Targeted-control Wnt/ β-catenin approach, and promote apoptosis of tumor cells, significantly can reduce the rate of propagation of multiple myeloma cells, finally complete the present invention.
Therefore, for solving the problems of the technologies described above, one of technical scheme that the present invention takes is: a kind of target polynucleotide of small interference ribonucleic acid of anti-multiple myeloma, described target polynucleotide is the polynucleotide of arbitrary shown sequence in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 in sequence table.
Target polynucleotide of the present invention, the polynucleotide be preferably made up of continuous 10 ~ 30 Nucleotide on SUMO-1 coding sequence are more preferably the polynucleotide that 15 ~ 27 Nucleotide are formed; The polynucleotide of arbitrary shown sequence in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 in sequence table best.
Target polynucleotide of the present invention also comprises the polynucleotide of hybridizing under stringent hybridisation conditions with described target polynucleotide, or with the polynucleotide of its complementation.Wherein said hybridization conditions is classified according to the stringency degree of condition used during measurement hybridization.Stringency degree can the melting temperature(Tm) Tm of nucleic acid binding complex or probe be foundation, or with the salt of hybridization solution or ionic strength conditions for foundation.Described hybridization is preferably carried out at the standard conditions; More preferably, polymerized nucleoside acid hybridization can carry out in accordance with the following steps, and the film and the label probe that one are loaded with transcribed DNA to be measured or RNA molecule are hybridized in hybridization buffer.The dilution inhibitor and 2 ~ 8 × SSC that consist of 0.1wt%SDS, 5wt% dextran glucosides, add 1/20 of hybridization buffer.20 × SSC is the solution of the citric acid composition of 3M sodium-chlor and 0.3M.Hybridization temperature is 50 ~ 70 DEG C.Cultivation several hours or after spending the night, clean film with cleaning buffer solution.Cleaning temperature is preferably room temperature, is preferably hybridization temperature.The composition of cleaning buffer solution is preferably 6 × SSC+0.1%SDS (wt) solution, is preferably 5 × SSC+0.1%SDS (wt).After having cleaned film with this cleaning buffer solution, just can by being identified DNA or RNA molecule by the mark on the probe of hybridizing in DNA or RNA molecule.Specific experiment step please refer to the mode described in " molecular cloning " and carries out: Cold Spring HarborLaboratory Press, the general scheme recorded in molecular biology part (Current Protocols inMolecular Biology).
The preparation method of target polynucleotide of the present invention is this area customary preparation methods, preferably for preparing by the mode of synthetic or by the mode increased from genome.
The present invention solves the problems of the technologies described above two of the technical scheme taked: a kind of small interference ribonucleic acid (siRNA) of anti-multiple myeloma, described small interference ribonucleic acid comprises just RNA fragment and antisense RNA fragment, described just RNA fragment is as shown in SEQ ID NO:4, and described antisense RNA fragment is as shown in SEQ ID NO:5 in sequence table.
Small interference ribonucleic acid of the present invention is a kind of oligo rna molecule, and its length is preferably 21 ~ 25 Nucleotide, and the major function of described small interference ribonucleic acid is the silence exciting target mRNA complementary with it.
Small interference ribonucleic acid of the present invention preferably comprises just RNA fragment and antisense RNA fragment, and the complementary pairing effect that described just RNA fragment and antisense RNA fragment pass through complementary base to each other forms double-stranded region.When just RNA fragment and antisense RNA fragment are positioned at different RNA chains, form double stranded rna molecule; When just RNA fragment and antisense RNA fragment are positioned at same RNA chain, then form the single strand RNA molecule with neck ring structure, described single strand RNA molecule can comprise and exceed more than one neck ring structure.
Small interference ribonucleic acid length of the present invention is for being preferably 8 ~ 50 Nucleotide, and between its sense fragment and antisense fragments, the base pair of complementary region is preferably 15 ~ 18 base pairs.Wherein said just RNA fragment is preferably as shown in SEQ ID NO:4, and described antisense RNA fragment is preferably as shown in SEQ ID NO:5 in sequence table.
The preparation method of the small interference ribonucleic acid described in the present invention is this area customary preparation methods, preferably for the mode by synthetic or obtain by preparing from the mode of genome amplification.
Small interference ribonucleic acid of the present invention is more preferably the double-chain interference RNA (dsRNA) of synthetic, processes the siRNA obtained after transfectional cell in cell by Dicer enzyme system; By the siRNA that synthetic directly obtains; Or by building corresponding lentiviral vectors, the siRNA that continuous expression obtains in target cell.
