CN103184223A - Si RNA (small interfering RNA) used for supressing tumors and application thereof - Google Patents
Si RNA (small interfering RNA) used for supressing tumors and application thereof Download PDFInfo
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Abstract
The invention discloses a si RNA used for supressing tumors and an application thereof. The si RNA comprises one type selected from the following nucleotide sequences: such as an RNA sequence presented by SEQ ID NO.7-12 or SEQ ID NO.15-126, and an RNA sequence which has higher than 90% of homology with the RNA sequence limited by SEQ ID NO.7-12 or SEQ ID NO.15-126 and has the same functions. The si RNA disclosed by the invention can restrain FAK (focal adhesion kinase) genetic expression of humans, can be applied to the preparation of antitumor medicines, and provides a new way for developing new drugs for treating or preventing and curing tumors (such as the liver cancer).
Description
Technical field
The present invention relates to a kind of siRNA and application thereof, particularly relate to a kind of for antineoplastic siRNA and application thereof.
Background technology
Focal adhesion kinase (focal adhesion kinase, FAK) be the non-receptor protein tyrosine kinase of a class kytoplasm, it is in very important position in cell signalling, mediate many signal paths, can regulate the cell growth and take place relevant with migration with fetal development, tumour.
The invasion by tumor cells growth is a complex process that multistep is rapid, has the multiple biological chemistry factor to participate.Tumour cell must attach to extracellular matrix, depends on the extracellular matrix signal transduction of PTK kinase activity by promotion, and then influences sticking, move and moving of cell.Wherein, FAK Mediated Signal Transduction system is one of wherein important cellular signal transduction pathways.
RNA disturbs (RNAi) to refer to the phenomenon of the efficient specificity degraded of high conservative, that brought out by double-stranded RNA (dsRNA), homologous mRNA during evolution.Owing to use the RNAi technology can specificity to reject or close the expression of specific gene, so this technology has been widely used in exploring the field of gene of gene function and communicable disease and malignant tumour.
Thereby, utilize the RNA perturbation technique, and in conjunction with the function of FAK, for tumor treatment provides good directive significance.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of for antineoplastic siRNA and application thereof.By utilizing the RNA perturbation technique, can effectively suppress the FAK expression of gene, thereby, can carry out oncotherapy more effectively.
For solving the problems of the technologies described above, of the present invention for antineoplastic siRNA(at the FAK gene), comprise being selected from a kind of in the following nucleotide sequences:
1) the RNA sequence shown in SEQ ID NO.7-8;
2) the RNA sequence shown in SEQ ID NO.9-10;
3) the RNA sequence shown in SEQ ID NO.11-12;
4) the RNA sequence shown in SEQ ID NO.15-16;
5) the RNA sequence shown in SEQ ID NO.17-18;
6) the RNA sequence shown in SEQ ID NO.19-20;
7) the RNA sequence shown in SEQ ID NO.21-22;
8) the RNA sequence shown in SEQ ID NO.23-24;
9) the RNA sequence shown in SEQ ID NO.25-26;
10) the RNA sequence shown in SEQ ID NO.27-28;
11) the RNA sequence shown in SEQ ID NO.29-30;
12) the RNA sequence shown in SEQ ID NO.31-32;
13) the RNA sequence shown in SEQ ID NO.33-34;
14) the RNA sequence shown in SEQ ID NO.35-36;
15) the RNA sequence shown in SEQ ID NO.37-38;
16) the RNA sequence shown in SEQ ID NO.39-40;
17) the RNA sequence shown in SEQ ID NO.41-42;
18) the RNA sequence shown in SEQ ID NO.43-44;
19) the RNA sequence shown in SEQ ID NO.45-46;
20) the RNA sequence shown in SEQ ID NO.47-48;
21) the RNA sequence shown in SEQ ID NO.49-50;
22) the RNA sequence shown in SEQ ID NO.51-52;
23) the RNA sequence shown in SEQ ID NO.53-54;
24) the RNA sequence shown in SEQ ID NO.55-56;
25) the RNA sequence shown in SEQ ID NO.57-58;
26) the RNA sequence shown in SEQ ID NO.59-60;
27) the RNA sequence shown in SEQ ID NO.61-62;
28) the RNA sequence shown in SEQ ID NO.63-64;
29) the RNA sequence shown in SEQ ID NO.65-66;
30) the RNA sequence shown in SEQ ID NO.67-68;
31) the RNA sequence shown in SEQ ID NO.69-70;
32) the RNA sequence shown in SEQ ID NO.71-72;
33) the RNA sequence shown in SEQ ID NO.73-74;
34) the RNA sequence shown in SEQ ID NO.75-76;
35) the RNA sequence shown in SEQ ID NO.77-78;
36) the RNA sequence shown in SEQ ID NO.79-80;
37) the RNA sequence shown in SEQ ID NO.81-82;
38) the RNA sequence shown in SEQ ID NO.83-84;
39) the RNA sequence shown in SEQ ID NO.85-86;
40) the RNA sequence shown in SEQ ID NO.87-88;
41) the RNA sequence shown in SEQ ID NO.89-90;
42) the RNA sequence shown in SEQ ID NO.91-92;
43) the RNA sequence shown in SEQ ID NO.93-94;
44) the RNA sequence shown in SEQ ID NO.95-96;
45) the RNA sequence shown in SEQ ID NO.97-98;
46) the RNA sequence shown in SEQ ID NO.99-100;
47) the RNA sequence shown in SEQ ID NO.101-102;
48) the RNA sequence shown in SEQ ID NO.103-104;
49) the RNA sequence shown in SEQ ID NO.105-106;
50) the RNA sequence shown in SEQ ID NO.107-108;
51) the RNA sequence shown in SEQ ID NO.109-110;
52) the RNA sequence shown in SEQ ID NO.111-112;
53) the RNA sequence shown in SEQ ID NO.113-114;
54) the RNA sequence shown in SEQ ID NO.115-116;
55) the RNA sequence shown in SEQ ID NO.117-118;
56) the RNA sequence shown in SEQ ID NO.119-120;
57) the RNA sequence shown in SEQ ID NO.121-122;
58) the RNA sequence shown in SEQ ID NO.123-124;
59) the RNA sequence shown in SEQ ID NO.125-126;
60) the RNA sequence with SEQ ID NO.7-12 or SEQ ID NO.15-126 restriction has 90% above homology, and has the RNA sequence of identical function.
Wherein, described siRNA is preferably the RNA sequence shown in SEQ ID NO.7-8,9-10 or 11-12.
In addition, the present invention also discloses the dna sequence dna that a kind of coding is used for antineoplastic siRNA as mentioned above.
The invention also discloses a kind of expression vector, this expression vector includes and is used for antineoplastic siRNA sequence as mentioned above.
