CN110055253A - A kind of siRNA molecule and its application for human cystatin E B - Google Patents
A kind of siRNA molecule and its application for human cystatin E B Download PDFInfo
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- CN110055253A CN110055253A CN201910351398.6A CN201910351398A CN110055253A CN 110055253 A CN110055253 A CN 110055253A CN 201910351398 A CN201910351398 A CN 201910351398A CN 110055253 A CN110055253 A CN 110055253A
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- 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
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- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
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Abstract
The present invention relates to a kind of siRNA molecules and its application for being directed to human cystatin E B (CSTB).The present invention devises the DNA double chain-ordering for striking and dropping effective siRNA and expression shRNA for people CSTB, and construct the slow virus carrier comprising above-mentioned DNA double chain-ordering, it is tested by ovarian cancer cell biological function, confirm that siRNA of the present invention and the construction of expression shRNA can significantly reduce CSTB mRNA and expressing quantity, human epithelial ovarian carcinoma cells proliferation is caused to inhibit, make cell block in the G2/M phase, and promote ovarian cellular apoptosis, therefore small molecule therapeutic, or the research and development reagent of research oophoroma pathomechanism be can be developed into.
Description
Technical field
The present invention relates to oncomolecularbiology technical fields, specifically, being related to a kind of for human cysteine albumen
The siRNA molecule and its application of enzyme inhibitor B.
Background technique
Oophoroma is tumor incidence ranking the 8th in global women tumour, but its lethality is highest
Gynecological cancer.Main cause is that oophoroma early symptom is unobvious, and after diagnosing clinical one, the overwhelming majority is in advanced stage, prognosis
Difference.The biomarker that searching can be used for diagnosing and treating oophoroma is highly desirable.
The applicant's previous work has been found that Cystatin B (Cystatin B, CSTB) is people's ovum
The tick mark object of nest cancer (Ovarian Cancer, OC), while finding that transforming growth factor (TGF-β) 1 can regulate and control CSTB
Expression (Int J Oncol 2014,44:1099).However function of the CSTB in OC, and specific work is regulated and controled by TGF-β 1
It is unclear with mechanism.
It is a Gene silence that RNA, which interferes (RNA interference, RNAi), is a kind of double-stranded RNA
(double-stranded RNA, dsRNA) molecule blocks the expression of specific gene in mRNA level in-site or makes the mistake of its silencing
Journey, the i.e. posttranscriptional gene silencing (Post-transcriptional gene silencing, PTGS) of sequence-specific.It adopts
With the important supplement that the function that RNAi technology research gene is exercised during tumor invasion is to Tumorigenesis research.And
And RNAi has become the available strategy of therapy of tumor at present.It can inhibit the suppression of proto-oncogene, mutation using RNAi technology
The expression such as oncogene, Cell cycle-related genes, anti-apoptotic related gene inhibit the occurrence and development of tumour.Periodical literature
(Oncology Research, 2016, Vol.24, pp.487-494) disclose CSTB gastric cancer generation in key effect and
Possible oversight mechanism, using Human gastric cancer SGC-7901 cells as external model 2000 transfected plasmids of Lipofectamine
PCDNA3.1-CSTB and siRNA-CSTB, carry out quantitatively real-time PCR (qRT-PCR) and western blot to determine CSTB gene
With the relative expression of albumen, cell Proliferation, migration and apoptosis are assessed by mtt assay, Transwell, flow cytometry respectively, tied
Fruit shows that compared with gastric epithelial cell, SGC-7901 cell CSTB is significantly lowered, and pc-CSTB and si-CSTB transfect cell respectively
Afterwards, CSTB is overexpressed or is suppressed, and compared with the control, the cell survival rate and mobility for transfecting pc-CSTB significantly reduce, cell
Apoptosis increases, and the cell survival rate and mobility for transfecting si-CSTB dramatically increase, and Apoptosis is reduced, the results showed that CSTB can
Using the potential treatment target as gastric cancer.However CSTB plays inhibiting effect to gastric cancer in the document, si-CSTB pairs in text
CSTB strike drop effect it is general.
In conclusion having no report of the CSTB in terms for the treatment of of ovarian cancer, also has no and strikes drop significant effect for CSTB,
The siRNA molecule for having drug development prospect.
Summary of the invention
The purpose of the present invention is aiming at the shortcomings in the prior art, provide for human cystatin E B, tool
SiRNA molecule, Related product and their purposes of standby drug and research and development reagent development prospect.
