CN108251425A - Inhibit the siRNA molecule of RIOK2 genes and its antitumor application - Google Patents
Inhibit the siRNA molecule of RIOK2 genes and its antitumor application Download PDFInfo
- Publication number
- CN108251425A CN108251425A CN201810043024.3A CN201810043024A CN108251425A CN 108251425 A CN108251425 A CN 108251425A CN 201810043024 A CN201810043024 A CN 201810043024A CN 108251425 A CN108251425 A CN 108251425A
- Authority
- CN
- China
- Prior art keywords
- riok2
- cell
- seq
- positive
- sirna
- 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.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
- C12N2310/141—MicroRNAs, miRNAs
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Oncology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a kind of for inhibiting the siRNA molecule of RIOK2 genes, it is made of the positive-sense strand and antisense strand of following sequence：Positive-sense strand is 5 ' CAAUCAAGCUUUAGAAGAANn 3 ', and antisense strand is 5 ' UUCUUCUAAAGCUUGAUUGNn 3 '.Wherein, positive-sense strand and the N in antisense strand are identical or different, and are cytimidine C, uracil U, guanine G, adenine A, dideoxycytosine dC, deoxy-guanine dG, deoxyadenine dA or deoxythymidine dT each independently；N represents the number of N, and n is 0,1 or 2.The siRNA molecule of the present invention can be used for preparing the drug for the treatment of non-small cell lung cancer.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to it is a kind of for inhibit RIOK2 genes siRNA molecule and its
Prepare the purposes in antitumor drug.
Background technology
Non-small cell lung cancer (non-small cell lung cancer, NSCLC) is most common type in lung cancer, greatly
The lung cancer of about 85-90% belongs to NSCLC.Since early diagnosis is difficult and lacks effective treatment means, NSCLC is in whole world model
Be considered as because cancer leads to dead Etiological in enclosing, it was reported that only less than 15% the NSCLC survival of patients times 5
Year or more.So it finds the biological marker of NSCLC early diagnosis and finds that new molecular therapy target spot is consistent as this is captured
The top priority of life property disease and the hot spot of lung cancer Molecular Pathogcnesis research.
RIOK2 is one of RIO (right open reading frame) family member, and RIO family proteins have kinases
Characteristic, but therefore be not referred to as SARS type kinase (PLoS One.2012,7 (5) with structural domain as typical kinases:
E37371), other members also have RIOK1 and RIOK3.Height in eucaryotes of the RIOK1 and RIOK2 from yeast to mammal
Conservative, RIOK3 exists only in multinuclear eukaryocyte (RNA Biol.2012,9 (2):162-74).RIOK1/2 passes through promotion
20S pre-rRNA play a crucial role to ripe 18S rRNA conversions in the synthesis of 40S ribosomal subunits
(Science.2014,345(6195):378-379；Nucleic Acids Res.2014,42(19):12189-12199).
RIO family molecules are in incidence cancer (Int J Radiat Oncol Biol Phys.2006；64(3):670-677), brain colloid
Knurl (PLoS Genet.2013,9 (2):E1003253), colon cancer (Cancer Immunol Immunother.2002,51
(10):574-582), (ProcNatl Acad Sci U S are A.2008,105 (49) for cancer of pancreas:It is 19372-19377) and pernicious
Melanoma (Melanoma Res.2003,13 (5):High expression in 503-509).Read etc. is further study showed that be overexpressed
RIOK1 and RIOK2 promotes the proliferation of glioma cell by RTK-PI3K-Akt accesses, and the expression for lowering RIOK1 and RIOK2 is led to
It crosses increase P53 activity and inhibits glioma, promote apoptosis (PLoS Genet.2013,9 (2):e1003253).This
Outside, RIOK family proteins can also regulate and control signaling molecules or the accesses such as ERK, Ras, NF- κ B, Hedgehog, and be oncogene Myc
Target gene (the Mol Cancer Ther.2007,6 (2) of GAP-associated protein GAP:542-551).Meanwhile inventor is it has also been found that RIOK2 high
It is expressed in non-small cell lung cancer cell, and has high correlation with the existence of patient and prognosis, TNM stage with tumour,
Lymphatic metastasis and closely related (the Sci Rep.2016,6 of differentiation:28666.), prompting RIOK2 molecules can be used as non-small cell
The potential target gene of lung cancer therapy.