The present invention discloses a kind of host cell again, and described host cell is a kind of cell that is used for antineoplastic siRNA sequence as mentioned above of expressing, as including aforesaid expression vector.
Of the present invention for antineoplastic siRNA the preparation antitumor drug or the preparation antineoplastic pharmaceutical compositions application.Described tumour comprises: liver cancer.
The present invention discloses a kind of antitumor drug again, and what contain significant quantity is used for antineoplastic siRNA and pharmaceutically acceptable carrier as mentioned above.
This antitumor drug can adopt ordinary method to be prepared into corresponding preparations, as being made into the injection form, for example is prepared by ordinary method with physiological saline or the aqueous solution that contains glucose and other assistant agents.Injection, solution etc. should be made under aseptic condition.
The mode administration easily of this antitumor drug is as by route of administration such as in part, intravenously, intramuscular, subcutaneous, intracutaneous, the nose.
The consumption of antitumor drug of the present invention and the course for the treatment of can be done suitably to adjust according to the light and heavy degree of formulation, patient's age, disease, be that concrete application dosage depends on multiple factor, as factors such as administering mode, medication person's healthy state, these all are in the skilled practitioners skill.
Moreover, the invention also discloses a kind of medicine box, contain the antitumor drug that is used for antineoplastic siRNA as mentioned above or contains the above.This medicine box can be according to the given indicative prompting of government authorities of making, using or selling medicine or biological products, and the government authorities that this prompting reflects production, uses or sells permits it to use at human body.
The present invention experimental results show that the expression of FAK gene in liver cancer tissue apparently higher than cancer beside organism, and therefore, the FAK gene can be used as the special marker gene of diagnosing liver cancer, makes diagnosing cancer of liver more accurately, fast.Simultaneously, of the present inventionly can suppress human FAK genetic expression for antineoplastic siRNA, reduce the liver cancer cell invasion and attack, therefore, siRNA of the present invention can be applicable in the preparation anti-tumor drug, as be applied to prepare in the medicine that suppresses liver cancer, for treatment or the exploitation of anti-curing oncoma (as liver cancer) new drug provide new approach.
Description of drawings
The present invention is further detailed explanation below in conjunction with accompanying drawing and embodiment:
Fig. 1 is the expression synoptic diagram of RT-PCR checking FAK gene in 10 routine hepatocarcinoma patient cancerous tissues and cancer beside organism, and wherein, " N " refers to cancer beside organism, and " C " refers to liver cancer tissue;
Fig. 2 is the expression figure of fluorescence quantitative PCR detection FAK gene in 95 routine hepatocarcinoma patients;
Fig. 3 is that Panomics analyzes the expression figure of FAK gene in 146 routine hepatocarcinoma patients;
Fig. 4 is the FAK expression figure behind the siRNA transfection SMMC7721 cell;
Fig. 5 is the FAK expression figure behind the siRNA transfection WRL68 cell;
Fig. 6 is the FAK expression figure behind the siRNA transfection QGY7701 cell;
Fig. 7 is the relative fluorescence value figure behind the siRNA transfection SMMC7721 cell;
Fig. 8 is the relative fluorescence value figure behind the siRNA transfection WRL68 cell;
Fig. 9 is the relative fluorescence value figure behind the siRNA transfection QGY7701 cell;
Figure 10 is the cell cut figure behind the siRNA transfection SMMC7721;
Figure 11 is the cell invasion figure behind siRNA transfection SMMC7721 and the SK-Hep1.
Embodiment
Following examples only are not used in for explanation the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions among the embodiment, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The expression of embodiment 1FAK gene in liver cancer tissue
1, separate tissue
Test with tissue-derived operation patients in primary hepatocarcinoma.The liver of excision cuts focus rapidly and reaches 5 centimeters outer cancer beside organisms on every side once exsomatizing, and is divided in immediately in the frozen pipe, puts into liquid nitrogen (80 ℃) and preserves.The other diagnosis of cancer and cancer is final foundation with pathological diagnosis all.
2, total RNA extracts
Adopt Trizol(Invitrogen company) reagent, the total RNA that organizes according to its specification sheets extracts.Concrete steps are as follows:
1) Homogenize homogenate: tissue homogenate: every 100mg tissue adds 1ml TRIzol reagent, and uses homogenizer homogenate.
2) chloroform of about 1/5 volume of adding turns upside down about abundant mixing 1min, and room temperature leaves standstill 5min.
3) 4 ℃, the centrifugal 15min of 12,000rpm.
4) carefully take out supernatant liquor, avoid touching the middle layer, change supernatant over to new 1.5ml centrifuge tube, add isopyknic Virahol, put upside down mixing gently, room temperature leaves standstill 5min.
5) 4 ℃, the centrifugal 15min of 12000rpm.
6) supernatant is removed in suction, keeps precipitation, and adds 1ml75% ethanol, 4 ℃, the centrifugal 15min washing precipitation of 12000rpm in precipitation.
7) supernatant is removed in suction, and the precipitation room temperature is dried the water that the back adds an amount of RNase-free naturally, with Tip pressure-vaccum, and abundant dissolution precipitation.
8) get 1-2 μ L, measure RNA concentration and A260/A280 value.General A260/A280 ratio is at 1.8-2.1.It is stand-by that RNA leaves-80 ℃ of refrigerators in.
3, the synthetic cDNA of reverse transcription
With PrimeScript RT Master Mix Perfect Real Time(TAKARA company) reverse transcription cDNA, reaction system (total amount is 10 μ l) is as shown in table 1.
Table 1 reaction system
Reaction conditions is as follows:
1) 37 ℃ 15 minutes;
2) 85 ℃ of 5 second;
3) 4 ℃ of preservations (20 ℃ of prolonged preservation).
4、RT-PCR
It is poor to adopt the relative quantification method to detect the expression of FAK gene in the liver cancer sample.
Use instrument: Thermal Cycler Dice
TMReal Time System TP800, Takara.
Template: above-mentioned reverse transcription product is pressed after the 1:10 dilution proportion template as the PCR reaction.
Primer is as shown in table 2:
Table 2 primer sequence
The system of PCR reaction:
The thermotolerance Taq archaeal dna polymerase (DR001) of TAKARA company is adopted in PCR reaction, and when being template with the people's gene group, 3.0kbp(p53 gene well can increase) dna fragmentation.Wherein, reagent and the consumption thereof of PCR reaction are as shown in table 3.
Reagent and the consumption thereof of table 3PCR reaction
| Reagent | Usage quantity |
| Takara Taq（5U/μl） | 0.1 |
| 10 * PCR damping fluid (Mg 2+Plus） | 2μl |
| DNTP mixture (every kind of 2.5mM) | 1.6μl |
| Primer mixture (each 5 μ M) | 1.6μl |
| Template | 0.5-4μl |
| ddH 2O | Benefit to 20 μ l |
The PCR reaction conditions:
β-actin：
94℃ 5 min.
