CN107354159A - Applications of the long-chain non-coding RNA SMAD5 AS1 siRNA in treatment of ovarian cancer - Google Patents
Applications of the long-chain non-coding RNA SMAD5 AS1 siRNA in treatment of ovarian cancer Download PDFInfo
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- CN107354159A CN107354159A CN201710657458.8A CN201710657458A CN107354159A CN 107354159 A CN107354159 A CN 107354159A CN 201710657458 A CN201710657458 A CN 201710657458A CN 107354159 A CN107354159 A CN 107354159A
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- 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
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- 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
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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- 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
Abstract
The invention discloses a kind of applications of long-chain non-coding RNA (long non coding RNA, lncRNA) the SMAD5 AS1 siRNA in treatment of ovarian cancer.The present invention suppresses SMAD5 AS1 siRNA sequence by designing and synthesizing special target, is transfected into Ovarian Cancer Cells, it was demonstrated that propagation, migration and the invasive ability of ovarian cancer cell can be significantly inhibited by suppressing SMAD5 AS1 expression.The present invention provides new method and new medicament research and development direction for the treatment of oophoroma.
Description
Technical field
The present invention relates to applications of the LncRNA SMAD5-AS1 siRNA in treatment of ovarian cancer, the invention belongs to biology
Field of engineering technology.
Background technology
Malignant tumor of ovary is the main cause of the death of gynaecologic reproductive system malignant tumor patient, and women the 5th is pernicious
Tumor mortality reason (CA Cancer J Clin.2017；67(1):7-30).U.S.'s current research data show that the whole world is annual
Newly-increased ovarian cancer diagnosis 225500, fatal rate is up to 62%.Although in the past few decades because the change of HRT makes
Obtain ovarian tumors rate to have declined, but the diagnosis of oophoroma and Therapy study do not obtain remarkable break-throughs.Ovarian epithelial
Cancer (Epithelial ovarian cancer, EOC), is commonly called as oophoroma, the 90% of malignant tumor of ovary is accounted for, due to ovary
Cancer early symptom unobvious and the effective early screening means of shortage, the early diagnosis of oophoroma is relatively difficult, the U.S. in 2016
National Cancer Institute data points out that oneself proceeds to late period to case of the U.S. more than 2/3 when being diagnosed as oophoroma, and
Advanced ovarian cancer prognosis is usually very poor, and 5 years survival rates of I phase oophoromas are only 25% (Eur J for 92.1%, III phases phase IV
Cancer.2015), also therefore oophoroma is often assigned the title of " lonely killer ".At present, the primary treatment regimen of oophoroma is
Tumour cell subtracts the standard chemotherapy regimen that operation of going out is combined based on platinum class and treated, and operation can reach maximum tumour and subtract
Go out, but some transfer stoves can not solve by surgery.Although the early stage of chemotherapy is efficient very high, most of oophoroma is suffered from
There is chemotherapy resistance phenomenon in person, and this is also Ovarian cancer rate, death rate height, and 5 annual survival rates only have for 30% or so the reason for.
Therefore it is particularly important to saving ovarian cancer patients life that sensitive oophoroma early diagnosis index and more effective therapeutic modality are found.
In recent years, epigenetics research was greatly enlarged our understanding to disease, and cancer is by former cancer base
Because and tumor suppressor gene missing or unconventionality expression caused by, and epigenetic regulation has played irreplaceable effect wherein
(Nat Rev Cancer.2016；16(11):694-707).Increasing evidence shows that the key factor of induced tumor is
The unconventionality expression of regulated and control network between multiple genes and gene.Long-chain non-coding RNA (long non-coding RNA, lncRNAs)
It is the non-coding RNA that transcript is more than 200bp, its biological function (Nat Rev can be played in several ways
Genet.2016；17(10):601-614).Numerous studies find that lncRNAs participates in activation after transcribing, X chromosome silence, turned
A variety of important regulating and controlling processes, these regulating and controlling effects such as interference caused the extensive concern of people, mammal base in recent years after record
Because in group sequence 4%~9% transcript is lncRNA, and encoding histone RNA ratio only accounts for 1%, but most
LncRNA function is not yet revealed (Nat Rev Genet.2016；17(1):47-62).
