CN104531706A - Small antisense oligonucleotide aiming at miR-92a seed sequence and application - Google Patents
Small antisense oligonucleotide aiming at miR-92a seed sequence and application Download PDFInfo
- Publication number
- CN104531706A CN104531706A CN201410764763.3A CN201410764763A CN104531706A CN 104531706 A CN104531706 A CN 104531706A CN 201410764763 A CN201410764763 A CN 201410764763A CN 104531706 A CN104531706 A CN 104531706A
- Authority
- CN
- China
- Prior art keywords
- mir
- antisense oligonucleotide
- cell
- small antisense
- antimir
- 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
Abstract
The invention belongs to the field of medicine preparation, and particularly relates to a small antisense oligonucleotide aiming at a miR-92a seed sequence and an application. A nucleotide sequence of the small antisense oligonucleotide is 5'-AGTGCAAT-3'; the small antisense oligonucleotide is subjected to perthio modification, targets on miR-92a in multiple myeloma cells RPMI-8266, and has an inhibiting effect on the multiple myeloma cells, so that the small antisense oligonucleotide can be applied to preparation of medicines for preventing or treating tumors, and has a wide prospect in clinical treatment.
Description
Technical field
The invention belongs to pharmaceutical formulating art, be specifically related to a kind of small antisense oligonucleotide for miR-92a Seed Sequences and application.
Background technology
Multiple myeloma (multiple myeloma, MM) is a kind of monoclonal the malignant plasma cell dyscrasia.The Thalidomide of recent appearance, the medicine such as Revlimid and Velcade effectively can extend the life-span of MM patient, but resistance or palindromia still receive much concern.The unconventionality expression of several genes and the imbalance of many signal paths is related at the pathogenic process of multiple myeloma.Research finds gene and the signal path of these imbalances, can be regulated by miRNA.
MicroRNA (miRNA) is a kind of noncoding RNA, and the non-coding region complementation that it can be held with 3 ' of said target mrna combines the translation or mRNA chain of degrading that suppress mRNA.Therefore, MiRNA by affecting the translation process of mRNA, thus can affect the several functions of cell.Bioinformatics Prediction finds, people's full genome of 30% can be regulated by miRNA, and the multiple bioprocesss such as the survival of miRNA participation cell, growth, differentiation, apoptosis and aging.Increasing evidence shows, in tumour, have some remarkable process LAN miRNA, similar oncogene function, is called as oncomirs.Research in recent years shows, the expression affecting oncomir may be a kind of new available strategy of Therapeutic cancer.
In MM cell, find that obvious otherness appears in the expression level of multiple miRNA, wherein the miRNA of high expression level comprises miR-21, miR-155, miR-17-92, miR-99a-125b and miR-106-25.Wherein miR-17-92 bunch as important oncogene bunch, in kinds cancer, cause attention.MiR-17-92 cluster gene is a kind of polycistron, can transcribe out miRNA in 6 (miR-17, miR-18, miR-19a, miR-20, miR-19b-1, miR-92-1).Previous research finds, miR-17-92 bunch has close associating with the expression of Myc gene, reticent Myc gene in multiple myeloma cells, and the down-regulated expression of miR-17-92 bunch can suppress or necrocytosis by Promote cell's growth.This shows that miR-17-92 bunch also may be a kind of very important oncogene in multiple myeloma.But definite effect is also not bery clear and definite.
Research confirms, has 7 ~ 8 highly conserved sequences, be called as Seed Sequences at the 5' end of miRNA.Seed Sequences, by carrying out the pairing of base complete complementary with the 3'UTR of its target gene mRNA, promotes target gene mRNA degraded or suppresses mRNA translation, thus the negative regulate after the expression level of target gene is transcribed.The pairing of miRNA and target gene can be disturbed for miRNA Seed Sequences antisense nucleic acid, thus stop miRNA to play function.
