CN103184223B

CN103184223B - For antineoplastic siRNA and application thereof

Info

Publication number: CN103184223B
Application number: CN201310084396.8A
Authority: CN
Inventors: 黄健; 刘星
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-03-15
Filing date: 2013-03-15
Publication date: 2015-11-11
Anticipated expiration: 2033-03-15
Also published as: CN105063051A; CN103184223A; CN105063051B; CN105063050A; CN105063050B

Abstract

The invention discloses a kind of for antineoplastic siRNA and application thereof, does this siRNA comprise the one be selected from following nucleotide sequences: as SEQ? ID? NO.7-12 or SEQ? ID? RNA sequence shown in NO.15-126, with SEQ? ID? NO.7-12 or SEQ? ID? the RNA sequence that NO.15-126 limits has more than 90% homology, and has the RNA sequence of identical function.SiRNA of the present invention can suppress human FAK genetic expression, can be applicable to preparing in anti-tumor drug, for the exploitation for the treatment of or anti-curing oncoma (as liver cancer) new drug provides new approach.

Description

For antineoplastic siRNA and application thereof

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 (focaladhesionkinase, FAK) be a class cytosolic non-receptor protein tyrosine kinase, it is in very important position in cell signalling, mediate many signal paths, can Growth of Cells be regulated and occur with migration relevant with fetal development, tumour.

The invasive growth of tumour cell is the complex process of a multi-step, has multiple biochemical factors to participate.Tumour cell must attach to extracellular matrix, by promoting the extracellular matrix signal transduction depending on PTK kinase activity, and then affects sticking, move and moving of cell.Wherein, the signal transduction system of FAK mediation is one of wherein important cellular signal transduction pathways.

RNA interference (RNAi) refer to high conservative during evolution, brought out by double-stranded RNA (dsRNA), the phenomenon of the efficient selective degradation of homologous mRNA.Owing to using RNAi technology can specific depletion or close the expression of specific gene, so this technology has been widely used in the field of gene exploring gene function and communicable disease and malignant tumour.

Thus, utilize RNA perturbation technique, and in conjunction with the function of FAK, the treatment for tumour provides good directive significance.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of for antineoplastic siRNA and application thereof.By utilizing RNA perturbation technique, effectively can suppress the expression of FAK gene, thus, more effectively can carry out oncotherapy.

For solving the problems of the technologies described above, of the present invention for antineoplastic siRNA (for FAK gene), comprise the one be selected from following nucleotide sequences:

1) the RNA sequence as shown in SEQIDNO.7-8;

2) the RNA sequence as shown in SEQIDNO.9-10;

3) the RNA sequence as shown in SEQIDNO.11-12;

4) the RNA sequence as shown in SEQIDNO.15-16;

5) the RNA sequence as shown in SEQIDNO.17-18;

6) the RNA sequence as shown in SEQIDNO.19-20;

7) the RNA sequence as shown in SEQIDNO.21-22;

8) the RNA sequence as shown in SEQIDNO.23-24;

9) the RNA sequence as shown in SEQIDNO.25-26;

10) the RNA sequence as shown in SEQIDNO.27-28;

11) the RNA sequence as shown in SEQIDNO.29-30;

12) the RNA sequence as shown in SEQIDNO.31-32;

13) the RNA sequence as shown in SEQIDNO.33-34;

14) the RNA sequence as shown in SEQIDNO.35-36;

15) the RNA sequence as shown in SEQIDNO.37-38;

16) the RNA sequence as shown in SEQIDNO.39-40;

17) the RNA sequence as shown in SEQIDNO.41-42;

18) the RNA sequence as shown in SEQIDNO.43-44;

19) the RNA sequence as shown in SEQIDNO.45-46;

20) the RNA sequence as shown in SEQIDNO.47-48;

21) the RNA sequence as shown in SEQIDNO.49-50;

22) the RNA sequence as shown in SEQIDNO.51-52;

23) the RNA sequence as shown in SEQIDNO.53-54;

24) the RNA sequence as shown in SEQIDNO.55-56;

25) the RNA sequence as shown in SEQIDNO.57-58;

26) the RNA sequence as shown in SEQIDNO.59-60;

27) the RNA sequence as shown in SEQIDNO.61-62;