4℃
Goal gene:
94℃ 5 min.
4℃
The result as shown in Figure 1, the expression level of the expression level of FAK gene in liver cancer tissue in the cancer beside organism.
5, quantitative fluorescent PCR
Adopt the differential expression of fluorescence quantitative PCR detection FAK gene in the liver cancer sample.
Use instrument: 7300 type quantitative real time PCR Instruments, ABI company.
Template: above-mentioned reverse transcription product is pressed after the 1:5 dilution proportion template as the PCR reaction.
Primer sequence is as shown in table 4:
Table 4 primer sequence
The system of PCR reaction: the SYBR Premix Ex Taq of TAKARA company is adopted in the PCR reaction.Wherein, reagent and the consumption thereof of PCR reaction are as shown in table 5.
Reagent and the consumption thereof of table 5PCR reaction
| Composition | Consumption |
| SYBR Premix EX Taq | 10.0μl |
| Upstream primer (10 μ M) | 0.5μl |
| Downstream primer (10 μ M) | 0.5μl |
| ROX Reference Dye（50*） | 0.4μl |
| cDNA | 1.0μl |
| ddH 2O | 7.6μl |
The PCR reaction conditions:
Pre-sex change: 95 ℃ 30 seconds
The PCR reaction:
Melting curve is measured:
95 ℃ 5 seconds
60 ℃ 60 seconds
95 ℃ 5 seconds
Fluorescence quantitative PCR detection shows, the expression level of the expression level of FAK gene in liver cancer tissue in the cancer beside organism, and through adopting Graphpad Prism5 statistical package (GraphPad Software, San Diego, CA USA) analyzes, and relatively checking with t of quantitative data analyzed, think that when P＜0.05 statistics is variant, the result shows that P＜0.0001(sees Fig. 2).
6, Panomics analyzes
1) key instrument
Bio-plex suspension chip system: Bio-plex100system(Biorad company)
Bio-plex washes the plate machine: Bio-plex pro II wash station(Biorad company)
Constant temperature vibration shaking table: Vortemp 56 shaking incubator(Labnet International)
2) operation steps
The suspension liquid chip inspecting reagent unit of people's 30 nucleic acid factor panel is Quantigene plex2.0Kit(Panomics, Affymetrix corp).Applied sample amount is 400ng.
Require to carry out following operation according to the test kit specification sheets:
1. the RNA concentration of specimens with tissue extraction is adjusted to 20ng/ μ l.
2. require preparation microballon probe suspension according to the test kit specification sheets, get 80 μ l and add in each hole of hybridization plate, again dummy or RNA sample 20 μ l are added in the hand-hole.After the pad pasting sealing, 600rpm in the constant temperature vibration shaking table, 54 ℃ of lucifuges are hatched 18-22h.
3. sample probe microballon Incubating Solution is transferred in the hole of magnetic bead spacer plate, utilizes Bio-plex to wash the plate machine and wash, every hole 100ul washs three times.
4. add the pre-liquid 100ul that amplifies, 50 ℃, 600rpm is hatched 1h.
5. after washing plate machine washing and washing three times, add 100 μ l and amplify liquid, 50 ℃, 600rpm is hatched 1h.
6. after the washing, add label probe liquid 100 μ l, 50 ℃, 600rpm is hatched 1h.
7. after the washing, add SAPE working fluid 100 μ l, room temperature 600rpm is hatched 30min.
8. behind the unnecessary SAPE of SAPE washings flush away, add 130 μ l SAPE washingss, send into the Bio-plex suspension chip system value of reading, and through adopting Graphpad Prism5 statistical package (GraphPad Software, San Diego, CA USA) analyzes, relatively analyzing with the t check of quantitative data is when P＜0.05 thinks that statistics is variant.
The result as shown in Figure 3, the expression level of the expression level of FAK gene in liver cancer tissue in the cancer beside organism, P＜0.0001(sees Fig. 3).
1, cell cultures
Adopt common hepatoma cell strain, namely preserve in SMMC7721, WRL68, QGY7701(laboratory) carry out the research of FAK expression of gene.Wherein, the culture condition of cell strain is for containing the 10%(percent by volume) the DMEM(dulbecco's modified eagle medium of foetal calf serum (Life Technologies)) nutrient solution, 37 ℃, contain 5%CO
2Cultivate in the incubator.
2, the design of siRNA
The free service device that provides according to some companies on the network, as the interface http://bioinfo2.clontech.com/rnaidesigner/sirnaSequenceDesign.d o of Clontech company, the interface https of Invitrogen company: //rnaidesigner.invitrogen.com/sirna/ and design corresponding siRNA according to InvivoGene server (network address http://www.sirnawizard.com).