The present invention solves the problems of the technologies described above three of the technical scheme taked: a kind of small interference ribonucleic acid of anti-multiple myeloma, described small interference ribonucleic acid comprises just RNA fragment and antisense RNA fragment, described just RNA fragment is as shown in SEQ ID NO:6, and described antisense RNA fragment is as shown in SEQ ID NO:7 in sequence table.
The present invention solves the problems of the technologies described above four of the technical scheme taked: a kind of small interference ribonucleic acid of anti-multiple myeloma, described small interference ribonucleic acid comprises just RNA fragment and antisense RNA fragment, described just RNA fragment is as shown in SEQ ID NO:8, and described antisense RNA fragment is as shown in SEQ ID NO:9 in sequence table.
The present invention solves the problems of the technologies described above five of the technical scheme taked: a kind of pharmaceutical composition for the treatment of multiple myeloma, and it comprises small interference ribonucleic acid as above and pharmaceutically acceptable carrier.
In pharmaceutical composition Chinese materia medica of the present invention, acceptable carrier is the conventional carrier in this area, and described pharmaceutically acceptable carrier is preferably vehicle, suspension agent, weighting agent and thinner.The formulation of pharmaceutical composition of the present invention is this area regular dosage form, is preferably the aqueous solution, suspension agent, wettable powder, emulsifiable concentrate, emulsion, can spray solution, aqueous dispersion, pulvis, granule, or microcapsule.
Pharmaceutical composition of the present invention at least comprises a kind of small interference ribonucleic acid of the present invention as activeconstituents and the pharmaceutically acceptable carrier of one.Wherein said pharmaceutical composition preparation method preferably takes the method for this area routine, using small interference ribonucleic acid of the present invention as activeconstituents, makes various formulation with pharmaceutically acceptable carrier.Wherein said pharmaceutically acceptable carrier is the pharmaceutical carrier of this area routine, as weighting agent, suspension agent, thinner and vehicle etc.In various preparation, be preferably the aqueous solution, the concentration of the described aqueous solution is preferably 80-120nM, is more preferably 100nM.
The present invention solves the problems of the technologies described above six of the technical scheme taked: the application of a kind of target polynucleotide as above in the anti-multiple myeloma medicine of preparation.
Application of the present invention is preferably for any one utilizing in target polynucleotide as above is prepared recombinant vectors or prepare virion.
Wherein said recombinant vectors is selected from the carrier derived by retrovirus of this area routine.The carrier that described virus derives comprises lentiviral vectors, adenovirus carrier, adenovirus related vector, herpesvirus vector or vaccinia virus vector etc., the preferred lentiviral vectors of the present invention.The advantage of lentiviral vectors is that they can make the polynucleotide passage of coding small interference ribonucleic acid (siRNA) be incorporated in the genome of host cell, produced the clone that specific gene is prevented by specificity, some virus vector can be used for transformant in body, thus the genetic expression realized in direct manipulation host and regulation and control thereof.
Wherein said virion is the virion of this area routine, be preferably based on human immunodeficiency virus (HIV), by virulent gene transformations such as removal env, vif, vpr, vpu wherein, the coating of HIV-1 is replaced to pack with vesicular stomatitis virus G glycoprotein, the host range of gained lentiviral vectors widely, significantly can increase the titre of virus.The virulent gene of lentiviral vectors is deleted and replaced by foreign gene, and belong to pseudotype virus, and can not infect other cell again after this virus infected cell, biological safety is high.Lentiviral vectors all has infection ability to somatoblast and Unseparated Cell, and can longer-term is stable in vivo expression.
Lentiviral particle preparation method of the present invention preferably comprises the following steps: be building up in lentiviral vectors by Target nucleotides of the present invention, then with package carrier cotransfection in eukaryotic cell, described eukaryotic cell is preferably 293T cell, after cultivating, carry out the recovery purifying of slow virus, obtain packaged lentiviral particle.
The application of target polynucleotide of the present invention in anti-multiple myeloma medicine is preferably for conduct it can be used as drug target.Described drug target refers to medicine effect binding site in vivo, selects to determine that novel active drug target spot is the top priority of new drug development.Rational drug design (rational drug design) can comprise the potential drug effect target position such as enzyme, acceptor, ionic channel, nucleic acid according to what disclose in life science, or the chemical structure characteristic of its endogenic ligand and natural substrate designs drug molecule, to find that selectively acting is in the new drug of target spot.By selection and the utilization of drug target, the efficiency of screening antineoplastic drugs can be significantly improved, effectively shorten the cycle of antitumor drug research.
The present invention solves the problems of the technologies described above seven of the technical scheme taked: the application of a kind of small interference ribonucleic acid as above in the anti-multiple myeloma medicine of preparation.