28) the RNA sequence as shown in SEQIDNO.63-64;

29) the RNA sequence as shown in SEQIDNO.65-66;

30) the RNA sequence as shown in SEQIDNO.67-68;

31) the RNA sequence as shown in SEQIDNO.69-70;

32) the RNA sequence as shown in SEQIDNO.71-72;

33) the RNA sequence as shown in SEQIDNO.73-74;

34) the RNA sequence as shown in SEQIDNO.75-76;

35) the RNA sequence as shown in SEQIDNO.77-78;

36) the RNA sequence as shown in SEQIDNO.79-80;

37) the RNA sequence as shown in SEQIDNO.81-82;

38) the RNA sequence as shown in SEQIDNO.83-84;

39) the RNA sequence as shown in SEQIDNO.85-86;

40) the RNA sequence as shown in SEQIDNO.87-88;

41) the RNA sequence as shown in SEQIDNO.89-90;

42) the RNA sequence as shown in SEQIDNO.91-92;

43) the RNA sequence as shown in SEQIDNO.93-94;

44) the RNA sequence as shown in SEQIDNO.95-96;

45) the RNA sequence as shown in SEQIDNO.97-98;

46) the RNA sequence as shown in SEQIDNO.99-100;

47) the RNA sequence as shown in SEQIDNO.101-102;

48) the RNA sequence as shown in SEQIDNO.103-104;

49) the RNA sequence as shown in SEQIDNO.105-106;

50) the RNA sequence as shown in SEQIDNO.107-108;

51) the RNA sequence as shown in SEQIDNO.109-110;

52) the RNA sequence as shown in SEQIDNO.111-112;

53) the RNA sequence as shown in SEQIDNO.113-114;

54) the RNA sequence as shown in SEQIDNO.115-116;

55) the RNA sequence as shown in SEQIDNO.117-118;

56) the RNA sequence as shown in SEQIDNO.119-120;

57) the RNA sequence as shown in SEQIDNO.121-122;

58) the RNA sequence as shown in SEQIDNO.123-124;

59) the RNA sequence as shown in SEQIDNO.125-126;

60) the RNA sequence limited with SEQIDNO.7-12 or SEQIDNO.15-126 has more than 90% homology, and has the RNA sequence of identical function.

Wherein, described siRNA is preferably the RNA sequence as shown in SEQIDNO.7-8,9-10 or 11-12.

In addition, the present invention also discloses a kind of coding as mentioned above for the DNA sequence dna of antineoplastic siRNA.

The invention also discloses a kind of expression vector, this expression vector includes as mentioned above for antineoplastic siRNA sequence.

The present invention again discloses a kind of host cell, and described host cell is a kind ofly expressed as mentioned above for the cell of antineoplastic siRNA sequence, as included expression vector as above.

Of the present inventionly preparing antitumor drug for antineoplastic siRNA or preparing the application in antineoplastic pharmaceutical compositions.Described tumour comprises: liver cancer.

The present invention again discloses a kind of antitumor drug, described above for antineoplastic siRNA and pharmaceutically acceptable carrier containing significant quantity.

This antitumor drug can adopt ordinary method to be prepared into corresponding preparations, as being made into injection form, such as, is prepared by ordinary method with physiological saline or the aqueous solution containing glucose and other assistant agents.Injection, solution etc. should aseptically manufacture.

This antitumor drug can administration in a convenient way, as by the route of administration such as in local, intravenously, intramuscular, subcutaneous, intracutaneous, nose.

The consumption of antitumor drug of the present invention and the course for the treatment of can appropriately adjust according to the light and heavy degree of the age of formulation, patient, disease, namely concrete application dosage depends on many factors, as factors such as administering mode, medication person's healthy state, these are all in skilled practitioners skill.

Moreover, the invention also discloses a kind of medicine box, containing described above for antineoplastic siRNA or containing upper described antitumor drug.This medicine box can indicative prompting given by the government authorities manufacturing, use or sell medicine or biological products, and this prompting reflects productions, use or the government authorities of sale permits that it uses on human body.