Wherein, the siRNA sequence at the FAK gene of design is as follows respectively:
FAK-siRNA1, positive-sense strand: 5 '-CAGGUGAAGAGCGAUUAUAtt-3 ' (SEQ ID NO.7)
FAK-siRNA1, antisense strand: 5 '-UAUAAUCGCUCUUCACCUGtt-3 ' (SEQ ID NO.8)
FAK-siRNA2, positive-sense strand: 5 '-CUCCAGUCUACAGAUUUGAtt-3 ' (SEQ ID NO.9)
FAK-siRNA2, antisense strand: 5 '-UCAAAUCUGUAGACUGGAGtt-3 ' (SEQ ID NO.10)
FAK-siRNA3, positive-sense strand: 5 '-CCCAGGUUUACUGAACUUAtt-3 ' (SEQ ID NO.11)
FAK-siRNA3, antisense strand: 5 '-UAAGUUCAGUAAACCUGGGtt-3 ' (SEQ ID NO.12)
Negative control siRNA, positive-sense strand: 5 '-UUCUCCGAACGUGUCACGUtt-3 ' (SEQ ID NO.13)
Negative control siRNA, antisense strand: 5 '-ACGUGACACGUUCGGAGAAtt-3 ' (SEQ ID NO.14)
FAK-siRNA4, positive-sense strand: 5 '-GCAUCUUCCAGUUACAAAUtt-3 ' (SEQ ID NO.15)
FAK-siRNA4, antisense strand: 5 '-AUUUGUAACUGGAAGAUGCtt-3 ' (SEQ ID NO.16)
FAK-siRNA5, positive-sense strand: 5 '-GACCCCAACUUGAAUCACAtt-3 ' (SEQ ID NO.17)
FAK-siRNA5, antisense strand: 5 '-UGUGAUUCAAGUUGGGGUCtt-3 ' (SEQ ID NO.18)
FAK-siRNA6, positive-sense strand: 5 '-GUGCAAUGGAGCGAGUAUUtt-3 ' (SEQ ID NO.19)
FAK-siRNA6, antisense strand: 5 '-AAUACUCGCUCCAUUGCACtt-3 ' (SEQ ID NO.20)
FAK-siRNA7, positive-sense strand: 5 '-GCAAUGGAGCGAGUAUUAAtt-3 ' (SEQ ID NO.21)
FAK-siRNA7, antisense strand: 5 '-UUAAUACUCGCUCCAUUGCtt-3 ' (SEQ ID NO.22)
FAK-siRNA8, positive-sense strand: 5 '-GAGCGAGUAUUAAAGGUCUtt-3 ' (SEQ ID NO.23)
FAK-siRNA8, antisense strand: 5 '-AGACCUUUAAUACUCGCUCtt-3 ' (SEQ ID NO.24)
FAK-siRNA9, positive-sense strand: 5 '-GGAGAUGCUACUGAUGUCAtt-3 ' (SEQ ID NO.25)
FAK-siRNA9, antisense strand: 5 '-UGACAUCAGUAGCAUCUCCtt-3 ' (SEQ ID NO.26)
FAK-siRNA10, positive-sense strand: 5 '-GGGCAUCAUUCAGAAGAUAtt-3 ' (SEQ ID NO.27)
FAK-siRNA10, antisense strand: 5 '-UAUCUUCUGAAUGAUGCCCtt-3 ' (SEQ ID NO.28)
FAK-siRNA11, positive-sense strand: 5 '-GCAUCAUUCAGAAGAUAGUtt-3 ' (SEQ ID NO.29)
FAK-siRNA11, antisense strand: 5 '-ACUAUCUUCUGAAUGAUGCtt-3 ' (SEQ ID NO.30)
FAK-siRNA12, positive-sense strand: 5 '-GUGGACAGUCACAAAGUAAtt-3 ' (SEQ ID NO.31)
FAK-siRNA12, antisense strand: 5 '-UUACUUUGUGACUGUCCACtt-3 ' (SEQ ID NO.32)
FAK-siRNA13, positive-sense strand: 5 '-GUGUGAGGGAGAAGUAUGAtt-3 ' (SEQ ID NO.33)
FAK-siRNA13, antisense strand: 5 '-UCAUACUUCUCCCUCACACtt-3 ' (SEQ ID NO.34)
FAK-siRNA14, positive-sense strand: 5 '-GAGGAGUGGAAAUAUGAAUtt-3 ' (SEQ ID NO.35)
FAK-siRNA14, antisense strand: 5 '-AUUCAUAUUUCCACUCCUCtt-3 ' (SEQ ID NO.36)
FAK-siRNA15, positive-sense strand: 5 '-GUGGAAAUAUGAAUUGAGAtt-3 ' (SEQ ID NO.37)
FAK-siRNA15, antisense strand: 5 '-UCUCAAUUCAUAUUUCCACtt-3 ' (SEQ ID NO.38)
FAK-siRNA16, positive-sense strand: 5 '-GUGAAGAGCGAUUAUAUGUtt-3 ' (SEQ ID NO.39)
FAK-siRNA16, antisense strand: 5 '-ACAUAUAAUCGCUCUUCACtt-3 ' (SEQ ID NO.40)
FAK-siRNA17, positive-sense strand: 5 '-GCGAUUAUAUGUUAGAGAUtt-3 ' (SEQ ID NO.41)
FAK-siRNA17, antisense strand: 5 '-AUCUCUAACAUAUAAUCGCtt-3 ' (SEQ ID NO.42)
FAK-siRNA18, positive-sense strand: 5 '-GAAAUACGGCGAUCAUACUtt-3 ' (SEQ ID NO.43)
FAK-siRNA18, antisense strand: 5 '-AGUAUGAUCGCCGUAUUUCtt-3 ' (SEQ ID NO.44)
FAK-siRNA19, positive-sense strand: 5 '-GAAGUCUAACUAUGAAGUAtt-3 ' (SEQ ID NO.45)
FAK-siRNA19, antisense strand: 5 '-UACUUCAUAGUUAGACUUCtt-3 ' (SEQ ID NO.46)
FAK-siRNA20, positive-sense strand: 5 '-GAUCCAACAAACAUUUAGAtt-3 ' (SEQ ID NO.47)
FAK-siRNA20, antisense strand: 5 '-UCUAAAUGUUUGUUGGAUCtt-3 ' (SEQ ID NO.48)
FAK-siRNA21, positive-sense strand: 5 '-GGUUCAAGCUGGAUUAUUUtt-3 ' (SEQ ID NO.49)
FAK-siRNA21, antisense strand: 5 '-AAAUAAUCCAGCUUGAACCtt-3 ' (SEQ ID NO.50)
FAK-siRNA22, positive-sense strand: 5 '-GCUGGAUUAUUUCAGUGGAtt-3 ' (SEQ ID NO.51)
FAK-siRNA22, antisense strand: 5 '-UCCACUGAAAUAAUCCAGCtt-3 ' (SEQ ID NO.52)
FAK-siRNA23, positive-sense strand: 5 '-GAAGGAAUCAGUUACCUAAtt-3 ' (SEQ ID NO.53)
FAK-siRNA23, antisense strand: 5 '-UUAGGUAACUGAUUCCUUCtt-3 ' (SEQ ID NO.54)
FAK-siRNA24, positive-sense strand: 5 '-GUGCAAACCAUUCAGUAUUtt-3 ' (SEQ ID NO.55)
FAK-siRNA24, antisense strand: 5 '-AAUACUGAAUGGUUUGCACtt-3 ' (SEQ ID NO.56)
FAK-siRNA25, positive-sense strand: 5 '-GCAAACCAUUCAGUAUUCAtt-3 ' (SEQ ID NO.57)
FAK-siRNA25, antisense strand: 5 '-UGAAUACUGAAUGGUUUGCtt-3 ' (SEQ ID NO.58)
FAK-siRNA26, positive-sense strand: 5 '-GGAGAAUAUGGCUGACCUAtt-3 ' (SEQ ID NO.59)
FAK-siRNA26, antisense strand: 5 '-UAGGUCAGCCAUAUUCUCCtt-3 ' (SEQ ID NO.60)
FAK-siRNA27, positive-sense strand: 5 '-GAAUAUGGCUGACCUAAUAtt-3 ' (SEQ ID NO.61)
FAK-siRNA27, antisense strand: 5 '-UAUUAGGUCAGCCAUAUUCtt-3 ' (SEQ ID NO.62)