The application method of small interference ribonucleic acid of the present invention is preferably the pharmaceutical composition of the anti-multiple myeloma of preparation.The character of described pharmaceutical composition as mentioned before.
The cell model of application method of the present invention preferably for utilizing described small interference ribonucleic acid to prepare SUMO-1 gene silencing.Wherein said cell model is the cell model of this area routine, is preferably eukaryotic cell, is more preferably tumour cell, is multiple myeloma cells best.The preparation method of the cell model of SUMO-1 gene silencing of the present invention is this area customary preparation methods, preferably for utilizing small interference ribonucleic acid transfection cell strain of the present invention or will comprising the lentiviral particle transfecting eukaryotic cells of described small interference ribonucleic acid.
Wherein said transfection method is this area ordinary method, it is preferably DEAE-dextran method, calcium phosphate method, cationic-liposome method, cationic polymers method, virus-mediated methods, biological particles passes method (particle gun Particle bombardment), microinjection, electroporation etc. are cationic-liposome method best.Wherein said clone is preferably eukaryotic cell lines, is more preferably multiple myeloma cells.
One preferred embodiments of the preparation method of the cell model of SUMO-1 gene silencing of the present invention comprises: by small interference ribonucleic acid transfection multiple myeloma cells, the tumor cell clone of picking screening, detects the situation of the SUMO-1 protein content of different tumor cell clones by western-blot method.The cell strain that wherein SUMO-1 expressing quantity significantly declines is the cell model of the SUMO-1 gene silencing successfully constructed.
On the basis meeting this area general knowledge, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
Positive progressive effect of the present invention is: the present invention sets up and screened the target polynucleotide of the small interference ribonucleic acid for SUMO-1 gene, and building corresponding small interference ribonucleic acid (siRNA), this siRNA significantly can lower the mrna expression of SUMO-1 gene and the expression of albumen.Utilize target polynucleotide and the small interference ribonucleic acid of small interference ribonucleic acid of the present invention, effectively can suppress the expression of SUMO-1 gene, significantly reduce the rate of propagation of multiple myeloma cells.The present invention contributes to, for the root of myelomatosis progress, illustrating prognosis and the regulation mechanism of multiple myeloma further; For the key of multiple myeloma recurrence, develop new more effective therapeutic targets, realize safer and more effective anti-myeloma treatment, finally avoid the recurrence of multiple myeloma, realize curing, be of great practical significance.
Accompanying drawing explanation
Fig. 1 is the expression level that in Primary bone marrow oncocyte and myeloma cell line, β-catenin and SUMOization are modified.Wherein Fig. 1 (A) and figure (C) expression level that is β-catenin and SUMO-1 in Primary bone marrow oncocyte; The expression level that Fig. 1 (B) and Fig. 1 (D) is β-catenin in myeloma cell line and SUMO-1.
Fig. 2 is the result figure that the β-catenin suppressing SUMOization to be modified after siRNA disturbs myeloma cell SUMO-1 to express expresses.The detected object that wherein Fig. 2 (A) represents is multiple myeloma NCI-H929 cell; Fig. 2 (B) adopts immunofluorescence coprecipitation method to detect the endogenous of β-catenin and SUMO-1 in myeloma cell and combines; Fig. 2 (C) chooses myeloma cell NCI-H929, detects the change level of β-catenin before and after different concns SUMO-siRNA process cell; Fig. 2 (D) chooses myeloma cell NCI-H929 and RPMI-822, detects heterogeneic expression level before and after SUMO-siRNA process.
Fig. 3 is the result figure of the siRNA interference effect in different cell strain.
Fig. 4 is the apoptosis result figure of inducing bone marrow oncocyte after siRNA disturbs myeloma cell SUMO-1 to express.Wherein Fig. 4 (A) apoptosis rate detected result that is myeloma cell strain; The statistical value that Fig. 4 (B) is Fig. 4 A result, wherein ordinate zou represents the apoptosis rate of cell; Fig. 4 (C) is MTS method detection degree of cell proliferation result.
Fig. 5 is the stability disturbing the expression of SUMO-1 can reduce β-catenin, promotes the result figure that β-catenin degraded increases.Wherein Fig. 5 (A) is for before and after multiple myeloma cells SUMO-1 interference, β-catenin mrna expression level; Fig. 5 (B) is for be set to SUMO-1 interference group and control group by myeloma cell, and 100 μ g/ml CHX (cycloheximide) process the synthesis of arrestin matter, detect the change of β-catenin expression level; Fig. 5 (C) is the result of immunoblotting analysis result in Fig. 5 (B) being carried out to semi-quantitative analysis.