The present invention tests proves that the expression of FAK gene in liver cancer tissue is apparently higher than cancer beside organism, and therefore, FAK gene can be used as the specific 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 liver cancer cell invasion and attack, therefore, siRNA of the present invention can be applicable to preparing in anti-tumor drug, suppress in the medicine of liver cancer as being applied to preparation, for the exploitation for the treatment of or anti-curing oncoma (as liver cancer) new drug provides new approach.

Accompanying drawing explanation

Below in conjunction with accompanying drawing and embodiment, the present invention is further detailed explanation:

Fig. 1 is that RT-PCR verifies the expression schematic diagram of 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 after siRNA transfection SMMC7721 cell;

Fig. 5 is the FAK expression figure after siRNA transfection WRL68 cell;

Fig. 6 is the FAK expression figure after siRNA transfection QGY7701 cell;

Fig. 7 is the relative fluorescence figure after siRNA transfection SMMC7721 cell;

Fig. 8 is the relative fluorescence figure after siRNA transfection WRL68 cell;

Fig. 9 is the relative fluorescence figure after siRNA transfection QGY7701 cell;

Figure 10 is the cell cut figure after siRNA transfection SMMC7721.

Embodiment

Following examples are only not used in for illustration of the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in embodiment, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.

The expression of embodiment 1FAK gene in liver cancer tissue

1, separate tissue

Test with the tissue-derived operation patients in primary hepatocarcinoma.The liver of excision, once in vitro, cuts rapidly focus and surrounding 5 centimeters of outer cancer beside organisms, is divided in immediately in cryopreservation tube, put into liquid nitrogen (-80 DEG C) and preserve.The other diagnosis of cancer and cancer is all final foundation with pathological diagnosis.

2, Total RNAs extraction

Adopt Trizol (Invitrogen company) reagent, carry out the Total RNAs extraction organized according to its specification sheets.Concrete steps are as follows:

1) Homogenize homogenate: tissue homogenate: every 100mg tissue adds 1mlTRIzol reagent, and uses homogenizer homogenate.

2) add the chloroform of about 1/5 volume, turn upside down fully mixing about 1min, and room temperature leaves standstill 5min.

3) 4 DEG C, the centrifugal 15min of 12,000rpm.

4) carefully take out supernatant liquor, avoid touching middle layer, supernatant is proceeded to new 1.5ml centrifuge tube, add isopyknic Virahol, put upside down mixing gently, room temperature leaves standstill 5min.

5) 4 DEG C, the centrifugal 15min of 12000rpm.

6) suck supernatant, retain precipitation, and add 1ml75% ethanol, 4 DEG C, the centrifugal 15min washing precipitation of 12000rpm in precipitation.

7) suck supernatant, precipitation room temperature adds the water of appropriate RNase-free after naturally drying, with Tip head 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 DEG C of refrigerators in.

3, reverse transcription synthesis cDNA

With PrimeScriptRTMasterMixPerfectRealTime (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 DEG C 15 minutes;

2) 85 DEG C of 5 second;

3) 4 DEG C of preservations (preserving for a long time for-20 DEG C).

4、RT-PCR

Relative quantification method is adopted to detect the expression of FAK gene in liver cancer sample poor.

Use instrument: ThermalCyclerDice ^tMrealTimeSystemTP800, Takara.

Template: the template that above-mentioned reverse transcription product reacts as PCR after pressing 1:10 dilution proportion.

Primer is as shown in table 2:

Table 2 primer sequence

The system of PCR reaction:

PCR reaction adopts the thermotolerance Taq DNA polymerase (DR001) of TAKARA company, when taking human genome as template, and the DNA fragmentation of 3.0kbp (the p53 gene) that can increase well.Wherein, PCR reaction reagent and consumption as shown in table 3.

The reagent of table 3PCR reaction and consumption thereof

Reagent	Usage quantity
		Takara Taq(5U/μl)	0.1μl
10 × PCR damping fluid (Mg ²⁺Plus)	2μl
		DNTP mixture (often kind of 2.5mM)	1.6μl
Primer mixture (each 5 μMs)	1.6μl
		Template	0.5-4μl
ddH ₂O	Mend to 20 μ l

PCR reaction conditions:

β-actin：

Goal gene:

As shown in Figure 1, the expression level of FAK gene in liver cancer tissue is apparently higher than the expression level in cancer beside organism for result.