Summary of the invention
In order to overcome the shortcoming of prior art with not enough, primary and foremost purpose of the present invention is to provide a kind of small antisense oligonucleotide for miR-92a Seed Sequences.
Another object of the present invention is to the application that the above-mentioned small antisense oligonucleotide for miR-92a Seed Sequences is provided.
Object of the present invention is achieved through the following technical solutions:
A kind of small antisense oligonucleotide for miR-92a Seed Sequences (tiny antimiR-92a, t-antimiR-92a), its nucleotide sequence is as follows: 5'-AGTGCAAT-3';
Described small antisense oligonucleotide is through full thio-modification;
The described small antisense oligonucleotide for miR-92a Seed Sequences is preparing the application in antitumor drug;
Described antitumor drug is preferably anti-multiple myeloma medicine;
The present invention has following advantage and effect relative to prior art:
(1) miR-92a of small antisense oligonucleotide t-antimiR-92a targeting in multiple myeloma cells RPMI-8266 for miR-92a Seed Sequences provided by the invention, the present invention has probed into small antisense oligonucleotide t-antimiR-92a to the retarding effect of multiple myeloma cells, for therapy of tumor finds new method, simultaneously also for the antitumor mechanism of traditional Chinese medicine provides new visual angle.
(2) for ripe miRNA, it is little that the small antisense oligonucleotide t-antimiR-92a for miR-92a Seed Sequences provided by the invention has molecular weight, and toxicity is little, high specificity, transfection efficiency advantages of higher.
Accompanying drawing explanation
Fig. 1 is the sequence diagram of miR-92a and t-antimiR-92a.
Fig. 2 is the interpretation of result figure of the signal path that miRFocus software analysis miR-92a participates in.
Fig. 3 is the interpretation of result figure of t-antimiR-92a to the effect of RPMI-8266 cell inhibitory effect.
Fig. 4 be transfection t-antimiR-92a to RPMI-8266 cell colony experimental result picture, wherein A is the number of cell clones statistical study of different treatment group, and B is the colony form under different treatment group microscope.
Fig. 5 is the interpretation of result figure that transfection t-antimiR-92a affects the invasion and attack of RPMI-8266 cell, and wherein, A is the cell invasion number statistical study of different treatment group, and B is the invasion and attack result under different treatment group microscope.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
The Design and synthesis of the small antisense oligonucleotide of embodiment 1
The Seed Sequences of people microRNA-92a is obtained from microRNA Families database, according to the small antisense oligonucleotide sequence (t-antimiR-92a) of complementary principle design for people microRNA-92a Seed Sequences, and BLAST software analysis is adopted to determine its small antisense oligonucleotide sequence and random controls sequence (Fig. 1).Small antisense oligonucleotide sequence: t-antimiR-92a (5'-AGTGCAAT-3'), stochastic sequence (scramble, SCR): 5 '-TCATACTA-3 '; By the synthesis of Shanghai biotechnology company limited, full thio-modification, high performance liquid chromatography (high performance liquidchromatography, HPLC) purifying.
Embodiment 2 cell cultures
By RPMI-8266 cell (multiple myeloma cells, buy in Shanghai cellular biochemical institute of the Chinese Academy of Sciences) to be inoculated in containing volume fraction be the foetal calf serum of 10%, in the RPMI-1640 substratum of antibiotic-free, be placed in 37 DEG C, volume fraction be 5% CO2 incubator, cultivate under saturated humidity.Within every 2 ~ 3 days, change liquid and go down to posterity.Experiment selects logarithmic phase, massfraction to be the cell of 0.2% trypan blue exclusion rate >95%.
The signal path analysis of embodiment 3 miRNA-92a
MiRFocus (http://mirfocus.org) software can be used for analyzing the target gene of miRNA and the signal path of its participation.
The signal path that miRFocus software analysis miR-92a participates in.Result such as Fig. 2, miR-92a participate in many important signal paths relevant with tumour.Such as mTOR signal path, cell cycle and other cancer paths etc.
