CN105462976A - Small interfering RNA for specific inhibition of TWIST1 gene expression and application thereof - Google Patents
Small interfering RNA for specific inhibition of TWIST1 gene expression and application thereof Download PDFInfo
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Abstract
The invention provides small interfering RNA for specific inhibition of TWIST1 gene expression and the application thereof. The positive-sense strand sequence and the antisense strand sequence of the small interfering RNA are shown in SEQ ID NO.1-2. The TWIST1 target sequence interfered by the small interfering RNA is shown in SEQ ID NO.3. The small interfering RNA inhibits TWIST1 gene expression by acting on the TWIST1 target sequence, can be used for preparing drugs for adjusting expression of TWIST1 genes in cells or organisms, can also be used for preparing drugs for treating tumor especially melanoma, and has broad application prospects.
Description
Technical field
The present invention relates to gene engineering technology field, relate in particular to siRNA and application thereof that a species specificity suppresses TWIST1 genetic expression.
Background technology
RNA disturbs (RNAinterference, RNAi) technology refer to high conservative during evolution, by double-stranded RNA (double-strandedRNA, dsRNA) phenomenon that bring out, the efficient selective degradation of homologous mRNA, RNA molecule is passed through to destroy specific mRNA, to suppress the biological procedures of certain genetic expression.This technology has some advantage following: 1 high efficiency; 2 specificitys; 3 position effects; 4 high stabilities; 5 propagabilities; 6 concentration-time dependencys.Owing to using RNAi technology can specific depletion or close the expression of specific gene, this technology becomes rapidly one of gene functional research and gene therapy research field research tool of greatest concern, has been widely used in the treatment field exploring gene function and communicable disease and malignant tumour.
Malignant melanoma (malignantmelanoma, MM): be a kind of malignant tumour stemming from melanocyte, be apt to occur in skin, grade malignancy is high, treatment difficulty is large.Although melanoma only accounts for 4% of whole dermatoma, accounting for 80% of dermatoma death toll, is a kind of malignant tumour that in dermatoma, mortality ratio is the highest.The data presentation of National Cancer Institute (NCI), between 2003-2007, number of patients is annual 20.1 people/10,000 people.By the end of 2010, estimate at 68130 patients and be diagnosed with melanoma, wherein therefore 8700 patients will die (http://www.cancer.gov/).The incidence of malignant melanoma constantly rises in worldwide, and the area that before comprising, sickness rate is low, it has become a kind of malignant disease of serious threat health of people.The trend increased year by year is had in Chinese melanoma patients.Beijing eight city statistical information display, within 2000, MM sickness rate is 0.2 example/100,000, has reached 1 example/100,000 by 2004.According to " China Dark melanoma diagnosis and treatment guide (2011 editions) " that Chinese Clinical tumour association (CSCO) the melanoma Committee of Experts delivers, the annual new cases of China about 20,000 example, has become one of disease seriously jeopardizing our people's health.
Twist1 is the transcription factor belonging to b helix-loop-helix family, is positioned at 7q21.2, comprises 2 exons and 1 intron.The molecular weight of this transcription factor is about 21kDa.The b helix-loop-helix domain of Twist1 is the mankind, and mouse, the frog, a lot of species camber such as fruit bat is guarded.In Twist1 albumen
109q-T
121be topmost structural domain, be responsible in conjunction with DNA.This gene is mainly expressed in the tissue of mesoderma origin.In human normal tissue, Twist1 at placenta, high expression level in cardiac muscle and the tissue such as skeletal muscle, at pancreas, kidney etc. organize in low expression, and in brain (ectodermal origin), not express in the tissue of lung and liver (endoderm origin).Twist1 can regulate and control a lot of downstream target gene, in this way, participates in controlling various biological processes and signal path part, such as FGF signal path, SHH signal path, and TGF signal β path etc.
Except playing a role in the normal tissue, Twist1 also with comprise mammary cancer, liver cancer, prostate cancer, cancer of the stomach, bladder cancer, esophageal squamous cell carcinoma is closely related with Several Kinds of Malignancies such as carcinoma of the pancreas.In these tumours, Twist1 can occur in tumour, plays the part of various rolls in development and transfer.Particularly can hinder cell aging and the apoptosis of tumor inducing, strengthen tumor stem cell colony, and cancer cell invasion and transfer can be helped, and an epithelium-leaf transforms (EMT).Wherein EMT is first step that transfer occurs tumour cell.Recent discovery, in mammary cancer, malignant melanoma and other tumours, Twist process LAN can be used as an index of prognosis mala, and many evidences show that Twist1 albumen also participates in regulating apoptosis.In human tumor, Twist1 activation may have very important clinical meaning, and Twist1 probably as the index instructing diagnosis, judging prognosis, and can be used as a novel targets of tumor biotherapy.