FAK-siRNA28, positive-sense strand: 5 '-GAAUGGAACCUCGCAGUCAtt-3 ' (SEQ ID NO.63)
FAK-siRNA28, antisense strand: 5 '-UGACUGCGAGGUUCCAUUCtt-3 ' (SEQ ID NO.64)
FAK-siRNA29, positive-sense strand: 5 '-GGAACCUCGCAGUCAUUUAtt-3 ' (SEQ ID NO.65)
FAK-siRNA29, antisense strand: 5 '-UAAAUGACUGCGAGGUUCCtt-3 ' (SEQ ID NO.66)
FAK-siRNA30, positive-sense strand: 5 '-GAACCUCGCAGUCAUUUAUtt-3 ' (SEQ ID NO.67)
FAK-siRNA30, antisense strand: 5 '-AUAAAUGACUGCGAGGUUCtt-3 ' (SEQ ID NO.68)
FAK-siRNA31, positive-sense strand: 5 '-GCAGUCAUUUAUCAUCAGAtt-3 ' (SEQ ID NO.69)
FAK-siRNA31, antisense strand: 5 '-UCUGAUGAUAAAUGACUGCtt-3 ' (SEQ ID NO.70)
FAK-siRNA32, positive-sense strand: 5 '-GAUGAUUAUGCUGAGAUUAtt-3 ' (SEQ ID NO.71)
FAK-siRNA32, antisense strand: 5 '-UAAUCUCAGCAUAAUCAUCtt-3 ' (SEQ ID NO.72)
FAK-siRNA33, positive-sense strand: 5 '-GGAUUAUGAGAUUCAAAGAtt-3 ' (SEQ ID NO.73)
FAK-siRNA33, antisense strand: 5 '-UCUUUGAAUCUCAUAAUCCtt-3 ' (SEQ ID NO.74)
FAK-siRNA34, positive-sense strand: 5 '-GUACAUCAAGGCAUUUAUAtt-3 ' (SEQ ID NO.75)
FAK-siRNA34, antisense strand: 5 '-UAUAAAUGCCUUGAUGUACtt-3 ' (SEQ ID NO.76)
FAK-siRNA35, positive-sense strand: 5 '-GUGAAGCUGAUUGGAGUCAtt-3 ' (SEQ ID NO.77)
FAK-siRNA35, antisense strand: 5 '-UGACUCCAAUCAGCUUCACtt-3 ' (SEQ ID NO.78)
FAK-siRNA36, positive-sense strand: 5 '-GCAAGUAAGGAAAUACAGUtt-3 ' (SEQ ID NO.79)
FAK-siRNA36, antisense strand: 5 '-ACUGUAUUUCCUUACUUGCtt-3 ' (SEQ ID NO.80)
FAK-siRNA37, positive-sense strand: 5 '-GGUGUCCUCAAAUGAUUGUtt-3 ' (SEQ ID NO.81)
FAK-siRNA37, antisense strand: 5 '-ACAAUCAUUUGAGGACACCtt-3 ' (SEQ ID NO.82)
FAK-siRNA38, positive-sense strand: 5 '-GGCUCCAGAGUCAAUCAAUtt-3 ' (SEQ ID NO.83)
FAK-siRNA38, antisense strand: 5 '-AUUGAUUGACUCUGGAGCCtt-3 ' (SEQ ID NO.84)
FAK-siRNA39, positive-sense strand: 5 '-GCUCCAGAGUCAAUCAAUUtt-3 ' (SEQ ID NO.85)
FAK-siRNA39, antisense strand: 5 '-AAUUGAUUGACUCUGGAGCtt-3 ' (SEQ ID NO.86)
FAK-siRNA40, positive-sense strand: 5 '-GAGUGAAGAACAAUGAUGUtt-3 ' (SEQ ID NO.87)
FAK-siRNA40, antisense strand: 5 '-ACAUCAUUGUUCUUCACUCtt-3 ' (SEQ ID NO.88)
FAK-siRNA41, positive-sense strand: 5 '-GUGAAGAACAAUGAUGUAAtt-3 ' (SEQ ID NO.89)
FAK-siRNA41, antisense strand: 5 '-UUACAUCAUUGUUCUUCACtt-3 ' (SEQ ID NO.90)
FAK-siRNA42, positive-sense strand: 5 '-GCCCAGGUUUACUGAACUUtt-3 ' (SEQ ID NO.91)
FAK-siRNA42, antisense strand: 5 '-AAGUUCAGUAAACCUGGGCtt-3 ' (SEQ ID NO.92)
FAK-siRNA43, positive-sense strand: 5 '-GCAGCAUCUAUCCAGGUCAtt-3 ' (SEQ ID NO.93)
FAK-siRNA43, antisense strand: 5 '-UGACCUGGAUAGAUGCUGCtt-3 ' (SEQ ID NO.94)
FAK-siRNA44, positive-sense strand: 5 '-GUGGAGGACUCUACAGUAUtt-3 ' (SEQ ID NO.95)
FAK-siRNA44, antisense strand: 5 '-AUACUGUAGAGUCCUCCACtt-3 ' (SEQ ID NO.96)
FAK-siRNA45, positive-sense strand: 5 '-GAGGGAUUGGGCAAGUGUUtt-3 ' (SEQ ID NO.97)
FAK-siRNA45, antisense strand: 5 '-AACACUUGCCCAAUCCCUCtt-3 ' (SEQ ID NO.98)
FAK-siRNA46, positive-sense strand: 5 '-GACUCUCUCGAGGCAGUAUtt-3 ' (SEQ ID NO.99)
FAK-siRNA46, antisense strand: 5 '-AUACUGCCUCGAGAGAGUCtt-3 ' (SEQ ID NO.100)
FAK-siRNA47, positive-sense strand: 5 '-GGUCGAAUGAUAAGGUGUAtt-3 ' (SEQ ID NO.101)
FAK-siRNA47, antisense strand: 5 '-UACACCUUAUCAUUCGACCtt-3 ' (SEQ ID NO.102)
FAK-siRNA48, positive-sense strand: 5 '-GCCUGGUGAAAGCUGUCAUtt-3 ' (SEQ ID NO.103)
FAK-siRNA48, antisense strand: 5 '-AUGACAGCUUUCACCAGGCtt-3 ' (SEQ ID NO.104)
FAK-siRNA49, positive-sense strand: 5 '-GGACAUUAUUGGCCACUGUtt-3 ' (SEQ ID NO.105)
FAK-siRNA49, antisense strand: 5 '-ACAGUGGCCAAUAAUGUCCtt-3 ' (SEQ ID NO.106)
FAK-siRNA50, positive-sense strand: 5 '-GCACAGAAGCUAUUGAACUtt-3 ' (SEQ ID NO.107)
FAK-siRNA50, antisense strand: 5 '-AGUUCAAUAGCUUCUGUGCtt-3 ' (SEQ ID NO.108)
FAK-siRNA51, positive-sense strand: 5 '-GCCCAGCAGUAUGUCAUGAtt-3 ' (SEQ ID NO.109)
FAK-siRNA51, antisense strand: 5 '-UCAUGACAUACUGCUGGGCtt-3 ' (SEQ ID NO.110)
FAK-siRNA52, positive-sense strand: 5 '-GCCUCCAGCAAGAGUACAAtt-3 ' (SEQ ID NO.111)
FAK-siRNA52, antisense strand: 5 '-UUGUACUCUUGCUGGAGGCtt-3 ' (SEQ ID NO.112)
FAK-siRNA53, positive-sense strand: 5 '-GCAUCAUGAAGAACAAUUUtt-3 ' (SEQ ID NO.113)
FAK-siRNA53, antisense strand: 5 '-AAAUUGUUCUUCAUGAUGCtt-3 ' (SEQ ID NO.114)
FAK-siRNA54, positive-sense strand: 5 '-GCUGCAUAGUGGAAGAGGAtt-3 ' (SEQ ID NO.115)
FAK-siRNA54, antisense strand: 5 '-UCCUCUUCCACUAUGCAGCtt-3 ' (SEQ ID NO.116)
FAK-siRNA55, positive-sense strand: 5 '-GAGCAUGAAGCAAAGAAUUtt-3 ' (SEQ ID NO.117)
FAK-siRNA55, antisense strand: 5 '-AAUUCUUUGCUUCAUGCUCtt-3 ' (SEQ ID NO.118)
FAK-siRNA56, positive-sense strand: 5 '-GCUAAUCCCACUUUACAAAtt-3 ' (SEQ ID NO.119)
