CN105462979B - One species specificity suppresses siRNA and its application of TWIST1 gene expressions - Google Patents
One species specificity suppresses siRNA and its application of TWIST1 gene expressions Download PDFInfo
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- CN105462979B CN105462979B CN201510959113.9A CN201510959113A CN105462979B CN 105462979 B CN105462979 B CN 105462979B CN 201510959113 A CN201510959113 A CN 201510959113A CN 105462979 B CN105462979 B CN 105462979B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
Abstract
The invention provides siRNA and its application that a species specificity suppresses TWIST1 genes.The positive-sense strand and antisense strand sequence of siRNA of the present invention are respectively as shown in SEQ ID NO.1 2.The TWIST1 target sequences of siRNA interference are as shown in SEQ ID NO.3, it suppresses TWIST1 gene expressions by acting on TWIST1 target sequences and realizing, available for the medicine for preparing TWIST1 gene expressions in regulation cell or organism, it can also be used for preparing the medicine for the treatment of tumour, especially melanoma, have broad application prospects.
Description
Technical field
The present invention relates to gene engineering technology field, relates in particular to a species specificity and suppresses TWIST1 gene expressions
SiRNA and its application.
Background technology
RNA interference (RNA interference, RNAi) technology refer to it is being highly conserved during evolution, by double
Phenomenon that chain RNA (double-stranded RNA, dsRNA) induces, the efficient selective degradation of homologous mRNA, RNA molecule are led to
Cross and destroy specific mRNA, to suppress the biological process of certain gene expression.The technology has some following advantage：1 is efficient
Property；2 specificity；3 position effects；4 high stabilities；5 propagabilities；6 concentration-time dependences.Due to can be with using RNAi technology
Specific depletion or the expression for closing specific gene, the technology rapidly become gene functional research and gene therapy research field most
One of concerned research tool, have been widely used for exploring gene function and the treatment neck of communicable disease and malignant tumour
Domain.
Malignant mela noma (malignant melanoma, MM)：A kind of malignant tumour of melanocyte is derived from,
Skin is apt to occur in, grade malignancy is high, and treatment difficulty is big.Although melanoma only accounts for the 4% of whole skin neoplasins, account for skin and swell
The 80% of knurl death toll, it is a kind of malignant tumour of death rate highest in skin neoplasin.National Cancer Institute (NCI)
Data show that between 2003-2007, number of patients is annual 20.1 people/10,000 people.By the end of 2010, it is estimated to be
68130 patients are diagnosed with melanoma, (the http wherein therefore 8700 patients will die://
www.cancer.gov/).The incidence of malignant mela noma constantly rises in worldwide, including hair in the past
The low area of sick rate, it turns into a kind of malignant disease of serious threat health of people.Have year by year in Chinese melanoma patients
The trend increased.The city statistics of Beijing eight shows that the MM incidences of disease are 0.2/100,000 within 2000, have been reached by 2004
1/100,000.Delivered according to (CSCO) the melanoma Committee of Experts of Chinese Clinical tumour association《Chinese melanoma diagnosis and treatment
Guide (2011 editions)》, the annual new cases in China about 20,000, have become the serious disease for jeopardizing the health of our people it
One.
Twist1 is the transcription factor for belonging to b helix-loop-helixs family, positioned at 7q21.2, includes 2 extrons and 1
Introne.The molecular weight of the transcription factor is about 21kDa.Twist1 b helix-loop-helix domains are in the mankind, mouse, the frog,
It is highly conserved in many species such as drosophila.In Twist1 albumen109Q-T121It is most important domain, is responsible for combining DNA.The base
Because mainly being expressed in the tissue of mesoderma origin.In human normal tissue, Twist1 is in placenta, the group such as cardiac muscle and skeletal muscle
Knit middle high expression, the low expression in the tissue such as pancreas, kidney, and in brain (ectodermal origin), lung and liver (endoderm origin)
Do not expressed in tissue.Twist1 can regulate and control many downstream target genes, in this way, to participate in controlling various lifes
Thing process and signal path part, such as FGF signal paths, SHH signal paths, and TGF signal beta paths etc..