Fig. 6 is that SUMOization modifies inhibitor to the exercising result figure of myeloma cell.Wherein Fig. 6 (A) is H
2o
2on SUMOization modification and the impact of β-catenin expression level after (100 μMs) process myeloma cell; Fig. 6 (B) processes the impact on SUMOization modification and β-catenin expression level after myeloma cell for anacardic acid (100 μMs); Fig. 6 (C) modifies inhibitor process myeloma cell, to the detected result of its growth-inhibiting effect for adopting SUMOization after process LAN β-catenin.
Fig. 7 is that the rate of propagation of siRNA on NCI-H929 cell strain affects result figure.
Fig. 8 is that the rate of propagation of siRNA on RPMI-8226 cell strain affects result figure.
Embodiment
Now in conjunction with the embodiments and accompanying drawing, the present invention is described in detail, but enforcement of the present invention is not limited only to this.
Agents useful for same of the present invention and raw material all commercially maybe can be prepared by literature method.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as the people such as Sambrook " molecular cloning: lab guide " (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or conveniently condition, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.
" room temperature " described in embodiment refers to the temperature of carrying out the operation room tested, and is generally 15-25 DEG C.
Embodiment 1: synthesize siRNA for SUMO-1 gene design
Design and synthesis SUMO-1 siRNA (siRNA), wherein said siRNA for SUMO-1 gene in target sequence as shown in table 1:
The siRNA action target sequence (CDNA) of table 1SUMO-1 gene
| Numbering | Sequence numbering | Sequence |
| siRNA-1 | SEQ ID NO:1 | CTGGGAATGGAGGAAGAAG |
| siRNA-2 | SEQ ID NO:2 | CAATGAATTCACTCAGGTT |
| siRNA-3 | SEQ ID NO:3 | GGACAGGATAGCAGTGAGA |
According to the sequence in above table, synthesize siRNA sequence by sharp rich (Guangzhou) company, use ddH
2the concentration that the siRNA nucleic acid molecule of synthesis is diluted to 100 μMs by O is for subsequent use.Sequence information and the structural information of gained siRNA are as described below:
The fragment sequence of siRNA-1: positive-sense strand 5 ' CUGGGAAUGGAGGAAGAAG dTdT3 ' (as shown in SEQ ID NO:4 in sequence table);
Antisense strand 3 ' dTdTGACCCUUACCUCCUUCUUC 5 ' (as shown in SEQ IDNO:5 in sequence table).Its structure is double-strand neck ring structure.
The fragment sequence of siRNA-2: positive-sense strand 5 ' CAAUGAAUUCACUCAGGUU dTdT 3 ' (as shown in SEQ ID NO:6 in sequence table);
Antisense strand 3 ' dTdT GUUACUUAAGUGAGUCCAA 5 ' (as shown in SEQ IDNO:7 in sequence table).Its structure is double-strand neck ring structure.
The fragment sequence of siRNA-3: positive-sense strand 5 ' GGACAGGAUAGCAGUGAGA dTdT3 ' (as shown in SEQ ID NO:8 in sequence table);
Antisense strand 3 ' dTdT CCUGUCCUAUCGUCACUCU 5 ' (as shown in SEQ IDNO:9 in sequence table).Its structure is double-strand neck ring structure.
Embodiment 2: utilize siRNA transfected into bone marrow oncocyte
To myeloma cell NCI-H929 (this cell is bought from ADCC company) and RPMI-8226 (this cell is bought from ADCC company) the centrifugal 5min of logarithmic phase be in, and abandon supernatant liquor, then use PBS liquid re-suspended cell, more centrifugal 5min.ELX800 microplate reader is utilized to adjust cell concn to 30 × 10
4individual/L, is inoculated into six orifice plates and spends the night.Cationic-liposome adopts lipo2000, and by embodiment 1 gained siRNA-3 water dissolution, the final concentration of obtained aqueous solution is adjusted to 100nM.Use optimum to dilute respectively cationic-liposome and siRNA solution, it is for subsequent use that room temperature leaves standstill 5min.Mixed-cation liposome solutions and siRNA solution, make liposome fully wrap up siRNA, room temperature stand for standby use.After 20 minutes, mixed solution is added in cell, put into CO
2in incubator transfection after 48 hours so that experiment detect.