In a first aspect, the siRNA molecule includes following sequence the present invention provides a kind of siRNA molecule:
Positive-sense strand: 5'-GGACAAACUACUUCAUCAA-3',
Antisense strand: 5'-UUGAUGAAGUAGUUUGUCC-3'.
Second aspect, the present invention provides a kind of for striking the DNA double chain of drop human cystatin E 1 B gene
Sequence, the DNA double chain-ordering include following sequence:
Upstream chain: 5'-ccggGGACAAACTACTTCATCAACTCGAGTTGATGAAGTAGTTTGTCCTTTTT Tg-3',
Downstream chain: 5'-aattcAAAAAAGGACAAACTACTTCATCAACTCGAGTTGATGAAGTAGTTTGT CC-3'.
The third aspect, the present invention provides a kind of construction, the construction includes following DNA double chain-ordering:
Upstream chain: 5'-ccggGGACAAACTACTTCATCAACTCGAGTTGATGAAGTAGTTTGTCCTTTTT Tg-3'；
Downstream chain: 5'-aattcAAAAAAGGACAAACTACTTCATCAACTCGAGTTGATGAAGTAGTTTGT CC-3'.
As a preference, the construction is slow virus carrier.
Fourth aspect, the present invention provides a kind of lentiviral particle, the lentiviral particle includes structure as described above
Build object.
5th aspect, the present invention provides a kind of cell model of human cystatin E 1 B gene silencing, institutes
The cell model stated includes lentiviral particle as described above.
6th aspect, the present invention provides the siRNA molecule, the DNA double chain-ordering, the construction,
The application of the lentiviral particle or the cell model in the drug of preparation treatment oophoroma.
7th aspect, the present invention provides the siRNA molecule, the DNA double chain-ordering, the construction,
The lentiviral particle or the cell model inhibit in human epithelial ovarian carcinoma cells proliferation, migration or the reagent of invasion in preparation
Using.
As a preference, the ovarian cancer cell is from Ovarian Cancer Cells OVCAR-3.
Eighth aspect, the present invention provides the siRNA molecule, the DNA double chain-ordering, the construction,
The lentiviral particle or the cell model are in the inhibition human epithelial ovarian carcinoma cells proliferation of non-treatment purpose, migration or invasion
Application.
The invention has the advantages that:
1, suitable siRNA is devised for people CSTB and express the DNA double chain-ordering of shRNA, and construct comprising upper
State the slow virus carrier of DNA double chain-ordering.Experiment shows that the construction of siRNA and expression shRNA of the invention can be significant
CSTB mRNA and expressing quantity are reduced, it is fairly obvious to strike drop effect, is significantly better than other siRNA, therefore can be developed into small point
Sub- therapeutic agent, or the research and development reagent of research oophoroma pathomechanism.
2, the treatment means for CSTB gene are put forward for the first time in oophoroma.
Detailed description of the invention
Attached drawing 1:si-CSTB-1, si-CSTB-2, si-CSTB-3 lower CSTB mRNA and albumen table in OVCAR-3 cell
It reaches.
Attached drawing 2: the human epithelial ovarian carcinoma cells proliferation situation of si-CSTB-2 is transfected.
Attached drawing 3:(A, B) CSTB protein expression situation；(C) ovarian cancer cell is in 72h proliferative conditions；(D, E, F) cell week
Phase testing result.
Attached drawing 4:(A, B, C) ovarian cellular apoptosis testing result；(D) the WB testing result of BAX albumen.
Specific embodiment
It elaborates with reference to the accompanying drawing to specific embodiment provided by the invention.
Embodiment 1
1, experimental method
1. the building of CSTB-shRNA slow virus carrier
ShRNA slow virus structure is carried out using slow virus carrier pLKO.1-TRC cloning vector (addgene article No. 10878)
It builds.Key step is as follows:
A) double digestion is carried out using AgeI and EcoRI restriction enzyme to pLKO.1-TRC carrier；
B) synthesis CSTB-shRNA and NC-shRNA sequence is annealed, and sequence is as follows:
CSTB-shRNA carrier insetion sequence:
sense 5'-ccggGGACAAACTACTTCATCAACTCGAGTTGATGAAGTAGTTTGTCCTTTTTTg-3'
(SEQ ID NO:1),
antisense 5'-aattcAAAAAAGGACAAACTACTTCATCAACTCGAGTTGATGAAGTAGTTTGTCC-
3'(SEQ ID NO:2)；
The insetion sequence of NC-shRNA carrier are as follows:
sense 5'-ccggTTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAATTTTTTg-3'
(SEQ ID NO:3),
antisense 5'-aattcAAAAAATTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAA-
3'(SEQ ID NO:4)。
C) the pLKO.1-TRC carrier of anneal sequence insertion linearisation is constructed CSTB-shRNA and NC-shRNA is slow again
Viral vectors.