Embodiment 4 mtt assay screens the best use of concentration of small antisense oligonucleotide
This experiment point t-antimiR-92a group, random (SCR) group and blank group, often group establishes 5 repeating holes.In t-antimiR-92a group, the nucleic acid final concentration of t-antimiR-92a is 0.2,0.3,0.4,0.5 and 0.6 μm of ol/L, and in random (SCR) group, the nucleic acid final concentration of stochastic sequence is 0.2,0.3,0.4,0.5 and 0.6 μm of ol/L.The RPMI-8266 cell of the logarithmic phase cultivated in Example 2, each group cell is with 1 × 10
5the density of cells/mL is inoculated in 96 orifice plates, every hole 50 μ L, transfection final volume 100 μ L (transfection method is with reference to Invitrogen company LipofectamineTM 2000 specification sheets), blank group adds the Opti-MEM substratum of the serum-free of 50 μ L.After transfection 6h, every hole adds the RPMI-1640 substratum that 100 μ L are 20% foetal calf serum containing volume fraction, makes every hole final volume be 200 μ L.After 48h, every hole adds MTT liquid 20 μ L, and cultivate 4h in incubator after, the centrifugal 10min of 1000r/min, abandons supernatant, and every hole adds 150 μ L DMSO, and concussion 10min, makes crystallization fully dissolve.Multi-functional microplate reader measures absorbance A
570nm.Experiment repetition 3 times, calculates proliferation inhibition rate.Proliferation inhibition rate=[1-(A experimental group/A control group)] × 100%.
T-antimiR-92a is effectively suppress RPMI-8266 proliferation activity between 0.2 μm of ol/L and 0.6 μm ol/L at final concentration, and its best use of concentration is 0.5 μm of ol/L, and there were significant differences compared with SCR group (P<0.05).T-antimiR-92a demonstrates nonspecific action (Fig. 3) at the final concentration of 0.5 μm of ol/L.
Embodiment 5 methylcellulose gum cell colony culture experiment observation of cell Colony forming situation
Experiment grouping is with embodiment 4, and the RPMI-8266 cell in vegetative period of taking the logarithm, the nucleic acid final concentration that t-antimiR-92a group, random (SCR) organize is set to the best use of concentration 0.5 μm of ol/L.Each group of cell is with 1 × 10
5the density of cells/mL is inoculated in 6 orifice plates, every hole 1500 μ L, transfection final volume 2000 μ L (transfection method is with reference to Invitrogen company LipofectamineTM 2000 specification sheets), blank group adds the Opti-MEM substratum of the serum-free of 500 μ L.After transfection 6h, take out cell counting, by 1 × 10
3it is in the semisolid medium of 20% foetal calf serum, 2mmol/L L-glutaminate, 5 μm of ol/L β-thin base ethanol and massfraction 0.9% methylcellulose gum that cells/well is inoculated in containing volume fraction, puts 37 DEG C, 5%CO
27d is cultivated with under saturated humidity condition.Under inverted microscope, colonies number and observation colony form, be 1 colony to be greater than the cell mass of 40 cells.Experiment repetition 3 times, calculates relative plating efficiency=(experimental group/blank group) × 100%.
The grade malignancy of Cell colony formation assay and cell has close relationship.In order to detect the impact that t-antimiR-92a is formed RPMI-8266 cell colony, Colony forming experiment is adopted to detect the grade malignancy of cancer cells.Transfection t-antimiR-92a cell is through the cultivation of 7d, there were significant differences compared with SCR group (P<0.05) (Fig. 4 A and Fig. 4 B) for the quantity of the colony of result display transfection t-antimiR-92a group, describe the Colony forming that t-antimiR-92a significantly can suppress RPMI-8266 cell, weaken the grade malignancy of multiple myeloma cells.Weaken the grade malignancy of multiple myeloma cells, significant for clinical treatment.