In recent years, RNA interfered (RNA interfering, RNAi) gene therapy technology new as one, disease-resistant
The field of medicaments such as malicious, antitumor and anti-inflammatory have a extensive future, and are rapidly developed, and part RNA drugs have been enter into clinic
Experimental stage, the disorders such as cancers especially multifactor for difficult and complicated cases, virus infection open completely new therapy approach.2016
On December 23, in, food and drug administration FDA have approved the first RNA drugs for being used to treat Duchenne-Arandisease
The application for quotation of Spinraza (Nusinersen) indicates that RNA drugs become a full member of drug main forces, become after chemicals,
The third-largest new drug type after biological protein medicament.Have tens of kinds of siRNA drugs in the world at present and enter clinical stage.
RNAi is the gene silencing effect after a kind of transcription, is referred in biological cell, exogenous or endogenic double-stranded RNA, is claimed
Have the mRNA of homologous sequence for siRNA (small interfering RNA, siRNA) identification, its specificity is cut
It cuts, so as to the process that it is blocked to translate, the expression of the final gene for inhibiting to transcribe the mRNA (Nature.1998,391:806-
811).RNAi drugs have been proved there is great potentiality in the treatment of a variety of diseases of viral infection and tumour, are reasons
The treatment means of blocking gene expression thought.
Invention content
The RNA drugs of tumour such as non-small cell lung cancer can be effectively treated in order to obtain, and inventor is using RIOK2 genes as target
Point designs and filters out hundreds of siRNA molecules, it is found that some of them can specifically inhibit RIOK2 gene expressions, wherein
One group of siRNA molecule can effectively inhibit RIOK2 genes.Therefore, of the invention first is designed to provide one kind for pressing down
The siRNA molecule of RIOK2 genes processed is made of the positive-sense strand and antisense strand of following sequence：
Positive-sense strand：5’-CAAUCAAGCUUUAGAAGAANn-3’(SEQ ID NO:1),
Antisense strand：5’-UUCUUCUAAAGCUUGAUUGNn-3’(SEQ ID NO:2),
Wherein, positive-sense strand and the N in antisense strand are identical or different, and be each independently cytimidine C, uracil U,
Guanine G, adenine A, dideoxycytosine dC, deoxy-guanine dG, deoxyadenine dA or deoxythymidine dT；N represents N
Number, n is 0,1 or 2.
In one embodiment, the n is 0, i.e.,
Positive-sense strand：5’-CAAUCAAGCUUUAGAAGAA-3’(SEQ ID NO:3),
Antisense strand：5’-UUCUUCUAAAGCUUGAUUG-3’(SEQ ID NO:4).
The siRNA molecule is the backbone sequences of this group of siRNA molecule.
In another preferred embodiment, the N be dT, n 2, i.e.,
Positive-sense strand：5’-CAAUCAAGCUUUAGAAGAAdTdT-3’(SEQ ID NO:5),
Antisense strand：5’-UUCUUCUAAAGCUUGAUUGdTdT-3’(SEQ ID NO:6).
Second object of the present invention is that providing above-mentioned siRNA molecule is preparing the drug for RIOK2 to be inhibited to express
In purposes.
Optionally, said medicine is antitumor drug.
Preferably, the drug is treatment non-small cell lung cancer drug.
Wherein siRNA molecule can be used to inhibit the growth of lung carcinoma cell, transfer as active ingredient or promote lung cancer
The apoptosis of cell.
In a preferred embodiment, said medicine applies to the dosage form or gelling agent of injection.
Preferably, said medicine also includes necessary adjuvant.
Experiment in vitro proves that siRNA molecule provided by the invention can specifically lower RIOK2 gene expressions, reach suppression
Non-small cell lung cancer cell processed growth, transfer and the technique effect for promoting cancer cell-apoptosis.
Description of the drawings
Fig. 1 is that the siRNA of the present invention inhibits the block diagram of RIOK2 target gene mRNA expressions in A549 lung carcinoma cells.
Compared with negative control group NC, siRNA1, siRNA2, siRNA3 and siRNA4 for targeting RIOK2 significantly suppress RIOK2 bases
The mRNA expressions of cause, wherein * P<0.05.
Fig. 2 is that the siRNA of the present invention inhibits the block diagram of RIOK2 target gene mRNA expressions in H1299 lung carcinoma cells.
Compared with negative control group NC, siRNA1, siRNA2, siRNA3 and siRNA4 for targeting RIOK2 significantly suppress RIOK2 bases
The mRNA expressions of cause, wherein * P<0.05.
