CN102641508A - Application of target up-regulation PAR-4 gene small ribonucleic acid (RNA) in preparing bladder cancer resisting drugs - Google Patents
Application of target up-regulation PAR-4 gene small ribonucleic acid (RNA) in preparing bladder cancer resisting drugs Download PDFInfo
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Abstract
The invention provides application of target up-regulation PAR-4 gene small ribonucleic acid (RNA) in preparing bladder cancer resisting drugs. The nucleotide sequence of the target up-regulation PAR-4 gene small RNA is composed of six positive sense strands and negative sense strands of 21 nucleotides. The 3' end of each strand is provided with two nucleotides in suspension mode (generally dTdT, and the 19 nucleotides in the middle are paired.). The target up-regulation PAR-4 gene small RNA comprises sa RNA of different sequences. By means of expression of target up-regulation PAR-4 genes in the bladder cancer cells, the small RNA can restrain tumor cell activity, induces cell apoptosis, and further can be applied to preparation of bladder cancer resisting drugs. The application of target up-regulation PAR-4 gene small RNA in preparing the bladder cancer resisting drugs is simple in operation of preparing the small RNA, low in cost, small in using amount, capable of achieving good activation effect when the transfection concentration is 50nM and accurate in activation action. The mRNA and protein level of the target genes are both improved. In addition, the sa RNA can induce genes to express without damaging completeness of gene groups, thereby being safe to use.
Description
Technical field
The invention belongs to biological technical field, relate to the application of little activator RNA in preparation anti-bladder cancer medicine that targeting raises the PAR-4 gene.
Background technology
DsRNA (the double-stranded RNA of report such as Li in 2006 target gene promoter region; Double-stranded RNA) can cause that sequence-specific genetic transcription activates; And gene activation (the RNA activation that causes this phenomenon called after RNA; RNAa), and such dsRNA call little activator RNA s (small activating RNAS, saRNAs).It is the universal phenomenon in the mammalian cell that the RNA activated gene is expressed; Its mechanism possibly be that short dsrna combines with the antisense transcription product of genes of interest; Promoter region is carried out epigenetics modify (acetylation of histone, demethylation etc.); Further raise the promoter region of transcription factor, thereby strengthen the expression of genes of interest to gene.
People PAR-4 gene is found in the prostate gland cancer cell of the inductive apoptosis of extrinsic factor first.The function of many research proof PAR-4 is that various apoptotic pathways are necessary, and the PAR-4 of excessive ectopic expression is enough to induce most of tumor cell generation apoptosis, but can not cause the apoptosis of normal or immortalized cells.PAR-4 can and suppress NF-kB through activation Fas death receptor signal transduction pathway, thereby the generation that guarantees caspase cascade reaction in the tumor cell is interference-free.No matter the more important thing is, be the carcinoma of prostate mouse model of subcutaneous dystopy or original position, and the adenovirus of intratumor injection ability overexpression PAR-4 all can cause tumor death and growth inhibited.Therefore, the proteic expression of PAR-4 is increased and is had significant oncotherapy prospect, is the ideal target gene of saRNAs.
Summary of the invention
The purpose of this invention is to provide the application of little RNA in preparation anti-bladder cancer medicine that a kind of targeting raises the PAR-4 gene.The present invention comprises the saRNA of multiple sequence, and these little RNA can raise PAR-4 expression of gene in the transitional cell bladder carcinoma cell line through targeting, suppress tumor cell activity, cell death inducing, and then can be applied to the preparation of anti-bladder cancer medicine.
Said targeting raises the nucleotides sequence of the little RNA of PAR-4 gene and classifies 3 positive-sense strand and antisense strand compositions to (6) 21 nucleotide as.Its sequence is respectively: dsPAR-4-433 S (SEQ ID NO:1): 5 '-AAU ACG GUC UUG UAC UUA A [dT] [dT]-3 ', dsPAR-4-433 AS (SEQ ID NO:2): 5 '-UUA AGU ACA AGA CCG UAU U [dT] [dT]-3 '; DsPAR-4-434 S (SEQ ID NO:3): 5 '-UAA UAC GGU CUU GUA CUU A [dT] [dT]-3 ', dsPAR-4-434 AS (SEQ ID NO:4): 5 '-UAA GUA CAA GAC CGU AUU A [dT] [dT]-3 '; DsPAR-4-435 S (SEQ ID NO:5): 5 '-AUA AUA CGG UCU UGU ACU U [dT] [dT]-3 ', dsPAR-4-435 AS (SEQ ID NO:6): 5 '-AAG UAC AAG ACC GUA UUA U [dT] [dT]-3 '.3 ' end of every chain is outstanding two nucleotide of suspension (be generally dTdT, middle 19 oligonucleotide ligands to) all.