Embodiment 6 Transwell cell method detects the invasive ability of small antisense oligonucleotide transfection RPMI-8266 cell
Experiment grouping is with embodiment 4, and the RPMI-8266 cell in vegetative period of taking the logarithm, the nucleic acid final concentration that t-antimiR-92a group, random (SCR) organize is set to the best use of concentration 0.5 μm of ol/L.Each group of cell is with 1 × 10
5the density of cells/mL is inoculated in 6 orifice plates, every hole 1500 μ L, transfection final volume 2000 μ L (transfection method is with reference to Invitrogen company LipofectamineTM 2000 specification sheets), blank group adds the Opti-MEM substratum of the serum-free of 500 μ L.Cell after transfection 6h carries out hunger and cultivates 12h.Centrifugal, be 3 × 10 with the RPMI-1640 substratum adjustment cell density of serum-free
6cell/mL, gets 100 μ L and adds in the upper room of Transwell cell, adds the RPMI-1640 substratum that 600 μ L are 20% foetal calf serum containing volume fraction in the lower room of cell.Put into 37 DEG C, volume fraction is the CO of 5%
28h is cultivated in incubator.After 8h, take out Transwell cell, inhale and abandon little indoor liquid, the cell of not attacking above film wiped gently by the cotton swab soaked with PBS, Transwell cell is added methyl alcohol internal fixtion l0min, haematoxylin dyeing 3 ~ 5min, tap water softly rinses out unnecessary dye liquor.Carefully cut film with knife blade, film is fixed on slide glass.Counted under microscope the cell count compared through film.
Cell invasion experiment has close relationship with the grade malignancy of cell.In order to detect the impact of t-antimiR-92a on the invasion and attack of RPMI-8266 cell, transwell Matrigel is adopted to detect the grade malignancy of cancer cells.Invasion and attack result is as Fig. 5 A and Fig. 5 B, there were significant differences compared with SCR group (P<0.05) for the invasive ability of transfection t-antimiR-92a cell, describe the invasive ability that t-antimiR-92a significantly can suppress RPMI-8266 cell, weaken the grade malignancy of multiple myeloma cells.Weaken the grade malignancy of multiple myeloma cells, significant for clinical treatment.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (4)
1. for a small antisense oligonucleotide for miR-92a Seed Sequences, it is characterized in that: the nucleotide sequence of described small antisense oligonucleotide is as follows: 5'-AGTGCAAT-3'.
2. the small antisense oligonucleotide for miR-92a Seed Sequences according to claim 1, is characterized in that: described small antisense oligonucleotide is through full thio-modification.
3. the small antisense oligonucleotide for miR-92a Seed Sequences described in claim 1 or 2 is preparing the application in antitumor drug.
4. the small antisense oligonucleotide for miR-92a Seed Sequences according to claim 3 is preparing the application in antitumor drug, it is characterized in that: described antitumor drug is anti-multiple myeloma medicine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410764763.3A CN104531706A (en) | 2014-12-12 | 2014-12-12 | Small antisense oligonucleotide aiming at miR-92a seed sequence and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410764763.3A CN104531706A (en) | 2014-12-12 | 2014-12-12 | Small antisense oligonucleotide aiming at miR-92a seed sequence and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104531706A true CN104531706A (en) | 2015-04-22 |
Family
ID=52847332
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410764763.3A Pending CN104531706A (en) | 2014-12-12 | 2014-12-12 | Small antisense oligonucleotide aiming at miR-92a seed sequence and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104531706A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090298916A1 (en) * | 2008-03-07 | 2009-12-03 | Santaris Pharma A/S | Pharmaceutical compositions for treatment of microRNA related diseases |