Except playing a role in the normal tissue, Twist1 also with including breast cancer, liver cancer, prostate cancer, stomach cancer, bladder
The Several Kinds of Malignancy such as cancer, esophageal squamous cell carcinoma and cancer of pancreas are closely related.In these tumours, Twist1 can be in tumour
Occur, play the part of various rolls in development and transfer.Cell ageing and the apoptosis of tumor inducing can be particularly hindered, strengthens tumour
Stem cell population, and cancer cell invasion and transfer can be helped, and an epithelium-leaf conversion (EMT).Wherein EMT is tumour
First step that cell shifts.It has recently been found that Twist crosses table in breast cancer, malignant mela noma and other tumours
Up to the index that can be used as prognosis mala, many evidences show that Twist1 albumen also assists in regulation Apoptosis.Swollen in the mankind
Twist1 activation may have a very important clinical meaning in knurl, Twist1 be likely to as instruct to diagnose, judging prognosis
Index, and can be as a novel targets of tumor biotherapy.
TWIST1 expression is lowered, the ability of tumour resistance aging and apoptosis can be effectively reduced, suppress EMT generation,
So as to suppress the migration of tumour cell and invasive ability.
The content of the invention
It is an object of the invention to provide a kind of siRNA for being capable of specificity suppression TWIST1 gene expressions
(siRNA) and its apply.
The present invention has designed and synthesized the siRNA for TWIST1 genes, its G/C content according to RNAi occurring principles
For 36.84%, and the length of positive-sense strand and antisense strand is 19nt.
Further, the positive-sense strand of siRNA duplex molecule of the invention and antisense strand sequence are respectively：
Positive-sense strand：5 '-CAUUCUGAUAGAAGUCUGAdTdT-3 ', (SEQ ID NO.1)
Antisense strand：5’-TdTdGUAAGACUAUCUUCAGACU-3’(SEQ ID NO.2).
Above-mentioned siRNA provided by the invention, can in specific downregulation cell or organism TWIST1 genes table
Up to amount.
Further, the target sequence that siRNA of the invention acts on TWIST1 genes is：5’-
CATTCTGATAGAAGTCTGA-3’(SEQ ID NO.3)。
A kind of DNA sequence dna for encoding siRNA of the present invention belongs to protection scope of the present invention.
The present invention provides a kind of expression vector, and it includes the DNA sequence dna for encoding above-mentioned siRNA.
A kind of host cell, it is the cell that can express 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, breast cancer, liver cancer, preceding
Row gland cancer, stomach cancer, carcinoma of urinary bladder, esophageal squamous cell carcinoma and cancer of pancreas.
Further, described medicine is immunotherapeutic agent.
A kind of cell line for expressing above-mentioned siRNA falls within protection scope of the present invention.
The invention provides TWIST1 gene expression medicine of the above-mentioned siRNA in regulation cell or organism is prepared
In purposes.
The invention provides purposes of the above-mentioned siRNA in the medicine for suppressing tumor cell migration and invasion and attack is prepared.
Preferably, the tumour cell is melanoma cells, breast cancer cell, liver cancer cells, prostate gland cancer cell, stomach
Cancer cell, transitional cell bladder carcinoma cell line, esophageal squamous cell carcinoma cell and pancreatic cancer cell.
The invention provides purposes of the above-mentioned siRNA in the medicine for preparing treatment disease, described disease is black
Melanoma, breast cancer, liver cancer, prostate cancer, stomach cancer, carcinoma of urinary bladder, esophageal squamous cell carcinoma and cancer of pancreas.
Effective targeted therapy is the auxiliary treating method constantly looked for both at home and abroad at present, specific target provided by the invention
To after the siRNA transfection melanoma cells for TWIST1 genes, it is found that siRNA can significantly lower the table of TWIST1 genes
Reach, effectively suppress migration and the invasive ability of tumour cell, for developing controlling for new Antioncogene medicine and tumour medicine
Therapeutic effect has important guiding effect, and antineoplastic is prepared available for exploitation, it will obtains huge social benefit and economy
Value.