It is simple to operate that the present invention prepares saRNA, and cost is lower, and consumption is less, and the transfection concentration of 50nM can reach good activation effect; Activation is definite, and target gene mRNA and protein level all have rising.The saRNA inducible gene expression belongs to the epigenetics adjusting in addition, does not destroy genomic integrity, therefore uses safer.
Description of drawings
Fig. 1 is the influence that different loci dsRNAs expresses PAR-4 mRNA in the T24 cell, and the GAPDH expression is as confidential reference items.
Fig. 2 is the influence degree that different loci dsRNAs expresses PAR-4 mRNA in the T24 cell, and data are PAR-4/GAPDH ratio, derive from the meansigma methods of 3 independent experiments.
Fig. 3 is that dsPAR-4-435 raises the proteic expression of PAR-4 in the T24 cell, and β-actin expression is as confidential reference items.
Fig. 4 is that dsPAR-4-435 raises the proteic expression of PAR-4 in the T24 cell, and data are PAR-4/ β-actin ratio, derive from the meansigma methods of 3 independent experiments.
Fig. 5 is that the proteic expression of PAR-4 is concentration dependent in the dsPAR-4-435 rise T24 cell, and β-actin expression is as confidential reference items.
Fig. 6 is that dsPAR-4-435 raises the proteic expression of PAR-4 in 5637 cells, and β-actin expression is as confidential reference items.
Fig. 7 is that dsPAR-4-435 raises the proteic expression of PAR-4 in 5637 cells, and data are PAR-4/ β-actin ratio, derive from the meansigma methods of 3 independent experiments.
Fig. 8 is the growth that dsPAR-4-435 suppresses the T24 cell, and cell image is taken under 100 times of situation of amplification by the Olympus inverted phase contrast microscope.
Fig. 9 is the growth that dsPAR-4-435 suppresses 5637 cells, and cell image is taken under 100 times of situation of amplification by the Olympus inverted phase contrast microscope.
Figure 10 is that mtt assay prompting dsPAR-4-435 can obviously suppress the T24 cytoactive, and is concentration and time dependence; Data are represented with mean+SD.
Figure 11 is that flow cytometer detection prompting dsPAR-4-435 can induce T24 cell generation apoptosis.
The specific embodiment
In order to understand essence of the present invention better, below in conjunction with accompanying drawing and embodiment the present invention is further described, but these specific embodiments are not to limit invention scope required for protection by any way.
Embodiment 1DsRNAs raises the expression of PAR-4
Experimental program:
1. cell line: human bladder cancer cell's strain T24,5637 (Shanghai cell institute)
2.dsRNAs synthetic: dsRNAs is by the chemosynthesis of Shanghai Ji Kai Bioisystech Co., Ltd.
3. experiment is divided into groups: the dsPAR-4 group of 50nM, matched group dsRNA (dsCon), and non-homogeneous with the known person genome sequence.
DsControl: positive-sense strand, 5 '-ACU ACU GAG UGA CAG UAG A [dT] [dT]-3 ' (SEQ ID NO:7)
Antisense strand, 5 '-UCU ACU GUC ACU CAG UAG U [dT] [dT]-3 ' (SEQ ID NO:8)
Blank group (Mock only adds liposome).