Even more important to be, compared to the mRNA of encoding proteins matter, most of lncRNAs have tissue specificity, only about
1% lncRNAs has expression in whole body is respectively organized.Recent research result indicate that lncRNAs expression or dysfunction
It is closely related with the generation of human diseases, including the serious harm mankind such as tumour, disease of cardiovascular system, nerve regression disease
Major disease (the Nat Rev Genet.2016 of health；17(10):601-614), however, works of the lncRNAs in oophoroma
With and regulatory mechanism research be still in starting stage (Cancer Lett.2016；383(1):28-40).In order to study lncRNAs
Latent effect in ovary carcinogenesis, we are first with biochip technology (Arraystar Human LncRNA
Microarry V3.0) it have detected lncRNAs express spectras in epithelial ovarian carcinoma patients (table 1) cancerous tissue.Choose rise or drop
Low more than 2 times of lncRNAs is as difference expression gene.Thus, we are filtered out significantly elevated 1945 in ovarian cancer patients
Individual lncRNAs and significantly reduced 1582 difference lncRNAs, as a result such as hierarchical clustering figure (Fig. 1) and volcano figure (Fig. 2) institute
Show, while it was found that the lncRNA of these unconventionality expressions is related to a plurality of tumor signal path (Fig. 3).Then, we use
Quantitative PCR demonstrates one group of lncRNAs significantly raised in chip results, including FOXN3-AS1, CATIP-AS2, RP11-
124N14.3, SMAD5-AS1 (Fig. 4).It is worth noting that, wherein SMAD5-AS1 is raised in ovarian cancer patients cancerous tissue
6.7 times, 6.0 times are raised in serum, this shows the potential key player that SMAD5-AS1 is played in oophoroma, and SMAD5-AS1
Whether played a role in disease still unknown, whether suppression SMAD5-AS1 gene expressions play therapeutic action to disease also has no
Research.Based on the present situation of existing gene therapy technology, the application has synthesized LncRNA SMAD5-AS1 two siRNAs, and
SMAD5-AS1siRNA is demonstrated in experimentation first and suppresses human epithelial ovarian carcinoma cells proliferation, migration and the effect of invasion and attack, is ovary
The treatment of cancer provides new drug target, also provides new approaches for the clinical diagnosis and treatment of oophoroma.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of method and medicine of new prevention and treatment oophoroma.
In order to achieve the above object, present invention employs following technological means：
The present invention have detected lncRNAs express spectras in ovarian cancer patients cancerous tissue using biochip technology.Using biology
Bioinformatics analysis method carries out data analysis, and more than 2 times lncRNAs are raised and lowered as difference expression gene in selection.Thus,
Filter out notable elevated 1945 lncRNAs and significantly reduced 1582 difference lncRNAs, difference in ovarian cancer patients
The lncRNAs that path analysis result related lncRNAs discloses these unconventionality expressions may participate in adjusting multiple oophoroma correlation
Signal path.The present invention applies realtime fluorescent quantitative PCR experiment, and to ovarian cancer patients cancerous tissue, cancer beside organism, health is suffered from
The lncRNAs that some in person, serum of ovarian cancer patients significantly raise has carried out the detection of expression, as a result shows lncRNA
SMAD5-AS1 significantly 6.7 times of rises in ovarian cancer patients cancerous tissue, significantly 6 times of the rise in serum of ovarian cancer patients.Connect
, the present invention is tested by CCK8, scratch experiment and Transwell experiment have detected SMAD5-AS1siRNA-1 (SEQ respectively
Shown in ID NO.2), the shadow of SMAD5-AS1siRNA-2 (shown in SEQ ID NO.3) cell proliferation, migration and invasive ability
Ring, as a result show that siRNA-1 and siRNA-2 can efficiently suppress the expression of SMAD5-AS1 in SKOV3 cells, have notable
Suppress the effect of ovarian cancer cell progress.
Therefore, on the basis of the studies above, the siRNA that the present invention proposes LncRNA SMAD5-AS1 is preparing prevention
With the application in treatment ovarian cancer, described SMAD5-AS1 sequence such as SEQ ID NO:Shown in 1.
Wherein, it is preferred that described LncRNA SMAD5-AS1 siRNA is by suppressing the propagation of ovarian cancer cell, moving
Move and invasion and attack reach the purpose for treating oophoroma.
Wherein, it is preferred that described LncRNA SMAD5-AS siRNA sequence such as SEQ ID NO:2 or SEQ ID
NO:Shown in 3.
Further, the invention also provides a kind of pharmaceutical preparation for being used to treat oophoroma, containing effective dose
LncRNA SMAD5-AS siRNA or its nucleotide sequence trim and the carrier pharmaceutically received, described LncRNA
SMAD5-AS siRNA sequence such as SEQ ID NO:2 or SEQ ID NO:Shown in 3.
Wherein, it is preferred that described nucleotide sequence trim is in SEQ ID NO:2 shown or SEQ ID NO:Shown in 3
One or more of modifications in ribose modification, base modification and the phosphate backbones modification of any nucleotides are carried out on the basis of sequence
Combination so as to obtain nucleotide sequence trim；Described carrier is virus, nano particle, cholesterol or liposome.