It will building slow virus carrier and pMD2.G (addgene article No. 12259) and pCMV-dR8.2 (addgene article No.
8455) cotransfection HEK293T packs out slow virus.
2. influence of the CSTB to human epithelial ovarian carcinoma cells proliferation
A) Ovarian Cancer Cells OVCAR-3 is selected, strikes low CSTB using slow virus technology, building stabilization strikes low CSTB cell
Strain OV-CSTB-shRNA, slow virus NC-shRNA construct cell strain OV-NC-shRNA as control.Pass through
Immunoblotting verifies CSTB and knocks out efficiency.
B) CCK-8 method: OV-NC-shRNA and OV-CSTB-shRNA are counted to (hole 4000cells/) respectively and are seeded in 96
Be incubated in orifice plate for 24 hours, 48h, 72h.CCK8 detection reagent is added at every point of time, measures OD extinction in 450nm after 2 hours
Degree.
3. influence of the CSTB to the ovarian cancer cell period
A) OV-NC-shRNA and OV-CSTB-shRNA cell Flow Cytometry: is laid in 6 orifice plates (cell respectively
300000/hole), until pancreatin digestion, complete medium terminates digestion, 1000rpm, 5min when cell confluency degree is up to 85%~90%；
PBS 2ml is washed 1 time；1000rpm, 5min；With 300 μ l PBS hang cell, be added 700 μ l be pre-chilled dehydrated alcohol, 4 DEG C of fixed 3h with
On；1000rpm, 5min；Abandon supernatant, PBS cleaning, 1000rpm, 5min；Supernatant is abandoned, every pipe adds 500 μ l PI dyestuff (BD, cat
550825), room temperature is protected from light 15min, flow cytometer cell cycle analysis, and experiment is repeated 3 times.
4. influence of the CSTB to ovarian cellular apoptosis
A) OV-NC-shRNA and OV-CSTB-shRNA cell Flow Cytometry: is laid in 6 orifice plates (cell respectively
300000/hole), until collecting cell culture medium when cell confluency degree is up to 85%~90%, digested with without EDTA pancreatin, is trained completely
It supports base and terminates digestion, 1000rpm, 5min；PBS 2ml is washed 2 times；1000rpm, 5min；Abandon supernatant, be added 100 μ l 1 ×
After Binding Buffer, every hole is separately added into 5 μ l PI and 1 μ l APC-AnnixV (this process is protected from light)；4 degree of dyeing 15min
Afterwards, every hole complements to 500 μ l 1 × Binding Buffer, is protected from light, mixes gently；Flow cytometer Apoptosis assay, it is real
It tests and is repeated 3 times.
B) protein blot is tested: detection pro apoptotic protein Bax.
2, experimental result
1. CSTB influences human epithelial ovarian carcinoma cells proliferation function
A variety of siRNA are devised, wherein the information of si-CSTB-1, si-CSTB-2, si-CSTB-3 are shown in Table 1.Compared to right
According to three of the above siRNA can lower CSTB mRNA and protein expression in OVCAR-3 cell, but si-CSTB-2 strikes drop effect
It is the most obvious, up to 90% or more, it is significantly higher than si-CSTB-1, si-CSTB-3 and others siRNA (P < 0.05) (Fig. 1).
Table 1.siRNA design
As shown in Fig. 2, significant Proliferation Ability occurs for the OVCAR-3 Ovarian Cancer Cells of transfection si-CSTB-2.
As shown in figure 3, (A, B) after low CSTB albumen is struck using the sh-RNA slow virus that si-CSTB-2 is constructed, relative to
Sh-NC group can cause ovarian cancer cell that significant Proliferation Ability (C) occurs in 72h；Cell cycle interpretation of result shows to strike low
CSTB albumen can cause cell block at G2/M phase (D, E), and three repeated experiments statistics is significant (F), and experiment repeats
Three times.* P < 0.01 P < 0.05, * *.