Fig. 3 is that the siRNA of the present invention inhibits the result figure of RIOK2 protein expression levels in A549 lung carcinoma cells.With the moon
Property control group NC is compared, and siRNA3 significantly suppresses RIOK2 protein expression levels, wherein * P<0.05.
Fig. 4 is that the siRNA of the present invention inhibits the result figure of RIOK2 protein expression levels in H1299 lung carcinoma cells.With the moon
Property control group NC is compared, and siRNA3 significantly suppresses RIOK2 protein expression levels, wherein * P<0.05.
Fig. 5 is that the siRNA of the present invention inhibits the growth curve chart of A549 proliferation of lung cancer cells.With negative control group NC phases
Than siRNA3 significantly suppresses the growth of cell, wherein * P in 48h, 72h and 96h<0.05.
Fig. 6 is that the siRNA of the present invention inhibits the growth curve chart of H1299 proliferation of lung cancer cells.With negative control group NC phases
Than siRNA3 significantly suppresses the growth of cell, wherein * P in 48h, 72h and 96h<0.05.
Fig. 7 is that the siRNA of the present invention inhibits the result figure of A549 lung carcinoma cells migration.Compared with negative control group NC,
SiRNA3 significantly suppresses the migration of cell, wherein * P<0.05.
Fig. 8 is that the siRNA of the present invention inhibits the result figure of H1299 lung carcinoma cells migration.Compared with negative control group NC,
SiRNA3 significantly suppresses the migration of cell, wherein * P<0.05.
Fig. 9 is that the siRNA of the present invention inhibits the result figure of A549 lung carcinoma cell invasions.Compared with negative control group NC,
SiRNA3 significantly suppresses the invasion of cell, wherein * P<0.05.
Figure 10 is that the siRNA of the present invention inhibits the result figure of H1299 lung carcinoma cell invasions.Compared with negative control group NC,
SiRNA3 significantly suppresses the invasion of cell, wherein * P<0.05.
Figure 11 is that the siRNA of the present invention promotes the result figure of A549 Increase Apoptosis of Lung Cancer Cells.Compared with negative control group NC,
SiRNA3 significantly promotes the apoptosis of cell, wherein * P<0.05.
Figure 12 is that the siRNA of the present invention promotes the result figure of H1299 Increase Apoptosis of Lung Cancer Cells.Compared with negative control group NC,
SiRNA3 significantly promotes the apoptosis of cell, wherein * P<0.05.
Specific embodiment
The present invention is described in further details below in conjunction with specific embodiment.It should be understood that following embodiment is only used for
The bright present invention is not for restriction the scope of the present invention.
Some siRNA molecules provided by the invention with selectively targeted RIOK2 genes, can block the mRNA of RIOK2 genes
Transcription, prevents the translation of gene, so as to fundamentally inhibit the expression of RIOK2, is finally reached the purpose for inhibiting tumour.
Several siRNA molecules that can target RIOK2 genes are specifically listed in embodiment, and number is respectively
SiRNA1, siRNA2, siRNA3 and siRNA4.They all have the activity for inhibiting RIOK2 genes.Wherein by positive-sense strand SEQ
IDNO:5 and antisense strand SEQ ID NO:The Double-stranded siRNA molecules of 6 compositions inhibit the effect of RIOK2 gene expressions especially prominent,
Number is " siRNA3 " in embodiment.
Herein, term " siRNA ", " siRNA sequence ", " siRNA molecule ", " double-strand siRNA " or " double-strand siRNA
Molecule " can be interchanged, and the meaning that they are represented is identical with range.Wherein, siRNA be positive-sense strand and antisense strand anneal to be formed it is double
Chain structure.
A variety of methods can be used in the preparation of siRNA molecule, such as：Chemical synthesis, in-vitro transcription, digestion long-chain dsRNA,
Carrier expression RNA, PCR synthesis rna expression element etc., the appearance of these methods provides selectable space for researcher, can
Preferably to obtain gene silencing efficiency.
The dosage form of the drug of the present invention can be diversified forms, as long as being suitable for the administration of corresponding disease and properly
Keep the activity of RNA molecule.For example, for injection drug delivery system, dosage form can be freeze-dried powder.The drug can also be gel
Agent.
Optionally, any pharmaceutically acceptable carrier and adjuvant can be included in said medicine dosage form, as long as it is suitble to
In corresponding drug delivery system and it is properly maintained the activity of RNA molecule.