Lower the expression of TWIST1, effectively can reduce the ability of tumour opposing aging and apoptosis, suppress the generation of EMT, thus the migration and invasion ability of inhibition tumor cell.
Summary of the invention
The object of the present invention is to provide a kind of specificity can the suppression siRNA (siRNA) of TWIST1 genetic expression and apply.
The present invention, according to RNAi occurring principle, has designed and synthesized the siRNA for TWIST1 gene, and its GC content is 52.63%, and the length of positive-sense strand and antisense strand is 19nt.
Further, the positive-sense strand of siRNA duplex molecule of the present invention and antisense strand sequence are respectively:
Positive-sense strand: 5 ' CCGGAGACCUAGAUGUCAUdTdT-3 ', (SEQIDNO.1)
Antisense strand: 5 '-TdTdGGCCUCUGGAUCUACAGUA-3 ' (SEQIDNO.2).
Above-mentioned siRNA provided by the invention, can the expression amount of TWIST1 gene in specific downregulation cell or organism.
Further, siRNA of the present invention acts on the target sequence of TWIST1 gene and is: 5 '-CCGGAGACCTAGATGTCAT-3 ' (SEQIDNO.3).
A kind of DNA sequence dna of encoding siRNA of the present invention belongs to protection scope of the present invention.
The invention provides a kind of expression vector, it comprises the DNA sequence dna of above-mentioned siRNA of encoding.
A kind of host cell, it is the cell can expressing siRNA of the present invention.
A kind of medicine containing above-mentioned siRNA belongs to protection scope of the present invention.
Further, described medicine is anti-tumor drug.Described tumour is melanoma, mammary cancer, liver cancer, prostate cancer, cancer of the stomach, bladder cancer, esophageal squamous cell carcinoma and carcinoma of the pancreas.
Further, described medicine is immunotherapeutic agent.
A kind of clone expressing above-mentioned siRNA also belongs to protection scope of the present invention.
The invention provides the purposes in the TWIST1 genetic expression medicine of above-mentioned siRNA in preparation adjustment cell or organism.
The invention provides the purposes of above-mentioned siRNA in the medicine preparing inhibition tumor cell migration and invasion.
Preferably, described tumour cell is melanoma cell, breast cancer cell, liver cancer cell, prostate cancer cell, stomach cancer cell, transitional cell bladder carcinoma cell line, esophageal squamous cell carcinoma cell and pancreatic cancer cell.
The invention provides the purposes of above-mentioned siRNA in the medicine preparing disease therapy, described disease is melanoma, mammary cancer, liver cancer, prostate cancer, cancer of the stomach, bladder cancer, esophageal squamous cell carcinoma and carcinoma of the pancreas.
Effective targeted therapy is the auxiliary treating method constantly found both at home and abroad at present, provided by the invention selectively targeted for after the siRNA transfection melanoma cell of TWIST1 gene, find that siRNA significantly can lower the expression of TWIST1 gene, the migration and invasion ability of effective inhibition tumor cell, result for the treatment of for the new Antioncogene medicine of exploitation and tumour medicine has important guiding effect, can be used for exploitation and prepare antitumor drug, huge social benefit and economic worth will be obtained.
Accompanying drawing explanation
Fig. 1 is that siRNA of the present invention strikes low effect to A375 cell TWIST1 genetic expression.The wherein A375 cell of 1: untransfected siRNA; 2: the A375 cell of transfection negative control sequence; The A375 cell of 3: transfection siRNA.
Fig. 2 is that in A375 cell, TWIST1 gene mRNA strikes low-level electrophoresis detection result, wherein swimming lane 1: molecular weight marker; Swimming lane 2: the A375 cell of transfection siRNA; Swimming lane 3: the A375 cell of transfection negative control sequence; Swimming lane 4: the A375 cell of untransfected siRNA.