FAK-siRNA56, antisense strand: 5 '-UUUGUAAAGUGGGAUUAGCtt-3 ' (SEQ ID NO.120)
FAK-siRNA57, positive-sense strand: 5 '-GUCGGGAACUAGCUGUAGAtt-3 ' (SEQ ID NO.121)
FAK-siRNA57, antisense strand: 5 '-UCUACAGCUAGUUCCCGACtt-3 ' (SEQ ID NO.122)
FAK-siRNA58, positive-sense strand: 5 '-GGGAACUAGCUGUAGAACAtt-3 ' (SEQ ID NO.123)
FAK-siRNA58, antisense strand: 5 '-UGUUCUACAGCUAGUUCCCtt-3 ' (SEQ ID NO.124)
FAK-siRNA59, positive-sense strand: 5 '-GUAGCAAUGUUAUUUCUCUtt-3 ' (SEQ ID NO.125)
FAK-siRNA59, antisense strand: 5 '-AGAGAAAUAACAUUGCUACtt-3 ' (SEQ ID NO.126)
3, siRNA transient transfection
Select SMMC7721, WRL68, the QGY7701 cell strain of the higher FAK gene of endogenous expression.Synthetic every kind of good siRNA(is respectively FAK-siRNA1, FAK-siRNA2, FAK-siRNA3 and negative control siRNA) use the DEPC water dissolution, obtain the solution of 20 μ M concentration.With 1 μ l Lipofectamine
TM2000(Invitrogen) and 1 μ l concentration be after every kind of siRNA of 20 μ M mixes respectively, mixed solution forwarded to carry among SMMC7721, the WRL68 and QGY7701 cell of passage cell density the day before yesterday about 30-40% (24 porocyte culture dish), change complete culture solution after 4 hours, continue to cultivate.Collecting cell behind the wink commentaries on classics 48h carries out follow-up total RNA then and extracts.
4, the extraction of cell total rna
Adopt Trizol(TAKARA company) reagent, extract total RNA of cell according to its specification sheets, step is as follows:
1) attached cell of Pei Yanging (24 hole culture dish), adopt above-mentioned " 3, siRNA transient transfection " method to carry out transfection after, cultivate 48h, inhale and remove nutrient solution, add 500 μ l TRIzol to culture dish, with the piping and druming of rifle head several times;
2) lysate is transferred in the centrifuge tube of 1.5ml, room temperature is placed 5min;
3) amount of pressing 0.2ml chloroform/ml TRIzol adds chloroform, builds the pipe lid, violent jolting 15s, and room temperature is placed 5min, 4 ℃ of centrifugal 15min of 12000g;
4) upper phase is transferred in the clean centrifuge tube, adds Virahol (0.5ml/mlTRIzol), put upside down mixing gently for several times, room temperature is placed 10min, 4 ℃ of centrifugal 15min of 12000g;
5) abandon supernatant, add 75% ethanol (1ml/ml TRIzol) mixing that fully vibrates, 4 ℃ of centrifugal 10min of 12000g;
6) remove supernatant, be deposited in room temperature and place 5～10min, make its natural airing (not complete drying); The DEPC water dissolution RNA that adds 30-50 μ l; Measure 260/280 ratio and quantitative with ultraviolet spectrophotometer;
7) remove DNA: adopt DNase I(RNase Free, TaKaRa company) remove DNA pollution possible among total RNA, operation steps is as follows:
1. in Eppendorf tube, prepare reaction solution (total amount 50 μ l) as shown in table 6, then, 37 ℃ of reaction 20min.
Table 6 reaction solution
| Composition | |
| Total RNA | |
| 20～50μg | |
| 10×DNase I Buffer | 5μl |
| DNase I（RNase-free，5U/μl） | 2μl |
| RNase Inhibitor（40U/μl） | 0.5μl |
| The H that DEPC handles 2O | Be 50 μ l up to the reaction solution total amount |
2. the H that adds the DEPC processing of 50 μ l
2O.
3. add 100 μ l(equivalent) phenol/chloroform/primary isoamyl alcohol (volume ratio is respectively 25:24:1), abundant mixing.
4. centrifugal 12000rpm, 15min gets upper strata (water layer) and moves in another Eppendorf tube.
5. add 100 μ l(equivalent) chloroform/primary isoamyl alcohol (volume ratio is 24:1), abundant mixing.
6. centrifugal 12000rpm, 15min gets upper strata (water layer) and moves in another Eppendorf tube.
7. add 10 μ l(1/10 amount) 3M sodium acetate (pH5.2).
8. adding 250 μ l(2.5 doubly measures) cold dehydrated alcohol, placed 30-60 minute for-20 ℃.
9. centrifugal recovery precipitates 12000rpm, 10min, the cold ethanol washing and precipitating with 70%, drying.
10. the H that handles with an amount of DEPC
2The O dissolving, quantitatively.
5, the synthetic cDNA of reverse transcription
With PrimeScript RT Master Mix Perfect Real Time(TAKARA company) reverse transcription cDNA, reaction system (total amount is 10 μ l) is as shown in table 7.
Table 7 reaction system
| | Consumption | |
| 5*PrimeScript RT Master Mix（for Real Time） | 2μl | |
| Total RNA | 500ng | |
| Remove the dH of RNase 2O | Complement to 10 μ l |
Reaction conditions is as follows:
1) 37 ℃ 15 minutes;
2) 85 ℃ of 5 second;
3) 4 ℃ of preservations (20 ℃ of prolonged preservation).