To be clearer and more comprehensible the present invention, hereby with preferred embodiment, and attached drawing is coordinated to be described in detail below.This field skill
Art personnel should be appreciated that following embodiments are only used for illustrating the present invention, not limit the invention.
Embodiment
SiRNA and PCR primer herein is synthesized by Biomics Bioisystech Co., Ltd.
Embodiment 1 synthesizes siRNA molecule and transfectional cell A549 and H1299
1. cell culture
Human lung carcinoma cell line A549 and H1299 (Hospital Attached to Nantong Univ.'s preservation) are trained in the DMEM/F12 containing 10%FBS
It supports in base (Thermo Fisher Scientific companies of the U.S.), in 37 DEG C, 5%CO2Incubator (U.S. Thermo
FisherScientific companies) in cultivate.
2. design and synthesize siRNA molecule
4 kinds of siRNA molecule sequences of targeting RIOK2 genes are designed and synthesized, referring to table 1, respectively：SiRNA1 has
Positive-sense strand SEQ ID NO:7 and antisense strand SEQ ID NO:8；SiRNA2 has positive-sense strand SEQ ID NO:9 and antisense strand SEQ
ID NO:10；SiRNA3 has positive-sense strand SEQ ID NO:5 and antisense strand SEQ ID NO:6；SiRNA4 has positive-sense strand SEQ
ID NO:11 and antisense strand SEQ ID NO:12.Positive-sense strand and corresponding antisense strand are annealed into siRNA double-strand respectively, before transfection
It is configured to a concentration of 20 μM.
Table 1. targets the siRNA sequence of RIOK2 genes
In addition, for the ease of comparing, NCs of the siRNA not homologous with people's gene as negative control has been designed and synthesized
(Negetive control) siRNA, sequence are：
Positive-sense strand：5’-UUCUCCGAACGUGUCACGUdTdT-3’(SEQ ID NO:13)；
Antisense strand：5’-ACGUGACACGUUCGGAGAAdTdT-3’(SEQ ID NO:14).
3. use siRNA transfectional cells
Plating cells simultaneously transfect：Step 1 gained cell is pressed 1 × 105A/hole is inoculated into 96 porocyte culture plates,
In antibiotic-free, the DMEM/F12 culture mediums containing 10%FBS, in 37 DEG C, 5%CO2Overnight incubation in incubator.Use cell transfecting
Reagent2000 (Thermo Fisher Scientific companies of the U.S.) carry out siRNA by its specification and turn
Dye.
4. the extraction of cell RNA
After cell transfecting 48h, cell is collected, RNA is extracted with RNA extracts reagents Trizol (Thermo companies of the U.S.), presses
Its specification carry out RNA extractions, gained RNA precipitate with 50 μ L without RNase water dissolutions, and take 2 μ L progress 1.5% Ago-Gel
Electrophoresis detection.The result shows that the purity and integrality of the cell total rna of purification are preferable, meet requirement of experiment.
5. real-time quantitative PCR
With gene-specific primer detect sample in RIOK2 gene mRNAs expression, while expand house-keeping gene β-
Actin is compareed as internal reference.Each sample expands RIOK2 genes and reference gene β-actin simultaneously, and each reaction is done 3 and put down
Row.Quantitative reaction is carried out with 2 × Master Mix (Thermo companies of the U.S.), establishes following reaction system：2 μ L RNA templates,
2 × Master Mix of 12.5 μ L, sense primer (10 μM) and downstream primer (10 μM) each 0.5 μ L, 50 × SYBR of 0.5 μ L
Green Solution supply system to 25 μ L with the water of no RNase.After mixing, it is anti-to put real-time quantitative PCR detecting system
It should.
Detect the real-time quantitative PCR primer of RIOK2 genes：
Sense primer is：5’-ACAACAGGCAAGATGGTCAG-3’(SEQ ID NO:15)；
Downstream primer is：5’-GACGACAAGGCAATTAGATGAG-3’(SEQ ID NO:16).
Detect the real-time quantitative PCR primer of house-keeping gene β-actin：
Sense primer is：5’-CCACACCTTCTACAATGAG-3’(SEQ ID NO:17)；
Downstream primer is：5’-ATAGCACAGCCTGGATAG-3’(SEQ ID NO:18).