SUMO-1 and β-catenin effect is detected: results myeloma cell by immunofluorescence coprecipitation method, add the RIPA reagent (RIPA reagent is bought from Sigma, St.Louis, MO company) containing proteinase inhibitor, cracking 30min on ice, collected after centrifugation supernatant.The lysate that takes a morsel is analyzed in order to Westernblot.1 μ g β-catenin antibody (this antibody is bought from BD Biosciences company) and 10-50 μ l protein A/G-beads are joined remaining cell lysate, and 4 DEG C are slowly rocked overnight incubation; 4 DEG C with after 3,000g centrifugation, by centrifugal for protein A/G-beads at the bottom of pipe; Carefully suck supernatant, protein A/G-beads 1ml lysis buffer is washed 3-4 time; Finally add 2 × SDS sample loading buffer of 15 μ l, boiling water boiling 10 minutes.Row SDS-PAGE analyzes, protein transduction is to pvdf membrane, after pvdf membrane being closed l h, add 3 μ g SUMO-1 antibody (this antibody is bought from Abcam company), incubated at room temperature l h, add after washing film 0.5 μ g horseradish peroxidase-labeled two anti-(described two anti-be that goat antirabbit two resists, this antibody is bought from sigma company), incubated at room temperature 45min, develop with ECL test kit (this test kit is bought from sigma company) after washing film, acquired results as shown in Figure 1.
Detect and more different myeloma cell strain, RPMI-8226 and NCI-H929 of result display β-catenin albumen high expression level, its corresponding SUMOization modification level is also apparently higher than normal healthy controls Fig. 1 (B) and Fig. 1 (D).The SUMO-1 that in figure, arrow marks is unconjugated SUMO-1 monomeric protein, and albumen more than arrow mark is the albumen that SUMOization modification occurs.Compared with the normal plasma cells of normal healthy controls group, in the primary plasmocyte of patients with malignant myeloma, β-catenin protein expression level obviously raises, corresponding SUMOization modification level is also higher, and both exist dependency, and result asks for an interview Fig. 1 (A) and Fig. 1 (C).
Use lysate trizol cracking gained myeloma cell NCI-H92 and RPMI-8226, extracted total RNA, reverse transcription becomes cDNA, carries out quantitative fluorescent PCR, detects the mRNA of SUMO-1 gene.
Wherein SUMO-1-5 ' primer for: sence:5'-AGG AGG CAA AAC CTT CAA CT-3'(is as shown in SEQ ID NO:10 in sequence table);
SUMO-1-3 ' primer for: antisence:5'-TTC TTC CTC CAT TCC CAG TT-3'(is as shown in SEQ ID NO:11 in sequence table).
Utilize fluorescence quantifying PCR method to detect siNRA-3 sequence exercising result described in embodiment 1, as described above, the PCR reaction system used is as shown in table 2 for the primer used in fluorescence quantitative PCR detection:
Table 2 quantitative fluorescent PCR reaction system
The PCR reaction conditions used is: (1) 95 DEG C 5 minutes; (2) 95 DEG C 30 seconds; (3) 60 DEG C 30 seconds, (4) 72 DEG C 30 seconds, wherein step (2)-(4) repeat 40 circulations; (5) 95 DEG C 1 minute, (6) 60 degrees Celsius 30 seconds, (7) 95 DEG C 30 seconds.Acquired results is as shown in Table 3 and Figure 2:
The statistical study of table 3 fluorescent quantitative PCR result
The detected object that wherein Fig. 2 (A) represents is multiple myeloma NCI-H929 cell, use trizol cracking multiple myeloma NCI-H929 cell (negative control group and different concns siRNA treatment group), extracted total RNA, reverse transcription becomes cDNA, carries out quantitative fluorescent PCR.Detect the expression level of the mRNA of SUMO-1 gene, wherein the 3rd group of interference sequence jamming effectiveness is the highest, can reach 80%.Wherein Fig. 2 (B) adopts immunofluorescence coprecipitation method to detect the endogenous combination of β-catenin and SUMO-1 in myeloma cell, and immunofluorescent coprecipitation method as mentioned before.Fig. 2 (C) chooses myeloma cell NCI-H929, adopts the method for western blot, detects the change level of β-catenin before and after different concns SUMO-siRNA process cell.Information expressed in Fig. 2 (D) is that the expression of the downstream gene of WNT signal path is suppressed.Utilize the method for Real-Time PCR, detect the expression level of WNT signal path downstream gene (downstream gene is survivin, cyclinD1 and c-myc respectively), judge whether WNT path is activated with this.After result display interference SUMO-1 genetic expression, the expression level of WNT signal path downstream gene (survivin, cyclinD1 and c-myc gene) significantly falls, and shows that WNT signal path is obviously suppressed.Experimental result according to Fig. 2 and table 3, the siRNA for the target polynucleotide of the small molecule disturbance ribonucleic acid of anti-multiple myeloma of the present invention significantly can lower the expression of SUMO-1 gene mRNA.