6, quantitative fluorescent PCR
Use ABI7300 real-time fluorescence quantitative PCR instrument and SYBR Premix Ex Taq test kit to carry out, with β-actin (ACTB) as interior reference, analyze the influence that the hepatoma cell strain FAK of transfection siRNA expresses, the primer is as shown in table 4, and reaction system (total amount is 20 μ l) is as shown in table 8.
Table 8PCR reaction system
The PCR reaction conditions is: 94 ℃ of pre-sex change 30s, 94 ℃ of 10s afterwards, 60 ℃ of 30s, carry out 40 circulations altogether after, carry out the detection of solubility curve.
Wherein, fluorescence background signal and threshold value generally adopt instrument acquiescence numerical value, generate automatically after each run finishes, and the cycle number that the fluorescent signal in each reaction tubes experiences when reaching preset threshold is defined as the Ct value; Data analysis is carried out as follows: every pair of primer (gene) is done 3 and is repeated pipe in each template, the Ct value that obtains is averaged; The Ct mean value of each goal gene deducts the Ct mean value of the internal control gene (ACTB) of corresponding templates, obtains Δ Ct.The Δ Ct of experimental group deducts the Δ Ct of control group, obtains Δ Δ Ct value, the multiple relation of the testing gene in control group and experimental group 2-Δ Δ Ct.
Experimental result shows, these three siRNA fragments of FAK-siRNA1, FAK-siRNA2 and FAK-siRNA3 are reticent FAK expression of gene effectively all, and the institute that prompting can be used for next step uses.Wherein, the experimental result in SMMC7721, WRL68, the QGY7701 cell is shown in Fig. 4-6.
7, the mensuration of cell growth curve
1) the day before yesterday is inoculated an amount of cell in transfection, and cell density control is between 30%～50% when making transfection;
2) adopt liposome Lipofectamine
TM2000(Invitrogen) transfection siRNA
Preparation siRNA and Lipofectamine
TM2000 mixed solution, the prescription of mixed solution are with 1 μ l Lipofectamine
TM2000 and 1 μ l concentration is that every kind of siRNA(FAK-siRNA1, FAK-siRNA2, FAK-siRNA3 and the negative control siRNA of 20 μ M uses the DEPC water dissolution respectively) mix respectively after, room temperature was placed about 20 minutes, formed siRNA-Lipofectamine
TM2000 mixtures; Then, with siRNA-Lipofectamine
TM2000 mixtures (2 μ l) add in the cell, mixing; Change normal nutrient solution after 4～6 hours;
3) after 24 hours, SMMC7721, WRL68, QGY7701 cell behind the digestion transfection siRNAs press 1 * 10 according to its growth characteristics
3The cell total amount is calculated in/100 μ l/ holes, and after fully diluting, plants in 96 orifice plates.Answer the hole every group of every days six, generally presses 7-8 days inoculating cells;
4) treat about half an hour cell adherent substantially back observation of cell state and number.With the chromogenic reagent reaction, to determine the initial density of cell reality, as the growth relative zero.Developer Cell counting kit-8(CCK-8, Dojindo, usage Japan): per 100 μ l training liquid adds 10 μ l CCK-8,37 ℃, 5%CO
2Place 1h, measure the absorbancy at 450nm place in microplate reader;
5) observation of cell form under the light microscopic.Regularly measure the record upgrowth situation on request;
6) generally need to survey 4 to 7 days.After data gathering, handle again, draw chart with Excel.
Found that these three siRNA fragments of FAK-siRNA1, FAK-siRNA2 and FAK-siRNA3 all can suppress the growth of liver cancer cell effectively.Wherein, the experimental result in SMMC7721, WRL68, the QGY7701 cell is shown in 7-9.
8, cell scratch experiment
The SMMC7721 cell is spread in the 24 porocyte culture dish with the density of 80%-90%.With 1 μ l Lipofectamine
TM2000 and 1 μ l concentration is that every kind of siRNA(FAK-siRNA1, FAK-siRNA2, FAK-siRNA3 and the negative control siRNA of 20 μ M uses the DEPC water dissolution respectively) mix respectively after, transfectional cell comes reticent FAK respectively.After 24 hours, use plastics rifle head to streak cell surface, 37 ℃, 5%CO
2Cell culture incubator in cultivated 4 days, in the time of 0,1,2,3 day, gather the image of same position respectively.The experiment triplicate.With the negative control cells transfected or without the cell of any processing in contrast.
Found that these three siRNA fragments of FAK-siRNA1, FAK-siRNA2 and FAK-siRNA3 can suppress the growth of SMMC7721 cell.Wherein, the experimental result of FAK-siRNA1 and FAK-siRNA3 is shown in 10.
9, cell invasion experiment
Inoculate an amount of cell (SMMC7721, SK-hep1), cell density control is between 30%～50% when making transfection; Adopt liposome Lipofectamine in 24 hours
TM2000(Invitrogen) transfection siRNA: preparation siRNA and Lipofectamine
TM2000 mixed solution is namely used 1 μ l Lipofectamine
TM2000 and 1 μ l concentration is that every kind of siRNA(FAK-siRNA1 and the negative control siRNA of 20 μ M uses the DEPC water dissolution respectively) mix respectively after, room temperature was placed about 20 minutes, formed siRNA-Lipofectamine
TM2000 mixtures; With siRNA-Lipofectamine
TM2000 mixtures (every kind of 2 μ l) add in the cell, mixing; Change normal nutrient solution after 4～6 hours.
24 hole Transwell cells (8-μ m aperture, BD Biosciences company) are all adopted in the cell invasion experiment, scribble artificial basilar membrane (Falcon354480; BD Biosciences company).Liver cancer cell in serum free medium (SMMC7721, SK-hep1) hunger is spent the night, and handles with tryptic digestion, and washs 3 times in containing the DMEM nutrient solution of 1%FBS.To contain 1 * 10
5500 μ l of individual cell contain in the DMEM nutrient solution of 1%FBS, join upper chamber.Simultaneously 750 μ l are contained the DMEM substratum of 10%FBS and the fibronectin of 10 μ g/ml (catalog number 356008, BD Biosciences company) and be placed at lower chamber.In contrast, will contain in the substratum of 1%FBS and add lower chamber.Through 48 hours latent period, remaining artificial basilar membrane and cell were removed with cotton swab in the upper chamber.Cell on the film lower surface is fixed with 4% Paraformaldehyde 96, and uses 0.5% violet staining.Cell is at least 6 countings and taking pictures in the field of microscope (multiplying power * 100) at random.All experiments are all repeating, and repeat 3 times, and adopt Graphpad Prism5 statistical package (GraphPad Software, San Diego, CA USA) analyzes, relatively analyzing with the t check of quantitative data is when P＜0.05 thinks that statistics is variant.
Found that FAK-siRNA1 can obviously suppress the invasion and attack (as shown in figure 11) of liver cancer cell SMMC7721, SK-hep1.