Reaction condition：42 DEG C of reverse transcriptions 30min, 95 DEG C of pre-degenerations 5min, 95 DEG C of denaturation 20sec, 58 DEG C of annealing 30sec,
72 DEG C of extension 30sec, recycle 45 times.And do solubility curve reaction：It 95 DEG C/5min, 58 DEG C/5min, is heated up with 0.5 DEG C/5sec
To 95 DEG C.
With 2-ΔΔCtMethod analyzes experimental result, and makees block diagram, as illustrated in fig. 1 and 2, compared with negative control NC, in A549
Its mRNA expression is inhibited with 4 kinds of siRNA molecules that RIOK2 is targeted in H1299 cells, siRNA1, siRNA2,
The mRNA level in-site inhibiting rate of siRNA3 and siRNA4 is respectively 63%, 67%, 74% and 76%, the statistically significant (P of difference
<0.05)。
Embodiment 2Western blot detect RIOK2 protein expression levels
Cell is pressed 1 × 106A/hole is taped against in 6 orifice plates, grows to the degree of converging of 70-80% afterwards for 24 hours, by embodiment 1
Method carries out cell transfecting.After handling 48h, with 1 × SDS buffer (the 50mM Tris-HCl, pH7.6 of precooling；1%SDS；
150mM NaCl；0.5%Triton-X 100；5mM EDTA；5% β-mercaptoethanol (BME)；1mM PMSF) albumen
Lysate lytic cell is placed in 4 DEG C, and 10,000rpm centrifugation 20min take supernatant to carry out polyacrylamide gel electricity after diluting
Swimming, and (Millipore companies of the U.S.) in transferring film to Kynoar (PVDF) film, and sealed with 5% skim milk room temperature
Close 2h, then with rabbit-anti people RIOK2 monoclonal antibodies (Abcam companies of the U.S., 1:200 dilutions), the anti-human β-actin monoclonals of mouse
Antibody (Abcam companies of the U.S., 1:200 dilutions) as internal reference.After TBST washings, then the secondary antibody with horseradish peroxidase
Be incubated (RIOK2 goat anti-rabbit igg-HRP, 1:1000 dilutions；β-actin sheep anti-mouse igg-HRP, 1:2000 dilutions), it uses
TBST washs 3 times (5min/ times).It is detected with BeyoECL Plus kits (green skies company).
As shown in Figures 3 and 4, compared with negative control NC, in A549 and H1299 cells target RIOK2 siRNA1,
SiRNA2, siRNA3 and siRNA4 show to inhibit the activity of RIOK2 protein expressions, and wherein siRNA3 is significantly inhibited
RIOK2 protein expression levels, inhibiting rate are respectively 75% and 84%, the statistically significant (P of difference<0.05).
Since siRNA3 protrudes the inhibition of RIOK2 genes, following experiments are as primary study object.
3 cell Proliferation of embodiment detects
Cell culture and siRNA transfections are carried out as described in Example 1.
Before transfection when cell confluency degree reaches 60-80%, the cell of exponential phase is taped against 96 porocyte culture plates
In, 3 holes are repeated, the sample (sample for transfecting 0h) as untransfected.
The cell of each experimental group of the transfection for 24 hours, after 48h, 72h and 96h is taken to carry out CCK8 cell proliferating determinings respectively, it is described
Assay method is：By the 100 μ L DMEM (Thermo companies of the U.S.) of mixing and 10 μ L CCK8 (Cell CountingKit-8,
Japanese Dojingo companies) it is added in each hole, incubator is incubated 4h, and with microplate reader, (Bio-Rad is public at wavelength 490nm
Department) OD values are measured, make growth curve.
As it can be seen in figures 5 and 6, compared with negative control NC, in A549 and H1299 cells, siRNA3 is significantly suppressed
The proliferation of cell.
4 cell migration of embodiment detects
Lung cell A549 and H1299 are taped against respectively in 24 porocyte culture plates, for 24 hours after as described in Example 1 into
Row is handled, and is resuspended after 48h with DMEM cultures, adjustment concentration to 1 × 106A cell/mL.Transwell plates (the U.S.
Corning companies) interior room DMEM culture medium preincubate 1h, upper chamber and lower room separate with 8 μm of polycarbonate membrane.100 μ L's
Cell re-suspension liquid and DMEM culture mediums of the 600 μ L containing 10%PBS or the conditioned medium (cell culture medium of above-mentioned 48h processing
Supernatant) it is added in each upper chamber.After for 24 hours, the cell stayed in upper chamber is removed with cotton swab, moves to cell in lower room with 10%
Formaldehyde fixes 30s.Finally, 0.5% violet staining 4min of cell, is then washed 3 times with PBS buffer solution.Cell is put at 200 times
It is counted in the big visual field, 5 visuals field is counted under the conditions of each.Migrating cell number is calculated, makes column diagram.