5, quantitative fluorescent PCR

Adopt the differential expression of fluorescence quantitative PCR detection FAK gene in liver cancer sample.

Use instrument: 7300 type quantitative real time PCR Instruments, ABI company.

Template: the template that above-mentioned reverse transcription product reacts as PCR after pressing 1:5 dilution proportion.

Primer sequence is as shown in table 4:

Table 4 primer sequence

The system of PCR reaction: PCR reaction adopts the SYBRPremixExTaq of TAKARA company.Wherein, PCR reaction reagent and consumption as shown in table 5.

The reagent of table 5PCR reaction and consumption thereof

Composition	Consumption
		SYBR Premix EX Taq	10.0μl
Upstream primer (10 μMs)	0.5μl
		Downstream primer (10 μMs)	0.5μl
ROX Reference Dye(50*)	0.4μl
		cDNA	1.0μl
ddH ₂O	7.6μl

PCR reaction conditions:

Denaturation: 95 DEG C 30 seconds

PCR reacts:

Melting curve measures:

95 DEG C 5 seconds

60 DEG C 60 seconds

95 DEG C 5 seconds

Fluorescence quantitative PCR detection shows, the expression level of FAK gene in liver cancer tissue is apparently higher than the expression level in cancer beside organism, and through adopting GraphpadPrism5 statistical package (GraphPadSoftware, SanDiego, CA, USA) to analyze, the comparison t inspection of quantitative data is analyzed, when P<0.05 thinks that statistics is variant, result display P<0.0001 (see Fig. 2).

6, Panomics analyzes

1) key instrument

Bio-plex suspension chip system: Bio-plex100system (Biorad company)

Bio-plex washes trigger: Bio-plexproIIwashstation (Biorad company)

Constant temperature oscillation shaking table: Vortemp56shakingincubator (LabnetInternational)

2) operation steps

The suspension liquid chip inspecting reagent unit of the 30 nucleic acid factor panel of people is Quantigeneplex2.0Kit (Panomics, Affymetrixcorp).Applied sample amount is 400ng.

Require to proceed as follows according to test kit specification sheets:

1. the RNA concentration of specimens of tissue extraction is adjusted to 20ng/ μ l.

2. require preparation microballon probe suspension according to test kit specification sheets, get 80 μ l and add in each hole of hybridization plate, then dummy or RNA sample 20 μ l are added in hand-hole.After pad pasting is closed, 600rpm in constant temperature oscillation shaking table, 54 DEG C of lucifuges hatch 18-22h.

3. sample probe microbeads for periods liquid is transferred in the hole of Beads enrichment plate, utilizes Bio-plex to wash trigger and wash, every hole 100ul, wash three times.

4. add pre-amplification liquid 100ul, 50 DEG C, 600rpm hatches 1h.

5. wash after trigger washs three times, add 100 μ l and amplify liquid, 50 DEG C, 600rpm hatches 1h.

6., after washing, add label probe liquid 100 μ l, 50 DEG C, 600rpm hatches 1h.

7., after washing, add SAPE working fluid 100 μ l, room temperature 600rpm hatches 30min.

8. after SAPE washings washes away unnecessary SAPE, add 130 μ lSAPE washingss, send into Bio-plex suspension chip system to carry out reading value, and through adopting GraphpadPrism5 statistical package (GraphPadSoftware, SanDiego, CA, USA) analyze, the comparison t inspection of quantitative data is analyzed, when P<0.05 thinks that statistics is variant.

Result as shown in Figure 3, the expression level of FAK gene in liver cancer tissue apparently higher than the expression level in cancer beside organism, P<0.0001 (see Fig. 3).

Embodiment 2 utilizes the expression of the reticent FAK gene of RNAi technology

1, cell cultures

Adopt common hepatoma cell strain, namely SMMC7721, WRL68, QGY7701 (laboratory preservation) carry out the expression study of FAK gene.Wherein, the culture condition of cell strain is DMEM (dulbecco'smodifiedeaglemedium) nutrient solution containing 10% (percent by volume) foetal calf serum (LifeTechnologies), 37 DEG C, containing 5%CO ₂cultivate in incubator.