SiRNA of the present invention verifies SUMO-1 gene inhibition effect in 8 strains (comprising above-mentioned RPMI-8226 and NCI-H929 mentioned) myeloma cell.Other six kinds of cell strains are that (above-mentioned six kinds of cell strains are all bought from ADCC company except CZ-1 for LP-1, SKO, CZ-1, ARH-77, KM-3 and U266.Wherein cell strain CZ-1 refers to document: the foundation of multiple myeloma cell line CZ-1 and biological characteristics Chinese Journal of Hematology 10 phases in 2002 thereof).
From the target polynucleotide sequence of the small interference ribonucleic acid of the anti-multiple myeloma described in embodiment 1 table 1, select SEQ ID NO:3 as the target of siRNA molecule, and select corresponding siRNA molecule (siRNA-3), RNA interference experiment is carried out in six kinds of multiple myeloma cell lines, as mentioned before, the result of gained siRNA interference effect is as shown in Fig. 3 and table 4 for its experimental technique.Wherein Fig. 3 is the result figure of the siRNA interference effect in different cell strain.
The siRNA interference effect of table 4 in different cell strain
| Cell category | Control group | Control group | siRNA-3 | siRNA-3 |
| LP-1 | 97.93716 | 102.0628 | 46.80829 | 36.9882 |
| SKO | 97.93716 | 102.0628 | 58.54928 | 48.46449 |
| CZ-1 | 97.93716 | 102.0628 | 56.23627 | 66.31177 |
| ARH-77 | 97.93716 | 102.0628 | 46.18164 | 45.36776 |
| KM-3 | 97.93716 | 102.0628 | 66.07523 | 66.05396 |
| U266 | 97.93716 | 102.0628 | 33.37096 | 23.30834 |
In table 4, secondary series and tertial content are two secondary orifices, represent cellular control unit use meaningless interference fragment disturb after SUMO-1mRNA expression level, 3rd row and fourth round are also two secondary orifices, and representative uses the ratio of siRNA-3 interference fragment interference its SUMO-1mRNA expression level rear and control group.Acquired results shows, siRNA-3 significantly can reduce the expression level of SUMO-1 in different multiple myeloma cell lines.
On the impact of different multiple myeloma cells apoptosis rate after embodiment 3:SUMO-1 gene expression inhibition
Adopt Annexin V-FITC-PI test kit (buying from Kai Ji company, Shanghai), flow cytometer carries out detecting (model is BD FACS calibur 4, the U.S.).
Flow cytometry: suspension cell centrifugal (the centrifugal 5min of 2000rpm) is collected; 1 ~ 5 × 10 are collected with PBS washed cell secondary (the centrifugal 5min of 2000rpm)
5cell; Add the BindingBuffer suspension cell of 500 μ L; After adding 5 μ L Annexin V-FITC mixings, add 5 μ L PropidiumIodide, mixing; Room temperature, lucifuge, reaction 5 ~ 15min; Flow cytomery.
MTS method: by cell by 10 × 10
3/ hole is inoculated in 96 orifice plates, after process in 48 hours, add 20 μ l MTS (buying from Promega company), 37 DEG C, 5%CO
2cell culture incubator uses Flex Station 3 (buying from Molecular Devices company) microplate reader to detect (490/690nm) after hatching 2 hours.
From the target polynucleotide sequence of the small interference ribonucleic acid of the anti-multiple myeloma described in embodiment 1 table 1, select SEQ ID NO:3 as the target of siRNA molecule, and select siRNA-3 molecule described in embodiment 1 to carry out siRNA interference experiment.Myeloma cell SUMO-1 expresses the apoptosis result of disturbed rear inducing bone marrow oncocyte as shown in Figure 4.Utilize flow cytometry to detect myeloma cell strain NCI-H929 and RPMI-8226 after siRNA interference, detection method comprises the following steps: by myeloma cell by 30 × 10
4/ hole fishplate bar (6 orifice plate), fishplate bar is in 6 orifice plates, and every strain cell connects 4 holes, difference transfection SUMO-1siRNA and β-catenin process LAN plasmid after spending the night.48 hours row Flow cytometry after transfection, specific experiment method as described above.