Fig. 3 is that A375 cell TWIST1 albumen strikes low-level Westernblot and detects, wherein swimming lane 1: the A375 cell of untransfected siRNA; Swimming lane 2: the A375 cell of transfection negative control sequence; Swimming lane 3: the A375 cell of transfection siRNA.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.
If do not specialize, chemical reagent used in embodiment is conventional commercial reagent, the conventional means that technique means used in embodiment is well known to those skilled in the art.
The design and synthesis of the siRNA of the selectively targeted TWIST1 gene of embodiment 1 and transfection melanoma cell
1, the design of the siRNA of selectively targeted TWIST1 gene
(network address is: http://sirna.wi.mit.edu/home.php to design website at public siRNA; Https: //www.thermofisher.com/cn/zh/home.html; Http:// sidirect2.rnai.jp/ etc.) the upper mRNA sequence inputting TWIST1, the siRNA sequence of low TWIST1mRNA expression can be struck according to website indication predicting.
The multiple website of comparison provide predict the outcome, select at least simultaneously at two all predicted siRNA arrived in website.
Screening does not have the siRNA sequence of bibliographical information.The siRNA sequence GC content of the present embodiment screening is 52.63%, and the length of positive-sense strand and antisense strand is 19nt.
Specifically, its positive-sense strand and antisense strand sequence are respectively:
Positive-sense strand: 5 ' CCGGAGACCUAGAUGUCAUdTdT-3 ' (SEQIDNO.1)
Antisense strand: 5 '-dTdTGGCCUCUGGAUCUACAGUA-3 ' (SEQIDNO.2).
The target sequence that siRNA of the present invention acts on TWIST1 gene is: 5 '-CCGGAGACCTAGATGTCAT-3 ' (SEQIDNO.3).
SiRNA and negative control sequence transfer to Rui Bo bio tech ltd, Guangzhou to synthesize.
2, siRNA transfection melanoma cell of the present invention
After melanoma cell A375 (purchased from ATCC, Americantypeculturecollection) is cultured to 80% in 10cm culture dish, with trysinization, add 6 orifice plates, make the final cell number in each hole be 5 × 10
5individual cell.
Transfection reagent prepares:
A.250 μ lOpti-MEM (Sai Mo flies generation, and you are scientific and technological, the U.S.)+5 μ lsiRNA (20uM), leave standstill 5 minutes.
B.250 μ lOpti-MEM (Sai Mo flies generation, and you are scientific and technological, the U.S.)+3 μ lLipofeactmine2000 (Sai Mo flies generation, and you are scientific and technological, the U.S.), leave standstill 5 minutes,
C. by after A and B gained reagent mix, 20 minutes are left standstill.Mixed transfection reagent is added in 6 orifice plates, mix with A375 cell.37 DEG C, cultivate after 48 hours, collecting cell.
The mrna expression level of TWIST1 gene is identified after embodiment 2siRNA transfection
1, real-time quantitative PCR (q-PCR) identifies the mrna expression level of the TWIST1 gene of the melanoma cell A375 of the siRNA of the present invention of transfection in embodiment 1
Table 1q-PCR reaction system
Front primer: AGCTACGCCTTCTCGGTCT
Rear primer: CCTTCTCTGGAAACAATGACATC.
Q-PCR response procedures: 95 DEG C of 5min; 95 DEG C of 15sec, 62 DEG C of 30sec, circulate 35 times.
Use real-time quantitative to carry the level of software analysis TWIST1 gene expression amount, the results are shown in Figure 1.Fig. 1 shows, and after A375 cell transfecting TWIST1 specific siRNA, TWIST1 expression amount is only equivalent to 52% of transfection negative control sequence (NC).The A375 cell of 1: untransfected siRNA; 2: the A375 cell of transfection negative control sequence; The A375 cell of 3: transfection siRNA.
PCR primer is carried out electrophoresis detection, and in A375 cell, TWIST1 gene mRNA strikes low-level electrophoresis detection and the results are shown in Figure after 2, Fig. 2 shows transfection siRNA, effectively can strike the expression of low TWIST1 gene.