As shown in FIG. 7 and 8, compared with negative control NC, siRNA3 is significantly suppressed carefully in A549 and H1299 cells
The migration of born of the same parents, the statistically significant (P of difference<0.05).
5 cell invasion of embodiment detects
Lung cell A549 and H1299 are taped against respectively in 24 porocyte culture plates, for 24 hours after as described in Example 1 into
Row is handled, and is resuspended after 48h with DMEM cultures, adjustment concentration to 1 × 106A cell/mL.Transwell plates (the U.S.
Corning companies) interior room DMEM culture medium preincubate 1h, the upper chamber and lower room Matirgel (0.5mg/ for being coated with 50 μ L
ML, U.S. company BD) 8 μm of polycarbonate membrane separate.The cell re-suspension liquid of 100 μ L is trained with DMEM of the 600 μ L containing 10%PBS
It supports base or conditioned medium (the cell culture medium supernatant of above-mentioned 48h processing) is added in each upper chamber.After for 24 hours, upper chamber is stayed in
In cell removed with cotton swab, the cell moved in lower room fixes 30s with 10% formaldehyde.Finally, 0.5% crystal violet of cell
4min is dyed, is then washed 3 times with PBS buffer solution.Cell counts in 200 times of magnification fields, and counting 5 under the conditions of each regards
It is wild.Invasion cell number is calculated, makes column diagram.
As shown in Figures 9 and 10, compared with negative control NC, siRNA3 is significantly suppressed in A549 and H1299 cells
The invasion of cell, the statistically significant (P of difference<0.05).
6 Apoptosis of embodiment detects
Cell culture and siRNA transfections are carried out as described in Example 1.
Annexin-V/ is carried out with Annexin V-FITC apoptosis assay kit (Sigam-Aldrich companies of the U.S.)
Propidium iodide (PI) double analysis.Lung cell A549 and H1299 transfection 48h after, with 0.25% pancreatin digestion and
PBS buffer solution is washed twice.1×105A cell is resuspended with 500 μ L combination buffers, and is marked by operating instruction with 5 μ L FITC
Annexin-V dyeing, add 5 μ L PI and room temperature be protected from light and be incubated 15min.Different experiments group cell BD FACS Calibur
(U.S. company BD) carries out fluidic cell test analysis, calculates apoptosis rate, makes column diagram.
As shown in FIG. 11 and 12, compared with negative control NC, siRNA3 is significantly promoted in A549 and H1299 cells
The apoptosis of cell, the statistically significant (P of difference<0.05).
By above-mentioned experiment as it can be seen that siRNA1, siRNA2, siRNA3 and siRNA4 of targeting RIOK2 show to inhibit
The activity of RIOK2 expression, wherein siRNA3 are especially prominent for the inhibition of RIOK2 genes；SiRNA3 molecules can inhibit
The proliferation of A549 and H1299 cells, migration, invasion, and the apoptosis of A549 cells and H1299 cells is remarkably promoted, thus have
Applied to antitumor, especially Treatment for Non-small Cell Lung potentiality.
Sequence table
<110>Hospital Attached to Nantong Univ.