Brief description of the drawings
The siRNA that Fig. 1 is the present invention strikes poorly efficient fruit to A375 cell TWIST1 gene expressions.Wherein 1：Untransfected
SiRNA A375 cells；2：Transfect the A375 cells of negative control sequence；3：Transfect siRNA A375 cells.
Fig. 2 is that TWIST1 gene mRNAs strike low-level electrophoresis detection result, wherein swimming lane 1 in A375 cells：Molecular weight
Mark；Swimming lane 2：Transfect siRNA A375 cells；Swimming lane 3：Transfect the A375 cells of negative control sequence；Swimming lane 4：Untransfected
SiRNA A375 cells.
Fig. 3 is that A375 cell TWIST1 albumen strikes low-level Western blot detections, wherein swimming lane 1：Untransfected
SiRNA A375 cells；Swimming lane 2：Transfect the A375 cells of negative control sequence；Swimming lane 3：Transfect siRNA A375 cells.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Without departing substantially from spirit of the invention
In the case of essence, the modifications or substitutions made to the inventive method, step or condition belong to the scope of the present invention.
Unless otherwise specified, chemical reagent used in embodiment is conventional commercial reagent, skill used in embodiment
The conventional meanses that art means are well known to those skilled in the art.
The siRNA of the selectively targeted TWIST1 genes of embodiment 1 design synthesis and transfection melanoma cells
1st, the siRNA of selectively targeted TWIST1 genes design
In public siRNA designs website, (network address is：http://sirna.wi.mit.edu/home.php；
https://www.thermofisher.com/cn/zh/home.html；http://sidirect2.rnai.jp/ etc.) on it is defeated
Enter TWIST1 mRNA sequence, the siRNA sequence of low TWIST1mRNA expression can be struck according to website indication predicting.
The prediction result provided of multiple websites is compared, selects at least while be predicted to two websites
siRNA。
Screening does not have the siRNA sequence of document report.The siRNA sequence G/C content of the present embodiment screening is 36.84%, 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 ' CAUUCUGAUAGAAGUCUGAdTdT-3 ', (SEQ ID NO.1)
Antisense strand：5’-dTdTGUAAGACUAUCUUCAGACU-3’(SEQ ID NO.2).
The siRNA of the present invention acts on the target sequences of TWIST1 genes：5’-GAGTATGTGATCAAGGTCA-3’
(SEQ ID NO.3)。
SiRNA and negative control sequence transfer to Guangzhou Rui Bo bio tech ltd to synthesize.
2nd, siRNA of the invention transfection melanoma cells
Melanoma cells A375 (being purchased from ATCC, American type culture collection) cultivates in 10cm
Cultivate to after 80% in ware, digested with pancreatin, add 6 orifice plates, the final cell number for making each hole is 5 × 105Individual cell.
Transfection reagent prepares：
A.250 μ l Opti-MEM (the silent winged generation that science and technology of match, the U.S.)+5 μ l siRNA (20uM), stand 5 minutes.
B.250 (the silent winged generation of match of μ l Opti-MEM (the silent winged generation that science and technology of match, the U.S.)+3 μ l Lipofeactmine 2000
You are scientific and technological, the U.S.), 5 minutes are stood,
C. after A is mixed with reagent obtained by B, 20 minutes are stood.Mixed transfection reagent is added in 6 orifice plates, with
A375 cells mix.37 DEG C, after cultivating 48 hours, collect cell.
The mRNA expressions of TWIST1 genes are identified after the siRNA of embodiment 2 transfections
1st, siRNA of the present invention melanoma cells A375 has been transfected in real-time quantitative PCR (q-PCR) identification embodiment 1
TWIST1 genes mRNA expressions
The q-PCR reaction systems of table 1
Preceding primer：AGCTACGCCTTCTCGGTCT
Primer afterwards：CCTTCTCTGGAAACAATGACATC.