-
2014
- 2014-12-12 CN CN201410764763.3A patent/CN104531706A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090298916A1 (en) * | 2008-03-07 | 2009-12-03 | Santaris Pharma A/S | Pharmaceutical compositions for treatment of microRNA related diseases |
Non-Patent Citations (2)
| Title |
|---|
| 丰茂晓: "miＲ-19a 和miＲ-92a 在多发性骨髓瘤中的19a 和miＲ-92a 在多发性骨髓瘤中的19a 和miＲ-92a 在多发性骨髓瘤中的功能及其信号通路分析", 《中国病理生理杂志》 * |
| 屈晓燕等: "多发性骨髓瘤患者微小RNA-92a表达水平及其临床意义", 《中华血液学杂志》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhang et al. | MicroRNA-221 and-222 regulate radiation sensitivity by targeting the PTEN pathway | |
| Sun et al. | ALKBH5 promotes lung fibroblast activation and silica-induced pulmonary fibrosis through miR-320a-3p and FOXM1 | |
| Sun et al. | MicroRNA-34a suppresses cell proliferation and induces apoptosis in U87 glioma stem cells | |
| Chen et al. | LncRNA LINC00342 regulated cell growth and metastasis in non-small cell lung cancer via targeting miR-203a-3p. | |
| Xu et al. | Down-regulation of ribosomal protein S15A mRNA with a short hairpin RNA inhibits human hepatic cancer cell growth in vitro | |
| WO2021022888A1 (en) | Aso targeting long-chain non-coding rna ddx11-as1, kit and application in treatment of liver cancer | |
| Liu et al. | miR-371-5p down-regulates pre mRNA processing factor 4 homolog B (PRPF4B) and facilitates the G1/S transition in human hepatocellular carcinoma cells | |
| Oliveira et al. | MicroRNA-100 acts as a tumor suppressor in human bladder carcinoma 5637 cells | |
| Qiao et al. | Microarray analysis of circRNAs expression profile in gliomas reveals that circ_0037655 could promote glioma progression by regulating miR-214/PI3K signaling | |
| Zhang et al. | MiR-520b suppresses proliferation of hepatoma cells through targeting ten-eleven translocation 1 (TET1) mRNA | |
| Sun et al. | Exosomal miR-181a-5p reduce Mycoplasma gallisepticum (HS strain) infection in chicken by targeting PPM1B and activating the TLR2-mediated MyD88/NF-κB signaling pathway | |
| CN102488903A (en) | Application of miR-224 to preparation of medicament for treating non-small cell lung cancer | |
| Wang et al. | MiR-371 promotes proliferation and metastasis in hepatocellular carcinoma by targeting PTEN | |
| Tian et al. | MicroRNA-497-5p is downregulated in hepatocellular carcinoma and associated with tumorigenesis and poor prognosis in patients | |
| CN104232647A (en) | miRNA (ribonucleic acid) with neuroglioma inhibition function, vector built by same and application | |
| CN103320444B (en) | For antisense oligonucleotide and the application of Microrna-21 Seed Sequences | |
| CN104531706A (en) | Small antisense oligonucleotide aiming at miR-92a seed sequence and application | |
| CN109966496A (en) | MiRNA-5571 is preparing the application in resistive connection rectal neoplasm drug | |
| CN101948859A (en) | shRNA expression vector expressed by specificity inhibitor SET protein, construction method and application thereof | |
| CN101921763B (en) | siRNA capable of inhibiting expression of PAR-1 gene and application thereof | |
| Cheng et al. | MicroRNA-294 Promotes Cell Proliferation, Migration and Invasion in SMMC-7721 Hepatoma Carcinoma Cells by Activating the JNK/ERK Signaling Pathway | |
| CN105561339B (en) | A kind of combination of miRNA prepares the application in anti-non-small cell lung cancer drug | |
| CN103305515B (en) | Family with sequence similarity 84, member B (FAM84B) antisense nucleotide and application thereof | |
| CN113652482B (en) | Application of LncRNA RXRG-1:3 in diagnosis or treatment of thyroid papilloma | |
| CN108192895B (en) | siRNA molecule targeting NOB1 gene and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150422 |
|
| RJ01 | Rejection of invention patent application after publication |