<120>Inhibit the siRNA molecule of RIOK2 genes and its antitumor application
<130> SHPI1711631
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> RNA
<213>Artificial sequence ()
<400> 1
caaucaagcu uuagaagaan 20
<210> 2
<211> 20
<212> RNA
<213>Artificial sequence ()
<400> 2
uucuucuaaa gcuugauugn 20
<210> 3
<211> 19
<212> RNA
<213>Artificial sequence ()
<400> 3
caaucaagcu uuagaagaa 19
<210> 4
<211> 19
<212> RNA
<213>Artificial sequence ()
<400> 4
uucuucuaaa gcuugauug 19
<210> 5
<211> 21
<212> RNA
<213>Artificial sequence ()
<400> 5
caaucaagcu uuagaagaad d 21
<210> 6
<211> 21
<212> RNA
<213>Artificial sequence ()
<400> 6
uucuucuaaa gcuugauugd d 21
<210> 7
<211> 21
<212> RNA
<213>Artificial sequence ()
<400> 7
gguugacaaa ugcaggauad d 21
<210> 8
<211> 21
<212> RNA
<213>Artificial sequence ()
<400> 8
uauccugcau uugucaacc 19
<210> 9
<211> 21
<212> RNA
<213>Artificial sequence ()
<400> 9
gagaauuagu gaaacauaad d 21
<210> 10
<211> 21
<212> RNA
<213>Artificial sequence ()
<400> 10
uuauguuuca cuaauucucd d 21
<210> 11
<211> 21
<212> RNA
<213>Artificial sequence ()
<400> 11
gcaaagaauc agauauuuad d 21
<210> 12
<211> 21
<212> RNA
<213>Artificial sequence ()
<400> 12
uaaauaucug auucuuugcd d 21
<210> 13
<211> 21
<212> RNA
<213>Artificial sequence ()
<400> 13
uucuccgaac gugucacgud d 21
<210> 14
<211> 21
<212> RNA
<213>Artificial sequence ()
<400> 14
acgugacacg uucggagaad d 21
<210> 15
<211> 20
<212> DNA
<213>Artificial sequence ()
<400> 15
acaacaggca agatggtcag 20
<210> 16
<211> 22
<212> DNA
<213>Artificial sequence ()
<400> 16
gacgacaagg caattagatg ag 22
<210> 17
<211> 19
<212> DNA
<213>Artificial sequence ()
<400> 17
ccacaccttc tacaatgag 19
<210> 18
<211> 18
<212> DNA
<213>Artificial sequence ()
<400> 18
atagcacagc ctggatag 18
Claims (7)
1. it is a kind of for inhibiting the siRNA molecule of RIOK2 genes, it is made of the positive-sense strand and antisense strand of following sequence：
Positive-sense strand：5’-CAAUCAAGCUUUAGAAGAANn-3’(SEQ ID NO:1),
Antisense strand：5’-UUCUUCUAAAGCUUGAUUGNn-3’(SEQ ID NO:2),
Wherein, positive-sense strand and the N in antisense strand are identical or different, and are that cytimidine C, uracil U, bird are fast each independently
Purine G, adenine A, dideoxycytosine dC, deoxy-guanine dG, deoxyadenine dA or deoxythymidine dT；N represents of N
Number, n is 0,1 or 2.
2. siRNA molecule as described in claim 1, which is characterized in that the n is 0, i.e.,
Positive-sense strand：5’-CAAUCAAGCUUUAGAAGAA-3’(SEQ ID NO:3),
Antisense strand：5’-UUCUUCUAAAGCUUGAUUG-3’(SEQ ID NO:4).
3. siRNA molecule as described in claim 1, which is characterized in that the N be dT, n 2, i.e.,
Positive-sense strand：5’-CAAUCAAGCUUUAGAAGAAdTdT-3’(SEQ ID NO:5),
Antisense strand：5’-UUCUUCUAAAGCUUGAUUGdTdT-3’(SEQ ID NO:6).
4. use of the siRNA molecule in the drug for inhibiting RIOK2 expression is prepared as described in any one of claim 1-3
On the way.
5. purposes as claimed in claim 4, which is characterized in that the drug is treatment non-small cell lung cancer drug.
6. purposes as claimed in claim 5, which is characterized in that the treatment non-small cell lung cancer drug is used to inhibit lung cancer thin
Growth, transfer or the apoptosis for promoting lung carcinoma cell of born of the same parents.