Wherein, it is preferred that described pharmaceutical preparation is ejection preparation and oral formulations.
Further, the invention also provides described pharmaceutical preparation in prevention and treatment ovarian cancer is prepared
Purposes.
The it is proposed of the present invention provides new drug target and therapeutic modality for the treatment of oophoroma.
Brief description of the drawings
Fig. 1 is that the hierarchical clustering figure of chip data shows that ovarian cancer patients cancerous tissue is expressed with lncRNAs in cancer beside organism
Situation；
Fig. 2 is ovarian cancer patients cancerous tissue and middle lncRNAs differential expressions volcano figure in cancer beside organism；
Wherein, vertical gray line represents to raise more than 2 times (positive x-axis) respectively and reduces by more than 50% (negative sense x-axis), horizontal
Gray line represents that p value has significant difference for 0.05.Right side stain represents the gene of expression conspicuousness up-regulation；Left side ash point represents
Express the gene that conspicuousness is lowered；
Fig. 3 is the related path analysises of difference lncRNAs；
Fig. 4 be in real-time fluorescence quantitative PCR detection chip data the lncRNAs that significantly raises in ovarian cancer patients tissue and
Expression (n=8) in serum；**p<0.01, * * * p<0.001, show that difference has statistical significance；
Fig. 5 is CCK8 experiments detection each group cell propagation and vigor (n=3)；***p<0.001vs SiRNA-NC, show
Difference has statistical significance；
Fig. 6 is that cell scratch experiment detects each group cell migration ability (n=3)；**p<0.01vs SiRNA-NC, show
Difference has statistical significance；
Fig. 7 is Transwell experiment detection each group cell migration invasive abilities (n=3)；***p<0.001vs SiRNA-
NC, show that difference has statistical significance.
Embodiment
Below by embodiment the present invention is described further checking, all embodiments be only used for illustration the present invention,
Do not limit the scope of the invention.For the change done in the claims in the present invention or equivalent change, the present invention is each fallen within
Protection domain within.
Embodiment 1 confirms LncRNA SMAD5-AS1 up-regulated expressions in oophoroma
We using biochip technology (the ST Arrays from Affymetrix of GeneChip Exon 1.0,
Inc. lncRNAs express spectras in ovarian cancer patients (table 1) cancerous tissue) be have detected.The lncRNA chips include coming from RNAdb,
The transcript that the authoritative databases such as Refseq, NCBI Gene, UCSC Gene annotation is lncRNA, data are comprehensively reliable.Using
Bioinformatic analysis method carries out data analysis, and we choose and more than 2 times lncRNAs are raised and lowered as differential expression base
Cause.Thus, we filter out notable elevated 1945 lncRNAs and significantly reduced 1582 difference in ovarian cancer patients
LncRNAs, as a result as shown in hierarchical clustering figure (Fig. 1) and volcano figure (Fig. 2).We have done difference lncRNAs correlations simultaneously
Path analysis.As a result disclosing the lncRNAs of these unconventionality expressions may participate in adjusting multiple oophoroma related signal path (figure
3)。
The ovarian cancer patients essential information that the detection of the chip of table 1. is included
Next, we apply realtime fluorescent quantitative PCR experiment, to ovarian cancer patients cancerous tissue, cancer beside organism, health is suffered from
The lncRNAs that some in person, serum of ovarian cancer patients significantly raise has carried out the detection of expression, as a result shows lncRNA
SMAD5-AS1 significantly 6.7 times of rises in ovarian cancer patients cancerous tissue, significantly 6 times of rise (is schemed in serum of ovarian cancer patients
4)。
Embodiment 2 confirms that LncRNA SMAD5-AS1 siRNA-1, siRNA-2 suppress ovarian cancer cell progress
We cultivate people's epithelial ovarian cancerous cell line SKOV3 and tested, and cellar culture uses the RPMI containing 10%FBS
1640 culture mediums, 37 DEG C, 5%CO2Cultivated in saturated humidity incubator.It is grouped as follows：SMAD5-AS1 siRNA-1(SEQ ID
Shown in NO.2) transfection group, SMAD5-AS1 siRNA-2 (shown in SEQ ID NO.3) transfection group, siRNA-NC (SEQ ID NO.4
It is shown) transfection group.SMAD5-AS1 expression in real-time fluorescence quantitative PCR detection each group cell.