2, the design of siRNA

According to the free service device that some companies on network provide, as Clontech company interface http://bioinfo2.clontech.com/rnaidesigner/sirnaSequenceDesign.d o, Invitrogen company interface https: //rnaidesigner.invitrogen.com/sirna/ and design corresponding siRNA according to InvivoGene server (network address http://www.sirnawizard.com).

Wherein, the siRNA sequence for FAK gene of design is as follows respectively:

FAK-siRNA1, positive-sense strand: 5 '-CAGGUGAAGAGCGAUUAUAtt-3 ' (SEQIDNO.7)

FAK-siRNA1, antisense strand: 5 '-UAUAAUCGCUCUUCACCUGtt-3 ' (SEQIDNO.8)

FAK-siRNA2, positive-sense strand: 5 '-CUCCAGUCUACAGAUUUGAtt-3 ' (SEQIDNO.9)

FAK-siRNA2, antisense strand: 5 '-UCAAAUCUGUAGACUGGAGtt-3 ' (SEQIDNO.10)

FAK-siRNA3, positive-sense strand: 5 '-CCCAGGUUUACUGAACUUAtt-3 ' (SEQIDNO.11)

FAK-siRNA3, antisense strand: 5 '-UAAGUUCAGUAAACCUGGGtt-3 ' (SEQIDNO.12)

Negative control siRNA, positive-sense strand: 5 '-UUCUCCGAACGUGUCACGUtt-3 ' (SEQIDNO.13)

Negative control siRNA, antisense strand: 5 '-ACGUGACACGUUCGGAGAAtt-3 ' (SEQIDNO.14)

FAK-siRNA4, positive-sense strand: 5 '-GCAUCUUCCAGUUACAAAUtt-3 ' (SEQIDNO.15)

FAK-siRNA4, antisense strand: 5 '-AUUUGUAACUGGAAGAUGCtt-3 ' (SEQIDNO.16)

FAK-siRNA5, positive-sense strand: 5 '-GACCCCAACUUGAAUCACAtt-3 ' (SEQIDNO.17)

FAK-siRNA5, antisense strand: 5 '-UGUGAUUCAAGUUGGGGUCtt-3 ' (SEQIDNO.18)

FAK-siRNA6, positive-sense strand: 5 '-GUGCAAUGGAGCGAGUAUUtt-3 ' (SEQIDNO.19)

FAK-siRNA6, antisense strand: 5 '-AAUACUCGCUCCAUUGCACtt-3 ' (SEQIDNO.20)

FAK-siRNA7, positive-sense strand: 5 '-GCAAUGGAGCGAGUAUUAAtt-3 ' (SEQIDNO.21)

FAK-siRNA7, antisense strand: 5 '-UUAAUACUCGCUCCAUUGCtt-3 ' (SEQIDNO.22)

FAK-siRNA8, positive-sense strand: 5 '-GAGCGAGUAUUAAAGGUCUtt-3 ' (SEQIDNO.23)

FAK-siRNA8, antisense strand: 5 '-AGACCUUUAAUACUCGCUCtt-3 ' (SEQIDNO.24)

FAK-siRNA9, positive-sense strand: 5 '-GGAGAUGCUACUGAUGUCAtt-3 ' (SEQIDNO.25)

FAK-siRNA9, antisense strand: 5 '-UGACAUCAGUAGCAUCUCCtt-3 ' (SEQIDNO.26)

FAK-siRNA10, positive-sense strand: 5 '-GGGCAUCAUUCAGAAGAUAtt-3 ' (SEQIDNO.27)

FAK-siRNA10, antisense strand: 5 '-UAUCUUCUGAAUGAUGCCCtt-3 ' (SEQIDNO.28)

FAK-siRNA11, positive-sense strand: 5 '-GCAUCAUUCAGAAGAUAGUtt-3 ' (SEQIDNO.29)

FAK-siRNA11, antisense strand: 5 '-ACUAUCUUCUGAAUGAUGCtt-3 ' (SEQIDNO.30)

FAK-siRNA12, positive-sense strand: 5 '-GUGGACAGUCACAAAGUAAtt-3 ' (SEQIDNO.31)

FAK-siRNA12, antisense strand: 5 '-UUACUUUGUGACUGUCCACtt-3 ' (SEQIDNO.32)