4. cell culture and transfection:
Cell culture is positioned over CO2 content and is in 5% the 37oC cell culture incubator in the RPMI1640 culture medium that contains 10% deactivation NBCS.Plant in culture plate density about 30～40% behind transfection passage the previous day.DsRNAs is transfected into cell after wrapping up through liposome Lipofectamine 2000; With 6 orifice plates is example (all the other cultivate vessel according to the adjustment of the ratio between floor space amount of reagent); 7.5 μ l 20mM dsRNAs storage liquid are got in every hole and 5 μ l liposomees are dissolved in 250 μ l serum-free mediums respectively, mix behind the 5min, and room temperature adds in the culture plate after leaving standstill 20min; Replenish and to contain blood serum medium to make the dsRNA final concentration be 50nM, effect continues 48 or 72 hours.
5. reverse-transcription polymerase chain reaction (RT-PCR):
Utilize Trizol Reagent with the total RNA of the cell extraction of each experimental group, ultraviolet spectrophotometer is accurately quantitative.Get the total RNA of 3 μ g and carry out reverse transcription.
GAPDH forward primer: 5 '-ATGGCACCGTCAAGGCTGAG-3 ' (SEQ ID NO:9)
Downstream primer: 5 '-GCAGTGATGGCATGGACTGT-3 ' (SEQ ID NO:10)
PAR-4 forward primer: 5 '-GCCGCAGAGTGCTTAGATGAG-3 ' (SEQ ID NO:11)
Downstream primer: 5 '-GCAGATAGGAACTGCCTGGATC-3 ' (SEQ ID NO:12).
The PCR reaction condition is: 94
oC degeneration 4min circulates 28 times by following parameter: 94
oC degeneration 45s, 58
oThe C 45s that anneals, 72
oC extends 60s, last 72
oC 10min, 4
oThe C stopped reaction, rear electrophoresis.
6. Western blot (Western Blot)
Behind cell transfecting 72 h, collecting cell utilizes the RIPA cell pyrolysis liquid to extract total protein, and BCA (bicinchoninic acid) method is measured protein concentration, and the protein content of adjustment sample to be tested.Every hole adds protein electrophoresis, commentaries on classics film, the sealing of 20ug, puts into one of dilution to film and resists, and gentle vibration 3h rearmounted 4 spends refrigerator overnight.Gentle again 2 h that vibrate reclaim an anti-back and in the Tris buffer, wash film 30 min, and middle replacing Tris buffer 2-3 time places film two anti-gentle vibration 1h of dilution respectively, and film 30 min are washed in the back in the Tris buffer, and liquid is changed 3-4 time in the centre.Exposure, developing and fixing with negative film scanner scanning X line film, are read the density scan value of purpose electrophoretic band through gel imaging system.
The result is following:
1. different loci dsRNAs is to the influence of PAR-4 mRNA expression in the T24 cell
Transitional cell bladder carcinoma cell line T24 is distinguished dsRNAs and the contrast dsRNA (dsCon) in the transfection 50nM table 3.2 and the blank group is set; Utilize RT-PCR to detect the mRNA expression of PAR-4 after 48 hours; Compare with matched group; DsPAR-4-433 ,-434, intracellular PAR-4 mRNA is organized in-435 effects obviously increases, be matched group more than 3 times (referring to Fig. 1, Fig. 2).
2.dsPAR-4-435 raise the proteic expression of PAR-4 in the T24 cell
Further detect T24 cell transfecting PAR-4 protein expression situation in each effect group cell after 72 hours through Western Blot; Compare with matched group; Intracellular PAR-4 albumen is organized in the dsPAR-4-435 effect obviously to be increased, be matched group more than 4 times (referring to Fig. 3, Fig. 4).
The proteic expression of PAR-4 is concentration dependent in the T24 cell 3.dsPAR-4-435 raise
DsPAR-4-435 and 25nM to the transfection 5,10 respectively of T24 cell, 25nM contrast dsRNA (dsCon) and the blank group are set; Utilize Western Blot to detect transfection PAR-4 protein expression situation in each effect group cell after 72 hours; Compare with matched group; 10, the dsPAR-4-435 of 25nM all can raise the expression of PAR-4 in the T24 cell in various degree, and this effect is concentration dependent (referring to Fig. 5).