Cell transfecting method：The adherent cell to 60-80% is changed into serum free medium, diluted respectively with Opti-MEM
X-tremeGENE siRNA transfection reagents and siRNAs, are mixed and incubation 15-20 minutes at room temperature, by transfection composite plus
Enter to cell culture supernatant so that the final concentration of 100nM of siRNAs, change complete medium within 6 hours after transfection, continue to cultivate
24-48h is tested.
CCK8 experiments detection cell propagation and vigor：Each group cell in exponential phase is inoculated in 96 hole cell trainings
Support (every milliliter 5 × 10 of plate4Individual cell), per the μ l of hole 100.After cultivating 24h, 10 μ l CCK solution are added per hole, after vibration mixes
Continue to be incubated 4h, after vibration mixes, with absorbance at ELIASA measure 450nm.
Scratch experiment detects cell migration ability：Six orifice plates are seeded cells into, when cell reaches 95% degree of converging, with
The 10ul pipette tips cut (0.5mm) of sterilization, serum free medium culture 48h is changed immediately；0h and 48h is respectively in phase after cut
Taken pictures under poor microscope, observe wound healing degree.During cell relative migration ability=0h during cut distance -48h cut away from
From.
Transwell experiment detection cell migration invasive abilities：Basilar memebrane is coated with, Matrigel aggregates into gel, aquation
Basilar memebrane.Serum starvation, the μ l of cell suspension 100 are taken to add Transwell cells, 0.1% violet staining 20min, observation is carefully
Born of the same parents count；Cell is pre-processed with 30 μ g Matrigel glue, every group of cell top adds 1 × 106Cell 80ul, cell bottom adds
Enter cell culture fluid culture；Take out upper chamber, the cell that 4% paraformaldehyde is fixed on lower room film, Trypan Blue, count under light microscopic
Through the cell number of filtration membrane.
As a result show that siRNA-1 and siRNA-2 can effectively suppress the expression of SMAD5-AS1 in SKOV3 cells, suppress efficiency
Up to more than 75%, compared to NC groups, transfection siRNA-1 and siRNA-2 reduces SKOV3 cell viabilities to 62%, 58% respectively, table
Bright two kinds of siRNAs can be by suppressing SMAD5-AS1 expression inhibiting human epithelial ovarian carcinoma cells proliferation (Fig. 5)；Cell scratch experiment result
It has been shown that, after siRNA-1 and siRNA-2 48h are transfected, transfer ability significantly reduces (Fig. 6) SKOV3 cells；Transwell is real
Test result to show, transfection siRNA-1 and siRNA-2 can significantly inhibit SKOV3 invasive ability (Fig. 7).Above experimental result table
Bright, siRNA-1 and siRNA-2 can efficiently suppress the expression of SMAD5-AS1 in SKOV3 cells, have and significantly inhibit ovary
The effect of cancer cell progress, illustrate the new direction that both siRNAs can develop as ovarian cancer.
Sequence table
<110>Harbin Medical University
<120>Applications of the long-chain non-coding RNA SMAD5-AS1 siRNA in treatment of ovarian cancer
<130> KLPI170522
<160> 4
<170> PatentIn 3.5
<210> 1
<211> 1827
<212> DNA
<213> SMAD5-AS1 sequence
<400> 1
tgatctgaaa ggaatggaag cacaaaatga tgaataaggt atttttaaca aagatacatg 60
ggtaaattaa cagcagtaat gtaaaaaaga ctgagggagc aacaatgtgg aaagggaagg 120
aaaggaagct gtataggaac aatcacacaa ataatcatca aaattaaact ttataaatct 180
aaacatagta tctacaacct ttatgaaggt taagttttgt taaattgtct aagttttgca 240
tttttacaat tttcactgta gagaaaaaaa tattaatagt taccaaagga ataaaagact 300
aaaataattt gttatacatc tgcaaatagc tccttaaatt tggaacacta atttcataat 360
gatatttgga tccataaatg tgaaactgtc acactgaatg taggtactgt tttccccaca 420
atggatacta ctgcattggc atctagcaga aattgtaagc tatttggaat gttactaaac 480
ctactgggac taaggttttc aatcttttgt ctttcaaatt aaaattaaaa actaattcac 540
tctgggcaat tcaggtactc taaggaaatt caagtactct agttttaaat acgaattttc 600
aaaaacgcaa cccaagtacc ctttcctctc aacttctgtg aggccaagcc acgacctcca 660