FAK-siRNA13, positive-sense strand: 5 '-GUGUGAGGGAGAAGUAUGAtt-3 ' (SEQIDNO.33)

FAK-siRNA13, antisense strand: 5 '-UCAUACUUCUCCCUCACACtt-3 ' (SEQIDNO.34)

FAK-siRNA14, positive-sense strand: 5 '-GAGGAGUGGAAAUAUGAAUtt-3 ' (SEQIDNO.35)

FAK-siRNA14, antisense strand: 5 '-AUUCAUAUUUCCACUCCUCtt-3 ' (SEQIDNO.36)

FAK-siRNA15, positive-sense strand: 5 '-GUGGAAAUAUGAAUUGAGAtt-3 ' (SEQIDNO.37)

FAK-siRNA15, antisense strand: 5 '-UCUCAAUUCAUAUUUCCACtt-3 ' (SEQIDNO.38)

FAK-siRNA16, positive-sense strand: 5 '-GUGAAGAGCGAUUAUAUGUtt-3 ' (SEQIDNO.39)

FAK-siRNA16, antisense strand: 5 '-ACAUAUAAUCGCUCUUCACtt-3 ' (SEQIDNO.40)

FAK-siRNA17, positive-sense strand: 5 '-GCGAUUAUAUGUUAGAGAUtt-3 ' (SEQIDNO.41)

FAK-siRNA17, antisense strand: 5 '-AUCUCUAACAUAUAAUCGCtt-3 ' (SEQIDNO.42)

FAK-siRNA18, positive-sense strand: 5 '-GAAAUACGGCGAUCAUACUtt-3 ' (SEQIDNO.43)

FAK-siRNA18, antisense strand: 5 '-AGUAUGAUCGCCGUAUUUCtt-3 ' (SEQIDNO.44)

FAK-siRNA19, positive-sense strand: 5 '-GAAGUCUAACUAUGAAGUAtt-3 ' (SEQIDNO.45)

FAK-siRNA19, antisense strand: 5 '-UACUUCAUAGUUAGACUUCtt-3 ' (SEQIDNO.46)

FAK-siRNA20, positive-sense strand: 5 '-GAUCCAACAAACAUUUAGAtt-3 ' (SEQIDNO.47)

FAK-siRNA20, antisense strand: 5 '-UCUAAAUGUUUGUUGGAUCtt-3 ' (SEQIDNO.48)

FAK-siRNA21, positive-sense strand: 5 '-GGUUCAAGCUGGAUUAUUUtt-3 ' (SEQIDNO.49)

FAK-siRNA21, antisense strand: 5 '-AAAUAAUCCAGCUUGAACCtt-3 ' (SEQIDNO.50)

FAK-siRNA22, positive-sense strand: 5 '-GCUGGAUUAUUUCAGUGGAtt-3 ' (SEQIDNO.51)

FAK-siRNA22, antisense strand: 5 '-UCCACUGAAAUAAUCCAGCtt-3 ' (SEQIDNO.52)

FAK-siRNA23, positive-sense strand: 5 '-GAAGGAAUCAGUUACCUAAtt-3 ' (SEQIDNO.53)

FAK-siRNA23, antisense strand: 5 '-UUAGGUAACUGAUUCCUUCtt-3 ' (SEQIDNO.54)

FAK-siRNA24, positive-sense strand: 5 '-GUGCAAACCAUUCAGUAUUtt-3 ' (SEQIDNO.55)

FAK-siRNA24, antisense strand: 5 '-AAUACUGAAUGGUUUGCACtt-3 ' (SEQIDNO.56)

FAK-siRNA25, positive-sense strand: 5 '-GCAAACCAUUCAGUAUUCAtt-3 ' (SEQIDNO.57)

FAK-siRNA25, antisense strand: 5 '-UGAAUACUGAAUGGUUUGCtt-3 ' (SEQIDNO.58)

FAK-siRNA26, positive-sense strand: 5 '-GGAGAAUAUGGCUGACCUAtt-3 ' (SEQIDNO.59)

FAK-siRNA26, antisense strand: 5 '-UAGGUCAGCCAUAUUCUCCtt-3 ' (SEQIDNO.60)