Q-PCR response procedures：95℃5min；95 DEG C of 15sec, 62 DEG C of 30sec, circulate 35 times.
The level of software analysis TWIST1 gene expression amounts is carried using real-time quantitative, as a result sees Fig. 1.Fig. 1 shows, A375
After cell transfecting TWIST1 specific siRNAs, TWIST1 expression quantity just corresponds to transfect the 32% of negative control sequence (NC).1：
Untransfected siRNA A375 cells；2：Transfect the A375 cells of negative control sequence；3：Transfect siRNA A375 cells.
PCR primer is subjected to electrophoresis detection, TWIST1 gene mRNAs strike low-level electrophoresis detection result in A375 cells
After seeing that Fig. 2, Fig. 2 displays transfect siRNA, the expression of low TWIST1 genes can be effectively struck.
TWIST1 protein expression levels are identified after the siRNA of embodiment 3 transfections
1st, Western blot identify the protein expression level of TWIST1 genes
Preparation of samples：Adherent melanoma cells A375：1 × PBS is washed twice, the digestion of 100 μ l pancreatin, 500 μ l cultures
Liquid is terminated and is put into 1.5ml EP pipes.Centrifuged 5 minutes with 14000rpm rotating speed.Supernatant is abandoned, 200 1 × PBS of μ l are washed once.Add
Enter 50 μ l cell lysis buffer solutions, acutely concussion, makes cell fully crack, -20 DEG C of preservations.Total protein concentration is detected with BCA methods.
5 × sample-loading buffer is diluted to 1 ×, it is added in the cell sample of cracking.95 DEG C of thermal denaturation 5min, then with 11500rpm's
Rotating speed centrifuges 5 minutes.
2nd, 12%SDS-PAGE glue is configured.
3rd, protein electrophorese：Point sample, each minimum 20 μ g of sample, the μ l of protein markers point 3.Constant pressure 100V, 30 points
Clock, subsequent adjustment voltage to 120V, 1 hour.Transferring film：Cellulose membrane is soaked with methanol in advance, is entered with the transferring film buffer solution of precooling
Row transferring film, constant current 300mA, 2 hours.Closing：Film is put into 5% skimmed milk power solution (1 × TTBS of 1g skimmed milk power+20ml)
In, vibrate 1 hour.Skimmed milk power solution is outwelled, adds primary antibody, is vibrated 1 hour.After removing primary antibody, washed three times with 1 × TTBS,
10 minutes every time.Secondary antibody is then added, is vibrated 1 hour.After removing secondary antibody, washed three times with 1 × TTBS, 10 minutes every time.Prepare
Nitrite ion is simultaneously uniformly dropped on film, exposure imaging.Three kinds of total protein of cell are subjected to Western blot detections, as a result see Fig. 3,
As a result after showing transfection siRNA, the expression of low TWIST1 albumen can effectively be struck.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (8)
1. a kind of siRNA, it is characterised in that its positive-sense strand and antisense strand sequence are respectively：
Positive-sense strand：5 '-CAUUCUGAUAGAAGUCUGAdTdT -3 ',
Antisense strand：5’-UCAGACUUCUAUCAGAAUGdTdT-3’；
The siRNA acts onTWIST1The target sequence of gene is：5’- CATTCTGATAGAAGTCTGA -3’.
A kind of 2. DNA fragmentation for encoding siRNA as claimed in claim 1.
3. a kind of expression vector, it includes the DNA fragmentation described in claim 2.
A kind of 4. medicine containing siRNA described in claim 1.
5. medicine as claimed in claim 4, it is antineoplastic.
6. the siRNA described in claim 1 is in regulation cell or organism is preparedTWIST1In gene expression medicine
Purposes.
7. purposes of the siRNA in the medicine for suppressing tumor cell migration and invasion and attack is prepared described in claim 1.
8. purposes of the siRNA in the medicine for preparing treatment disease described in claim 1, described disease is melanin
Knurl, breast cancer, liver cancer, prostate cancer, stomach cancer, carcinoma of urinary bladder, esophageal squamous cell carcinoma or cancer of pancreas.
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