TWIST1 protein expression level is identified after embodiment 3siRNA transfection
1, Westernblot identifies the protein expression level of TWIST1 gene
Preparation of samples: adherent melanoma cell A375:1 × PBS washes twice, 100 μ l trysinizations, 500 μ l nutrient solutions stop putting into 1.5mlEP pipe.With the rotating speed of 14000rpm centrifugal 5 minutes.Abandon supernatant, 200 μ l1 × PBS wash once.Add 50 μ l cell lysis buffer solution, concuss, make the abundant cracking of cell ,-20 DEG C of preservations.Total protein concentration is detected by BCA method.5 × sample-loading buffer is diluted to 1 ×, join in the cell sample of cracking.95 DEG C of thermally denature 5min, use the rotating speed of 11500rpm centrifugal 5 minutes subsequently.
2,12%SDS-PAGE glue is configured.
3, protein electrophorese: point sample, the minimum 20 μ g of each sample, protein markers point 3 μ l.Constant voltage 100V, 30 minutes, adjusts voltage to 120V, 1 hour subsequently.Transferring film: cellulose membrane soaks with methyl alcohol in advance, carries out transferring film, constant current 300mA, 2 hours with the transferring film damping fluid of precooling.Close: film is put into 5% skim milk powder solution (1g skim-milk+20ml1 × TTBS), vibrate 1 hour.Outwell skim milk powder solution, add primary antibodie, vibrate 1 hour.After removing primary antibodie, wash three times with 1 × TTBS, each 10 minutes.Add two subsequently to resist, vibrate 1 hour.Remove two anti-after, wash three times with 1 × TTBS, each 10 minutes.Preparation nitrite ion also evenly drops on film, exposure imaging.Three kinds of total protein of cell are carried out Westernblot detection, the results are shown in Figure 3, after result display transfection siRNA, effectively can strike the expression of low TWIST1 albumen.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. a siRNA, is characterized in that, its GC content is 52.63%, and the length of positive-sense strand and antisense strand is 19nt.
2. siRNA as claimed in claim 1, its positive-sense strand and antisense strand sequence are respectively:
Positive-sense strand: 5 ' CCGGAGACCUAGAUGUCAUdTdT-3 ',
Antisense strand: 5 '-dTdTGGCCUCUGGAUCUACAGUA-3 '.
3. siRNA as claimed in claim 2, it is characterized in that, the target sequence that siRNA acts on TWIST1 gene is: 5 '-CCGGAGACCTAGATGTCAT-3 '.
4. a coding as arbitrary in claim 1-3 as described in the DNA sequence dna of siRNA.
5. an expression vector, it comprises DNA sequence dna according to claim 4.
6. the medicine containing the arbitrary described siRNA of claim 1-3.
7. medicine as claimed in claim 6, it is antitumor drug.
8. the purposes in the TWIST1 genetic expression medicine of the arbitrary described siRNA of claim 1-3 in preparation adjustment cell or organism.
9. the arbitrary described purposes of siRNA in the medicine preparing inhibition tumor cell migration and invasion of claim 1-3.
10. the arbitrary described purposes of siRNA in the medicine preparing disease therapy of claim 1-3, described disease is melanoma, mammary cancer, liver cancer, prostate cancer, cancer of the stomach, bladder cancer, esophageal squamous cell carcinoma and carcinoma of the pancreas.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113897357A (en) * | 2020-07-06 | 2022-01-07 | 北京大学 | Twist1 gene editing system and application thereof in preparation of product for treating triple negative breast cancer |
| CN117757947A (en) * | 2024-02-21 | 2024-03-26 | 上海金翌生物科技有限公司 | Primer group, probe group, kit and method for detecting methylation level of bladder cancer biomarker |
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| US20110044895A1 (en) * | 2007-07-12 | 2011-02-24 | Berry David A | Methods and compositions for reducing stemness in oncogenesis |
| US20140314854A1 (en) * | 2012-04-19 | 2014-10-23 | Silenseed Ltd. | METHODS AND COMPOSITIONS FOR RNAi-BASED CANCER TREATMENT |
Non-Patent Citations (2)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113897357A (en) * | 2020-07-06 | 2022-01-07 | 北京大学 | Twist1 gene editing system and application thereof in preparation of product for treating triple negative breast cancer |
| CN117757947A (en) * | 2024-02-21 | 2024-03-26 | 上海金翌生物科技有限公司 | Primer group, probe group, kit and method for detecting methylation level of bladder cancer biomarker |
| CN117757947B (en) * | 2024-02-21 | 2024-07-09 | 上海金翌生物科技有限公司 | Primer group, probe group, kit and method for detecting methylation level of bladder cancer biomarker |
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Application publication date: 20160406 |