4.dsPAR-4-435 raise the proteic expression of PAR-4 in 5637 cells
DsPAR-4-435 and the 50nM of transfection 50nM contrast dsRNA (dsCon) and the blank group are set respectively to transitional cell bladder carcinoma cell line 5637; Utilize Western Blot to detect transfection PAR-4 protein expression situation in each effect group cell after 72 hours; Compare with matched group; Intracellular PAR-4 albumen is organized in the dsPAR-4-435 effect obviously to be increased, be matched group more than 2 times (referring to Fig. 6, Fig. 7).
Experimental program:
1. cell line: with embodiment 1.
2.dsRNAs it is synthetic: with embodiment 1.
3. experiment is divided into groups: with embodiment 1.
4. cell culture and transfection: with embodiment 1.
5. tetrazolium salts (MTT) colorimetry
The take the logarithm bladder cancer T24 cell of trophophase; Single cell suspension is processed in trypsinization; Density with 2000～5000 cells in every hole (serves as the accuracy that guarantees the result, before experiment, measures the growth curve of adherent rate, doubling time and the different vaccination number cell of cell, confirm the inoculation number of every porocyte again in 96 well culture plates the cell kind; Cell is unlikely to overfill when stopping to guarantee to cultivate), culture plate is placed 37
oC, 5% CO
2And after the interior overnight incubation of the cell culture incubator of saturated humidity, inhale and remove old culture fluid, the adding variable concentrations (1～50nM) dsP21-306 or dsPAR-4-435, and set up negative control (dsCon) and blank (Mock).Act on after 24～72 hours, every hole adds 20 μ l MTT solution (5 mg/ml), 37
oC is hatched after 4 hours and is abandoned culture medium, and every hole adds DMSO 150 μ l, and room temperature was placed 10 minutes, and dissolving to be crystallized back is measured its absorbance (OD) value with ELIASA in 490 nm wavelength.Calculate cell survival rate by following formula: survival rate=treatment group OD value/matched group OD value * 100%;
6. flow cytometer detects apoptosis
Cell after collecting 50nM dsP21-306 or dsPAR-4-435, dsCon and Mock processed group 24,48 or 72h after the trypsinization, 4
oCentrifugal 5 minutes of C, 2000rpm abandon supernatant, with PBS washing and cell counting, get and contain 1 * 10
5The suspension of individual cell, 4
oCentrifugal 5 minutes of C, 2000rpm abandon supernatant, and each sample adds 1 * Annexin V binding buffer liquid of 100 μ l, and concentration is every milliliter 1 * 10
6Individual cell, respectively add 5 μ l PI and Annexin V after room temperature kept in Dark Place 15 minutes, and then add 1 * Annexin V binding buffer liquid of 400 μ l, detect with Beckman Coulter FC500 flow cytometer, data are directly read in software.The result judges:
Normal survivaling cell: Annexin V (-), PI (-); Be positioned at left lower quadrant;
Viable apoptotic cell: Annexin V (+), PI (-); Be positioned at right lower quadrant (LR);
Non-viable apoptotic cell: Annexin V (+), PI (+); Be positioned at right upper quadrant (UR);
Complete dead cell: Annexin V (-), PI (+); Be positioned at left upper quadrant.
The result is following:
1. dsPAR-4-435 suppresses the growth of T24 cell
DsPAR-4-435 and contrast dsRNA (dsCon) to transitional cell bladder carcinoma cell line T24,5637 difference transfection 50nM; And blank group (Mock is set; Only add liposome), transfection 48 or after 72 hours utilizes Olympus inverted phase contrast microscope observation of cell and Taking Pictures recording.(referring to Fig. 8, Fig. 9).
Fig. 8-9 shows that aspect morphology, the cell of Mock and dsCon group is kept fit and grown apace after transfection; And the cell of transfection dsPAR-4-435 is along with the prolongation of time, and cell loses activity gradually, in transfection 48 with after 72 hours; Cell quantity obviously is less than matched group; The attached cell major part division that stops growing, and shrinkage, distortion even death occur, but in the culture medium buoyant dead cell not have dsP21-306 to do the time spent obvious.