2. striking low CSTB causes ovarian cellular apoptosis
As shown in figure 4, after striking low CSTB albumen using the sh-CSTB slow virus that si-CSTB-2 is constructed, relative to sh-NC
Group, 48h can cause ovarian cancer cell viable apoptotic cell to increase (A, B, C).WB detection in 48 hours, after striking low CSTB albumen, phase
For sh-NC group, BAX protein upregulation (D).Experiment is repeated three times.*P<0.05.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, under the premise of not departing from the method for the present invention, can also make several improvement and supplement, these are improved and supplement also should be regarded as
Protection scope of the present invention.
SEQUENCE LISTING
<110>Jinshan Hospital Fudan University
<120>a kind of siRNA molecule and its application for human cystatin E B
<130> /
<160> 10
<170> PatentIn version 3.3
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Claims (10)
1. a kind of siRNA molecule, which is characterized in that the siRNA molecule includes following sequence:
Positive-sense strand: 5'-GGACAAACUACUUCAUCAA-3',
Antisense strand: 5'-UUGAUGAAGUAGUUUGUCC-3'.
2. a kind of for striking the DNA double chain-ordering of drop human cystatin E 1 B gene, which is characterized in that described
DNA double chain-ordering includes following sequence:
Upstream chain: 5'-ccggGGACAAACTACTTCATCAACTCGAGTTGATGAAGTAGTTTGTCCTTTTT Tg-3',
Downstream chain: 5'-aattcAAAAAAGGACAAACTACTTCATCAACTCGAGTTGATGAAGTAGTTTGT CC-3'.
3. a kind of construction, which is characterized in that the construction includes following DNA double chain-ordering:
Upstream chain: 5'-ccggGGACAAACTACTTCATCAACTCGAGTTGATGAAGTAGTTTGTCCTTTTT Tg-3'；
Downstream chain: 5'-aattcAAAAAAGGACAAACTACTTCATCAACTCGAGTTGATGAAGTAGTTTGT CC-3'.
4. construction according to claim 3, which is characterized in that the construction is slow virus carrier.
5. a kind of lentiviral particle, which is characterized in that the lentiviral particle includes construction as claimed in claim 3.
6. a kind of cell model of human cystatin E 1 B gene silencing, which is characterized in that the cell model
Include lentiviral particle as claimed in claim 5.
7. siRNA molecule described in claim 1, DNA double chain-ordering as claimed in claim 2, building as claimed in claim 3
Lentiviral particle described in object, claim 5 or cell model as claimed in claim 6 are in the drug of preparation treatment oophoroma
Application.
8. siRNA molecule described in claim 1, DNA double chain-ordering as claimed in claim 2, building as claimed in claim 3
Lentiviral particle described in object, claim 5 or cell model as claimed in claim 6 increase in preparation inhibition ovarian cancer cell
The application in reagent grown, migrate or invaded.
9. application according to claim 8, which is characterized in that the ovarian cancer cell is from Ovarian Cancer Cells
OVCAR-3。
10. siRNA molecule described in claim 1, DNA double chain-ordering as claimed in claim 2, structure as claimed in claim 3
Lentiviral particle described in object, claim 5 or cell model as claimed in claim 6 are built in the inhibition ovary of non-treatment purpose
Application in cancer cell multiplication, migration or invasion.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113025579A (en) * | 2021-04-22 | 2021-06-25 | 河南农业大学 | ST-KDABHD16A cell line for stably knocking down pig abhd16a gene and construction method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103397019A (en) * | 2003-11-21 | 2013-11-20 | 雷维维科公司 | Use of interfering RNA in production of transgenic animals |
| US20170307616A1 (en) * | 2014-10-02 | 2017-10-26 | Dana-Farber Cancer Institute, Inc. | Compositions and methods for treating malignancies |
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2019
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103397019A (en) * | 2003-11-21 | 2013-11-20 | 雷维维科公司 | Use of interfering RNA in production of transgenic animals |
| US20170307616A1 (en) * | 2014-10-02 | 2017-10-26 | Dana-Farber Cancer Institute, Inc. | Compositions and methods for treating malignancies |
Non-Patent Citations (2)
| Title |
|---|
| XINGXING WANG等: "Cystatin B is a progression marker of human epithelial ovarian tumors mediated by the TGF-β signaling pathway", 《INTERNATIONAL JOURNAL OF ONCOLOGY》 * |
| 汪星星: "半胱氨酸蛋白酶抑制素B在人上皮性卵巢肿瘤中的作用", 《中国优秀硕士论文全文库医药卫生辑》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113025579A (en) * | 2021-04-22 | 2021-06-25 | 河南农业大学 | ST-KDABHD16A cell line for stably knocking down pig abhd16a gene and construction method thereof |
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Application publication date: 20190726 |