cctattcaat ctttgctgct agataacaac ccttttggta gtgctaggca acaggcatga 720
tgtgacaaac ctagacatca aaacttttcc acagaaagat aaggtaaaca agatatactt 780
agcgccacac ccatttaaat gcacaagcaa ccaaaattgc tccctccccc cgcaacaccc 840
cccccccccc cccaaagctc cagctggtca ggaaacattg tctttacaat caagataaac 900
atctggctca gggtgttcag ccattcctca cctacaggct tgcctaagcc ccattctcca 960
atgccatcac caccagaacc ggaacattct gttgggaagc cggcaaacgt ccaaataccc 1020
caagtatcct cgcccgagtt ctgcaaccaa aaatctgttt tggctactga gcatgctcag 1080
acttaagacg ggcgcgccac atgggagttt tcaaagctta ggctccaaac tccacgtagt 1140
aacagcagct gaaaggagga gaggggaaaa ggggagggga agagactgga atattaattt 1200
gagccctgat attgaagtat aggacccggc cagcggctga atatggtgaa ggcaaaaaga 1260
cgctgctttg gcattctctt tgcagcacag agaagtttac atgaagtaga agaaatcctt 1320
gcagtggtga aaactgacat ggcaagtggc agaaaccagg tctaactacc agctgattgc 1380
agtagccggt cacaactgcc cagagtgtaa ttcccaccga taagcgcgga tccttatctt 1440
tgaaacacaa agcgacggcg gcacggattc atcaagccat tggacagctt cgcagtgcag 1500
cagcccaggc tgcccggccg agcaccgcag tcctatcaag gtttgctgta gctcagaagg 1560
ctctgcctac atgactactg cataccctac tgggagaatt ggaagaatgt gatgatggta 1620
ggagtcaccc agcatagaag catgagtgaa tgacatccca gagatctgca aaggatcctg 1680
gtagccactt caaagacttt ttctcccttc ctcagacaat catggcacat ggacatcaga 1740
ggaagaaatg gcgaagtttg tatgactcct ccagcagagt ccttggaaac aataaagctt 1800
cctttccaaa aaaaaaaaaa aaaaaaa 1827
<210> 2
<211> 23
<212> DNA
<213> siRNA-1
<400> 2
ccgataagcg cggatcctta tct 23
<210> 3
<211> 23
<212> DNA
<213> siRNA-2
<400> 3
tggcagaaac caggtctaac tac 23
<210> 4
<211> 21
<212> DNA
<213> siRNA-NC
<400> 4
uucuccgaac gugucacgut t 21
Claims (7)
1.LncRNA SMAD5-AS1 siRNA is preparing the application in preventing and treating ovarian cancer, it is characterised in that institute
The SMAD5-AS1 stated sequence such as SEQ ID NO:Shown in 1.
2. application as claimed in claim 1, it is characterised in that described LncRNA SMAD5-AS1 siRNA passes through suppression
Propagation, migration and the invasion and attack of ovarian cancer cell reach the purpose for the treatment of oophoroma.
3. application as claimed in claim 1, it is characterised in that described LncRNA SMAD5-AS siRNA sequence is such as
SEQ ID NO:2 or SEQ ID NO:Shown in 3.
4. a kind of pharmaceutical preparation for being used to treat oophoroma, it is characterised in that described pharmaceutical preparation is containing effective dose
LncRNA SMAD5-AS siRNA or its nucleotide sequence trim and the carrier pharmaceutically received, described LncRNA
SMAD5-AS siRNA sequence such as SEQ ID NO:2 or SEQ ID NO:Shown in 3.
5. pharmaceutical preparation as claimed in claim 4, it is characterised in that described nucleotide sequence trim is in SEQ ID NO:
2 shown or SEQ ID NO:Ribose modification, base modification and the phosphate backbones of any nucleotides are carried out on the basis of sequence shown in 3
The combination of one or more of modifications in modification is so as to the nucleotide sequence trim obtained；Described carrier is virus, nanometer
Grain, cholesterol or liposome.
6. the pharmaceutical preparation as described in claim 4 or 5, it is characterised in that described pharmaceutical preparation is for ejection preparation and orally
Preparation.
7. the pharmaceutical preparation as described in claim any one of 4-6 is preparing the purposes in preventing and treating ovarian cancer.
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| CN112826826A (en) * | 2021-02-03 | 2021-05-25 | 上海兰天生物医药科技有限公司 | Application of siRNA sequence in preparation of medicine for treating ovarian cancer |
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| CN112826826A (en) * | 2021-02-03 | 2021-05-25 | 上海兰天生物医药科技有限公司 | Application of siRNA sequence in preparation of medicine for treating ovarian cancer |
| CN112826826B (en) * | 2021-02-03 | 2023-03-17 | 上海兰天生物医药科技有限公司 | Application of siRNA sequence in preparation of medicine for treating ovarian cancer |
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