2.dsPAR-4-435 can obviously suppress the T24 cytoactive
For growth and the active inhibition degree of clear and definite dsPAR-4-435 to the T24 cell, we have carried out the MTT test.In 96 orifice plates, respectively to the dsPAR-4-435 of the various concentration of T24 cell transfecting (5,10,25,50nM), the dsCon of 50nM and blank group Mock is set, and each group is provided with 8 multiple holes altogether.Referring to Figure 10, dsPAR-4-435 occurs within 48 hours the inhibitory action of T24, and the dsPAR-4-435 that is low to moderate 5nM also has certain inhibitory action.The inhibitory action of dsPAR-4-435 is dosage and time dependence, and is consistent with the adjusting rule of target gene.The dsPAR-4-435 of 5～50nM suppression ratio to the T24 cytoactive when transfection 24h is 1.0% to 10.3%, and when transfection 48h and 72h, its suppression ratio reaches 12.1% to 32.3% and 21.9% to 62.3% (Figure 10, table 1) respectively.
3.dsPAR-4-435 can induce T24 cell generation apoptosis
Utilize Annexin V and PI dyeing, detect research dsPAR-4-435 through flow cytometer and whether suppress the T24 cytoactive with apoptosis-related.To the dsPAR-4-435 and contrast dsRNA (dsCon) of transitional cell bladder carcinoma cell line T24 difference transfection 50nM, and blank group (Mock only adds liposome) is set.Shown in figure 11, dsPAR-4-435 can induce part T24 cell generation apoptosis.After 72 hours, the early stage cell proportion (LR) and the apoptotic cells ratio in late period (UR) of dying of transferring is increased to 7.1% and 20.9% respectively in transfection, and apoptotic cell ratio (LR+UR) reaches 28.0%, than matched group showed increased.In addition, dsP21-306 transfection T24 cell after 24 hours the non-viable non-apoptotic cell ratio also reach 6.2%, survivaling cell only has only 65.9% (referring to Figure 11).
< 110>Zhejiang University
< 120>targeting raises the application of the little RNA of PAR-4 gene in preparation anti-bladder cancer medicine
<160>?12
<210>?1
<211>?21
<212>?RNA
< 213>artificial sequence
< 223>last two is deoxyribonucleotide
<400>?1
AAU?ACG?GUC?UUG?UAC?UUA?ATT
<210>?2
<211>?21
<212>?RNA
< 213>artificial sequence
< 223>last two is deoxyribonucleotide
<400>?2
UUA?AGU?ACA?AGA?CCG?UAU?UTT
<210>?3
<211>?21
<212>?RNA
< 213>artificial sequence
< 223>last two is deoxyribonucleotide
<400>?3
UAA?UAC?GGU?CUU?GUA?CUU?ATT
<210>?4
<211>?21
<212>?RNA
< 213>artificial sequence
< 223>last two is deoxyribonucleotide
<400>?4
UAA?GUA?CAA?GAC?CGU?AUU?ATT
<210>?5
<211>?21
<212>?RNA
< 213>artificial sequence
< 223>last two is deoxyribonucleotide
<400>?5
AUA?AUA?CGG?UCU?UGU?ACU?UTT
<210>?6
<211>?21
<212>?RNA
< 213>artificial sequence
< 223>last two is deoxyribonucleotide
<400>?6
AAG?UAC?AAG?ACC?GUA?UUA?UTT
<210>?7
<211>?21
<212>?RNA
< 213>artificial sequence
< 223>last two is deoxyribonucleotide
<400>?7
ACU?ACU?GAG?UGA?CAG?UAG?ATT
<210>?8
<211>?21
<212>?RNA
< 213>artificial sequence
< 223>last two is deoxyribonucleotide
<400>?8
UCU?ACU?GUC?ACU?CAG?UAG?UTT
<210>?9
<211>?20
<212>?DNA
< 213>artificial sequence
< 223>as the forward primer of GAPDH pcr amplification
<400>?9
ATG?GCA?CCG?TCA?AGG?CTG?AG
<210>?10
<211>?20
<212>?DNA
< 213>artificial sequence
< 223>as the downstream primer of GAPDH pcr amplification
<400>?10
GCA?GTG?ATG?GCA?TGG?ACT?GT
<210>?11
<211>?21
<212>?DNA
< 213>artificial sequence
< 223>downstream primer that increases as the PAR-4 gene PCR
<400>?11
GCC?GCA?GAG?TGC?TTA?GAT?GAG
<210>?12
<211>?22