7. the purposes as described in any one of claim 4-6, which is characterized in that the drug is injection type or gelling agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810043024.3A CN108251425A (en) | 2018-01-17 | 2018-01-17 | Inhibit the siRNA molecule of RIOK2 genes and its antitumor application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810043024.3A CN108251425A (en) | 2018-01-17 | 2018-01-17 | Inhibit the siRNA molecule of RIOK2 genes and its antitumor application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108251425A true CN108251425A (en) | 2018-07-06 |
Family
ID=62740784
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810043024.3A Pending CN108251425A (en) | 2018-01-17 | 2018-01-17 | Inhibit the siRNA molecule of RIOK2 genes and its antitumor application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108251425A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114085832A (en) * | 2021-10-25 | 2022-02-25 | 南通大学附属医院 | siRNA molecule for inhibiting PRR14 gene |
| WO2023097119A3 (en) * | 2021-11-29 | 2023-07-13 | Dana-Farber Cancer Institute, Inc. | Methods and compositions to modulate riok2 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2015173601A (en) * | 2014-03-13 | 2015-10-05 | 大日本住友製薬株式会社 | Screening method for cancer stem cell proliferation inhibition material |
-
2018
- 2018-01-17 CN CN201810043024.3A patent/CN108251425A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2015173601A (en) * | 2014-03-13 | 2015-10-05 | 大日本住友製薬株式会社 | Screening method for cancer stem cell proliferation inhibition material |
Non-Patent Citations (2)
| Title |
|---|
| KUN LIU等: "High Expression of RIOK2 and NOB1 Predict Human Non-small Cell Lung Cancer Outcomes", 《SCIENTIFIC REPORTS》 * |
| 谢小冬主编: "《现代生物技术概论》", 31 January 2007, 军事医学科学出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114085832A (en) * | 2021-10-25 | 2022-02-25 | 南通大学附属医院 | siRNA molecule for inhibiting PRR14 gene |
| CN114085832B (en) * | 2021-10-25 | 2024-05-17 | 南通大学附属医院 | SiRNA molecules for inhibiting PRR14 gene |
| WO2023097119A3 (en) * | 2021-11-29 | 2023-07-13 | Dana-Farber Cancer Institute, Inc. | Methods and compositions to modulate riok2 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yang et al. | miR-200b regulates epithelial-mesenchymal transition of chemo-resistant breast cancer cells by targeting FN1 | |
| Youn et al. | Modification of cardiac progenitor cell-derived exosomes by miR-322 provides protection against myocardial infarction through nox2-dependent angiogenesis | |
| Yao et al. | Tumor suppressive role of miR-1224-5p in keloid proliferation, apoptosis and invasion via the TGF-β1/Smad3 signaling pathway | |
| CN109837306A (en) | Contain the excretion body and its preparation method and application of miRNA-204-5p | |
| Yi et al. | Co-treatment with therapeutic neural stem cells expressing carboxyl esterase and CPT-11 inhibit growth of primary and metastatic lung cancers in mice | |
| CN107541513B (en) | Small interfering RNA (ribonucleic acid) for silencing CD317, recombinant vector, medicament and application thereof | |
| CN107586781A (en) | Liver cancer marker lncRNA ENST00000620463.1 and its application | |
| CN107828888A (en) | Circular rna circ PTPRA purposes | |
| Tittarelli et al. | Hypoxic melanoma cells deliver microRNAs to dendritic cells and cytotoxic T lymphocytes through connexin-43 channels | |
| CN106222141B (en) | NK cell culture fluids and cell culture processes | |
| CN108251425A (en) | Inhibit the siRNA molecule of RIOK2 genes and its antitumor application | |
| Lin et al. | Influenza A virus infection induces indoleamine 2, 3-dioxygenase (IDO) expression and modulates subsequent inflammatory mediators in nasal epithelial cells | |
| CN110251529A (en) | MiR-124-3p and its analog are preparing the application in anti-breast cancer disease medicament | |
| CN107988230A (en) | Suppress the siRNA molecule of NOB1 genes | |
| CN109091493B (en) | Application of LncRNA ZEB1-AS1 in preparation of drug for improving ovarian epithelial cancer drug resistance | |
| CN107913284A (en) | Application of the Microrna of 367 clusters of miRNA302 in targeted inhibition angiogenesis and tumour growth | |
| CN107893115B (en) | ALKBH1 gene and application of expression product thereof in preparation of kit for diagnosing tumors and drugs for treating tumors | |
| CN110075122A (en) | A kind of liver cancer treatment excretion body drug | |
| CN108192895A (en) | Target siRNA molecule and its application of NOB1 genes | |
| CN109745335A (en) | MiR-218 is preparing the application in mammary cancer chemotherapy drug sensitizer | |
| Huang et al. | Effects of hTERT antisense oligodeoxynucleotide on cell apoptosis and expression of hTERT and bcl-2 mRNA in keloid fibroblasts. | |
| CN110468202A (en) | It is a kind of target TIGIT miR-206 as diagnosing cancer of liver and treat novel molecular purposes | |
| CN103266111A (en) | Dual-target siRNA (small interfering ribonucleic acid) molecule and application thereof | |
| Premadasa et al. | Cannabinoid enhancement of lncRNA MMP25-AS1/MMP25 interaction reduces neutrophil infiltration and intestinal epithelial injury in HIV/SIV infection | |
| CN108144061A (en) | Application of the microRNA-210 inhibitor on treatment inflammatory skin medicine is prepared |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20180706 |