Myeloma cell strain NCI-H929 and RPMI-8226 after utilizing MTS method to detect siRNA interference, MTS detection method comprises the following steps: by myeloma cell by 30 × 10
4/ hole fishplate bar (6 orifice plate), fishplate bar is in 6 orifice plates, and every strain cell connects 4 holes, respectively transfection negative control plasmids after spending the night, SUMO-1siRNA, β-catenin process LAN plasmid, and transfection SUMO-1siRNA and β-catenin process LAN plasmid simultaneously.After 24 hours, cell is carried out fishplate bar after transfection, 10 × 10
3/ hole is inoculated in 96 orifice plates, and incubator utilizes MTS method to read value and detects its propagation degree after cultivating 48 hours, as indicated earlier, acquired results is as shown in Fig. 4 (C) for specific experiment method.After detected result shows that siRNA disturbs myeloma cell SUMO-1 to express, the apoptosis rate of myeloma cell strain significantly rises, wherein NCI-H929 cell strain rises to 17%, RPMI-8226 cell strain from 5% and rises to 18% from 10%, and its result is as shown in the Fig. 4 (A) in Fig. 4.After siRNA disturbs myeloma cell SUMO-1 to express, the growth of myeloma cell strain is obviously suppressed, be 58.7% to the inhibiting rate of myeloma cell strain NCI-H929, be 42.9% to the inhibiting rate of myeloma cell strain RPMI-8226, its result is as shown in Fig. 4 (C).
The expression of interference SUMO-1 can reduce the stability of β-catenin, and promote that its degraded increases, its result as shown in Figure 5.Wherein Fig. 5 (A) is for before and after multiple myeloma cells SUMO-1 interference, β-catenin mrna expression level.Fig. 5 (B) is for be set to SUMO-1 interference group and control group by myeloma cell, and 100 μ g/ml CHX (cycloheximide) process the synthesis of arrestin matter, detect the change of β-catenin expression level.Fig. 5 (C) is for carry out semi-quantitative analysis to immunoblotting analysis result in Fig. 5 (B) group.
Detect and compare β-catenin expression level before and after SUMO-1 interference, result shows, and the mRNA level in-site of the β-catenin of SUMO-1 interference group and control group does not change.In order to seek the reason that β-catenin protein content reduces, process cell further by 100 μ g/ml CHX (cycloheximide) to synthesize with arrestin matter, different time points collecting cell, crack protein, western blot detects β-catenin expression amount, the effect played wherein of research proteolytic degradation.Carry out semi-quantitative analysis to westernblot result subsequently, result display SUMO-1 interference group β-catenin obviously shortens biological half-life.Although the expression of acquired results prompting interference SUMO-1 gene does not affect the expression synthesis of β-catenin, the degraded of β-catenin albumen can be promoted.
Embodiment 4:SUMOization modifies inhibitor to the impact of different multiple myeloma cells apoptosis rate
Hydrogen peroxide (buying from sigma, USA), anacardic acid (buying from sigma, USA).
A. myeloma cell NCI-H929 cell is pressed 30 × 10
4/ hole fishplate bar (6 orifice plate), administration after spending the night: control group or 100 μMs of hydrogen peroxide, cell (0 is received after process different time, 3h, 6h), PBS washes one time, loading buffer lysing cell, the expression of 100 DEG C of water-bath pan boiling sample row western blot, detection SUMO-1 and β-catenin after 10 minutes.
B. myeloma cell NCI-H929 cell is pressed 30 × 10
6/ hole fishplate bar (6 orifice plate), administration after spending the night: contrast or 100 μMs of anacardic acid, cell (0 is received after process different time, 3h, 6h), PBS washes one time, loading buffer lysing cell, the expression of 100 DEG C of water-bath pan boiling sample row westernblot, detection SUMO-1 and β-catenin after 10 minutes.
In order to confirm that SUMOization is modified at the effect in myeloma cell further, we adopt SUMOization to modify inhibitor H respectively
2o
2process myeloma cell with anacardic acid, suppress its SUMOization modification level, result shows: suppress SUMOization modification can reduce β-catenin protein expression; Process LAN β-catenin partly can recover SUMOization and modify inhibitor to the growth-inhibiting of myeloma cell, points out β-catenin to carry out self-protection to reduce degraded by SUMOization modification further, promotes myelomatosis propagation.Suppress its SUMO to modify and can suppress tumor cell proliferation.
SUMOization modifies inhibitor to the exercising result of myeloma cell as shown in Figure 6.Wherein Fig. 6 (A) is H
2o
2on SUMOization modification and the impact of β-catenin expression level after (100 μMs) process myeloma cell.After result display uses hydrogen peroxide process myeloma cell, SUMOization modification level and the β-catenin level of cell all reduce, and in time-dependent manner.Fig. 6 (B) processes the impact on SUMOization modification and β-catenin expression level after myeloma cell for anacardicacid (100 μMs); After result display uses anacardic acid process myeloma cell, SUMOization modification level and the β-catenin level of cell all reduce, and in time-dependent manner.