In sum, the present invention utilizes the expression of the reticent FAK of RNAi technology can suppress cell growth, motion and the invasion and attack of liver cancer.Thereby siRNA of the present invention can be applicable to preparation treatment or anti-curing oncoma (as liver cancer) medicine, is tumor treatment, and new approach is provided.
Claims (10)
1. one kind is used for antineoplastic siRNA, it is characterized in that, comprises being selected from a kind of in the following nucleotide sequences:
1) the RNA sequence shown in SEQ ID NO.7-8;
2) the RNA sequence shown in SEQ ID NO.9-10;
3) the RNA sequence shown in SEQ ID NO.11-12;
4) the RNA sequence shown in SEQ ID NO.15-16;
5) the RNA sequence shown in SEQ ID NO.17-18;
6) the RNA sequence shown in SEQ ID NO.19-20;
7) the RNA sequence shown in SEQ ID NO.21-22;
8) the RNA sequence shown in SEQ ID NO.23-24;
9) the RNA sequence shown in SEQ ID NO.25-26;
10) the RNA sequence shown in SEQ ID NO.27-28;
11) the RNA sequence shown in SEQ ID NO.29-30;
12) the RNA sequence shown in SEQ ID NO.31-32;
13) the RNA sequence shown in SEQ ID NO.33-34;
14) the RNA sequence shown in SEQ ID NO.35-36;
15) the RNA sequence shown in SEQ ID NO.37-38;
16) the RNA sequence shown in SEQ ID NO.39-40;
17) the RNA sequence shown in SEQ ID NO.41-42;
18) the RNA sequence shown in SEQ ID NO.43-44;
19) the RNA sequence shown in SEQ ID NO.45-46;
20) the RNA sequence shown in SEQ ID NO.47-48;
21) the RNA sequence shown in SEQ ID NO.49-50;
22) the RNA sequence shown in SEQ ID NO.51-52;
23) the RNA sequence shown in SEQ ID NO.53-54;
24) the RNA sequence shown in SEQ ID NO.55-56;
25) the RNA sequence shown in SEQ ID NO.57-58;
26) the RNA sequence shown in SEQ ID NO.59-60;
27) the RNA sequence shown in SEQ ID NO.61-62;
28) the RNA sequence shown in SEQ ID NO.63-64;
29) the RNA sequence shown in SEQ ID NO.65-66;
30) the RNA sequence shown in SEQ ID NO.67-68;
31) the RNA sequence shown in SEQ ID NO.69-70;
32) the RNA sequence shown in SEQ ID NO.71-72;
33) the RNA sequence shown in SEQ ID NO.73-74;
34) the RNA sequence shown in SEQ ID NO.75-76;
35) the RNA sequence shown in SEQ ID NO.77-78;
36) the RNA sequence shown in SEQ ID NO.79-80;
37) the RNA sequence shown in SEQ ID NO.81-82;
38) the RNA sequence shown in SEQ ID NO.83-84;
39) the RNA sequence shown in SEQ ID NO.85-86;
40) the RNA sequence shown in SEQ ID NO.87-88;
41) the RNA sequence shown in SEQ ID NO.89-90;
42) the RNA sequence shown in SEQ ID NO.91-92;
43) the RNA sequence shown in SEQ ID NO.93-94;
44) the RNA sequence shown in SEQ ID NO.95-96;
45) the RNA sequence shown in SEQ ID NO.97-98;
46) the RNA sequence shown in SEQ ID NO.99-100;
47) the RNA sequence shown in SEQ ID NO.101-102;
48) the RNA sequence shown in SEQ ID NO.103-104;
49) the RNA sequence shown in SEQ ID NO.105-106;
50) the RNA sequence shown in SEQ ID NO.107-108;
51) the RNA sequence shown in SEQ ID NO.109-110;
52) the RNA sequence shown in SEQ ID NO.111-112;
53) the RNA sequence shown in SEQ ID NO.113-114;
54) the RNA sequence shown in SEQ ID NO.115-116;
55) the RNA sequence shown in SEQ ID NO.117-118;
56) the RNA sequence shown in SEQ ID NO.119-120;
57) the RNA sequence shown in SEQ ID NO.121-122;
58) the RNA sequence shown in SEQ ID NO.123-124;
59) the RNA sequence shown in SEQ ID NO.125-126;
60) the RNA sequence with SEQ ID NO.7-12 or SEQ ID NO.15-126 restriction has 90% above homology, and has the RNA sequence of identical function.
2. siRNA as claimed in claim 1 is characterized in that: described siRNA is the RNA sequence shown in SEQ ID NO.7-8,9-10 or 11-12.
One kind the coding dna sequence dna for antineoplastic siRNA as claimed in claim 1.
4. expression vector, it is characterized in that: described expression vector includes as claimed in claim 1 for antineoplastic siRNA sequence.
5. host cell, it is characterized in that: described host cell is a kind of cell for antineoplastic siRNA sequence as claimed in claim 1 of expressing.
6. host cell as claimed in claim 5, it is characterized in that: described host cell includes the described expression vector of claim 4.
One kind as claimed in claim 1 for the application of antineoplastic siRNA at preparation antitumor drug or antineoplastic pharmaceutical compositions.
8. application as claimed in claim 7 is characterized in that: described tumour comprises: liver cancer.
9. an antitumor drug is characterized in that: contain the as claimed in claim 1 for antineoplastic siRNA and pharmaceutically acceptable carrier of significant quantity.
10. medicine box is characterized in that: contain as claimed in claim 1 for antineoplastic siRNA or contain antitumor drug as claimed in claim 9.
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| CN101358200A (en) * | 2008-09-27 | 2009-02-04 | 四川大学 | RNA interference expression plasmid- cationic liposome-heparin antineoplastic complexes targeting human FAK and PLK1 gene |
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| CN101358200A (en) * | 2008-09-27 | 2009-02-04 | 四川大学 | RNA interference expression plasmid- cationic liposome-heparin antineoplastic complexes targeting human FAK and PLK1 gene |
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| Title |
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| JING-SONG CHEN ET AL.: "FAK is involved in invasion and metastasis of hepatocellular carcinoma", 《CLIN EXP METASTASIS》, vol. 27, no. 2, 24 February 2010 (2010-02-24), pages 71 - 82, XP019788058 * |
| JING-SONG CHEN ET AL.: "Sonic hedgehog signaling pathway induces cell migration and invasion through focal adhesion kinase/AKT signaling-mediated activation of matrix metalloproteinase (MMP)-2 and MMP-9 in liver cancer", 《CARCINOGENESIS》, vol. 34, no. 1, 4 September 2012 (2012-09-04), pages 1 - 10 * |
| 陈劲松: "Sonic Hedgehog信号通路在干细胞癌侵袭转移中的作用及其机制", 《中山大学博士学位论文》, 31 December 2010 (2010-12-31) * |
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