<212>?DNA
< 213>artificial sequence
< 223>downstream primer that increases as the PAR-4 gene PCR
<400>?12
GCA?GAT?AGG?AAC?TGC?CTG?GAT?C
Claims (3)
1. the application of little RNA in preparation anti-bladder cancer medicine of a targeting rise PAR-4 gene; The nucleotides sequence of the little RNA of said targeting rise PAR-4 gene is classified the positive-sense strand and the antisense strand of 6 21 nucleotide as and is formed; Its sequence is respectively: dsPAR-4-433 S:5 '-AAU ACG GUC UUG UAC UUA A [dT] [dT]-3 ', dsPAR-4-433 AS:5 '-UUA AGU ACA AGA CCG UAU U [dT] [dT]-3 '; DsPAR-4-434 S:5 '-UAA UAC GGU CUU GUA CUU A [dT] [dT]-3 ', dsPAR-4-434 AS:5 '-UAA GUA CAA GAC CGU AUU A [dT] [dT]-3 '; DsPAR-4-435 S:5 '-AUA AUA CGG UCU UGU ACU U [dT] [dT]-3 ', dsPAR-4-435 AS:5 '-AAG UAC AAG ACC GUA UUA U [dT] [dT]-3 '.
2. a kind of targeting according to claim 1 raises the application of little RNA in preparation anti-bladder cancer medicine of PAR-4 gene, it is characterized in that 3 ' end of every chain is outstanding two the nucleotide dTdT of suspension all, and middle 19 oligonucleotide ligands are right.
3. a kind of targeting according to claim 1 raises the application of little RNA in preparation anti-bladder cancer medicine of PAR-4 gene, it is characterized in that the excipient that said medicine also has preparation to allow.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2016145608A1 (en) * | 2015-03-17 | 2016-09-22 | 中国医学科学院北京协和医院 | Small activating rna, manufacturing method and application thereof |
| CN107446927A (en) * | 2017-09-15 | 2017-12-08 | 三峡大学 | Activate microRNA, screening technique and its application that LRIG1 is expressed in glioma U251 cells |
| CN111000858A (en) * | 2019-05-10 | 2020-04-14 | 常州市第二人民医院 | Application of NUPR1 inhibitor in preparation of bladder cancer treatment drug |
| CN113584027A (en) * | 2018-04-10 | 2021-11-02 | 中美瑞康核酸技术(南通)研究院有限公司 | Method for activating expression of p21 gene |
| US11730750B2 (en) | 2020-02-17 | 2023-08-22 | University Of Kentucky Research Foundation | Drugs for GRP78 cell surface translocation and Par-4 secretion |
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| CN102373204A (en) * | 2010-08-27 | 2012-03-14 | 复旦大学附属金山医院 | Method for transcriptional gene expression regulation and application |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016145608A1 (en) * | 2015-03-17 | 2016-09-22 | 中国医学科学院北京协和医院 | Small activating rna, manufacturing method and application thereof |
| CN107446927A (en) * | 2017-09-15 | 2017-12-08 | 三峡大学 | Activate microRNA, screening technique and its application that LRIG1 is expressed in glioma U251 cells |
| CN107446927B (en) * | 2017-09-15 | 2020-06-23 | 三峡大学 | Small molecular RNA for activating LRIG1 expression in glioma U251 cell, screening method and application thereof |
| CN113584027A (en) * | 2018-04-10 | 2021-11-02 | 中美瑞康核酸技术(南通)研究院有限公司 | Method for activating expression of p21 gene |
| CN111000858A (en) * | 2019-05-10 | 2020-04-14 | 常州市第二人民医院 | Application of NUPR1 inhibitor in preparation of bladder cancer treatment drug |
| CN111000858B (en) * | 2019-05-10 | 2021-04-09 | 常州市第二人民医院 | Application of NUPR1 inhibitor in preparation of bladder cancer treatment drug |
| US11730750B2 (en) | 2020-02-17 | 2023-08-22 | University Of Kentucky Research Foundation | Drugs for GRP78 cell surface translocation and Par-4 secretion |
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Application publication date: 20120822 |