Embodiment 5:SUMOization modifies the impact of inhibitor on different multiple myeloma cells increment degree
By myeloma cell NCI-H929 and RPMI-8226 respectively by 30 × 10
4/ hole fishplate bar (6 orifice plate), fishplate bar is in 6 orifice plates, every strain cell connects 2 holes, transfection negative control plasmids select SEQ ID NO:3 as the target of siRNA molecule in the target polynucleotide sequence of the small interference ribonucleic acid of anti-multiple myeloma from embodiment 1 described in table 1 respectively after spending the night, and select corresponding siRNA molecule (siRNA-3) transfected into bone marrow oncocyte, transfection method is as described in Example 2.Described negative control plasmids is human β-catenin pcDNA3.0, this plasmid has identical plasmid backbone with β-catenin process LAN plasmid, uniquely unlike not expressing β-catenin albumen, being used as negative control, avoiding occurring because transfected plasmids produces false positive results.After 24 hours, gained cell is carried out fishplate bar after transfection, 10 × 10
3/ hole is inoculated in 96 orifice plates, and incubator is cultivated different time and adopted rear MTS to read its propagation degree of value detection, and specific experiment method as described in Example 3.Result is as shown in table 5 and table 6 and Fig. 7 and Fig. 8:
The rate of propagation of table 5siRNA on NCI-H929 cell strain affects result
The rate of propagation of table 6siRNA on RPMI-8226 cell strain affects result
Wherein Fig. 7 is that the rate of propagation of siRNA on NCI-H929 cell strain affects result.Fig. 8 is that the rate of propagation of siRNA on RPMI-8226 cell strain affects result.Acquired results shows, siRNA sequence disclosed by the invention significantly can reduce the rate of propagation of multiple myeloma cells.SUMO-1siRNA sequence can the growth of suppression myeloma cell of time-dependent manner, to interference the 4th day, compared with cellular control unit, and still can up to 50% to the growth-inhibiting effect of myeloma cell.
Below the preferred embodiment of the invention is illustrated, but the invention is not limited to described embodiment, those of ordinary skill in the art also can make all equivalent modification or replacement under the prerequisite without prejudice to the invention spirit, and these equivalent modification or replacement are all included in the application's claim limited range.
Claims (10)
1. a target polynucleotide for the small interference ribonucleic acid of anti-multiple myeloma, is characterized in that, the sequence of described target polynucleotide is as shown in arbitrary in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3.
2. the small interference ribonucleic acid of an anti-multiple myeloma, it is characterized in that, described small interference ribonucleic acid comprises just RNA fragment and antisense RNA fragment, described just RNA fragment is as shown in SEQ ID NO:4 in sequence table, and described antisense RNA fragment is as shown in SEQ ID NO:5 in sequence table.
3. the small interference ribonucleic acid of an anti-multiple myeloma, it is characterized in that, described small interference ribonucleic acid comprises just RNA fragment and antisense RNA fragment, described just RNA fragment is as shown in SEQ ID NO:6 in sequence table, and described antisense RNA fragment is as shown in SEQ ID NO:7 in sequence table.
4. the small interference ribonucleic acid of an anti-multiple myeloma, it is characterized in that, described small interference ribonucleic acid comprises just RNA fragment and antisense RNA fragment, described just RNA fragment is as shown in SEQ ID NO:8 in sequence table, and described antisense RNA fragment is as shown in SEQ ID NO:9 in sequence table.
5. a pharmaceutical composition, is characterized in that, its comprise as arbitrary in claim 2 to 4 as described in small interference ribonucleic acid and pharmaceutically acceptable carrier.
6. pharmaceutical composition according to claim 5, is characterized in that, described pharmaceutically acceptable carrier is pharmaceutically acceptable vehicle, suspension agent, weighting agent and/or thinner.
7. pharmaceutical composition according to claim 6, is characterized in that, described thinner is water.
8. pharmaceutical composition according to claim 7, is characterized in that, the formulation of described pharmaceutical composition is the concentration 100nM of the aqueous solution.
9. the application of target polynucleotide as claimed in claim 1 in the anti-multiple myeloma medicine of preparation.
10. the application of the small interference ribonucleic acid as described in as arbitrary in claim 2 to 4 in the anti-multiple myeloma medicine of preparation.
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| CN103505745A (en) * | 2013-09-25 | 2014-01-15 | 深圳大学 | Application of micromolecule RNA used as immunosuppressor |
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| CN103923925A (en) * | 2014-05-07 | 2014-07-16 | 杨举伦 | siRNA (small interfering ribonucleic acid) for inhibiting ER81 gene expression and application of siRNA in breast cancer cells |
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| DRISCOLL ET AL: "The sumoylation pathway is dysregulated in multiple myeloma and is associated with adverse patient outcome", 《BLOOD》 * |
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