FAK-siRNA27, positive-sense strand: 5 '-GAAUAUGGCUGACCUAAUAtt-3 ' (SEQIDNO.61)

FAK-siRNA27, antisense strand: 5 '-UAUUAGGUCAGCCAUAUUCtt-3 ' (SEQIDNO.62)

FAK-siRNA28, positive-sense strand: 5 '-GAAUGGAACCUCGCAGUCAtt-3 ' (SEQIDNO.63)

FAK-siRNA28, antisense strand: 5 '-UGACUGCGAGGUUCCAUUCtt-3 ' (SEQIDNO.64)

FAK-siRNA29, positive-sense strand: 5 '-GGAACCUCGCAGUCAUUUAtt-3 ' (SEQIDNO.65)

FAK-siRNA29, antisense strand: 5 '-UAAAUGACUGCGAGGUUCCtt-3 ' (SEQIDNO.66)

FAK-siRNA30, positive-sense strand: 5 '-GAACCUCGCAGUCAUUUAUtt-3 ' (SEQIDNO.67)

FAK-siRNA30, antisense strand: 5 '-AUAAAUGACUGCGAGGUUCtt-3 ' (SEQIDNO.68)

FAK-siRNA31, positive-sense strand: 5 '-GCAGUCAUUUAUCAUCAGAtt-3 ' (SEQIDNO.69)

FAK-siRNA31, antisense strand: 5 '-UCUGAUGAUAAAUGACUGCtt-3 ' (SEQIDNO.70)

FAK-siRNA32, positive-sense strand: 5 '-GAUGAUUAUGCUGAGAUUAtt-3 ' (SEQIDNO.71)

FAK-siRNA32, antisense strand: 5 '-UAAUCUCAGCAUAAUCAUCtt-3 ' (SEQIDNO.72)

FAK-siRNA33, positive-sense strand: 5 '-GGAUUAUGAGAUUCAAAGAtt-3 ' (SEQIDNO.73)

FAK-siRNA33, antisense strand: 5 '-UCUUUGAAUCUCAUAAUCCtt-3 ' (SEQIDNO.74)

FAK-siRNA34, positive-sense strand: 5 '-GUACAUCAAGGCAUUUAUAtt-3 ' (SEQIDNO.75)

FAK-siRNA34, antisense strand: 5 '-UAUAAAUGCCUUGAUGUACtt-3 ' (SEQIDNO.76)

FAK-siRNA35, positive-sense strand: 5 '-GUGAAGCUGAUUGGAGUCAtt-3 ' (SEQIDNO.77)

FAK-siRNA35, antisense strand: 5 '-UGACUCCAAUCAGCUUCACtt-3 ' (SEQIDNO.78)

FAK-siRNA36, positive-sense strand: 5 '-GCAAGUAAGGAAAUACAGUtt-3 ' (SEQIDNO.79)

FAK-siRNA36, antisense strand: 5 '-ACUGUAUUUCCUUACUUGCtt-3 ' (SEQIDNO.80)

FAK-siRNA37, positive-sense strand: 5 '-GGUGUCCUCAAAUGAUUGUtt-3 ' (SEQIDNO.81)

FAK-siRNA37, antisense strand: 5 '-ACAAUCAUUUGAGGACACCtt-3 ' (SEQIDNO.82)

FAK-siRNA38, positive-sense strand: 5 '-GGCUCCAGAGUCAAUCAAUtt-3 ' (SEQIDNO.83)

FAK-siRNA38, antisense strand: 5 '-AUUGAUUGACUCUGGAGCCtt-3 ' (SEQIDNO.84)

FAK-siRNA39, positive-sense strand: 5 '-GCUCCAGAGUCAAUCAAUUtt-3 ' (SEQIDNO.85)

FAK-siRNA39, antisense strand: 5 '-AAUUGAUUGACUCUGGAGCtt-3 ' (SEQIDNO.86)

FAK-siRNA40, positive-sense strand: 5 '-GAGUGAAGAACAAUGAUGUtt-3 ' (SEQIDNO.87)

FAK-siRNA40, antisense strand: 5 '-ACAUCAUUGUUCUUCACUCtt-3 ' (SEQIDNO.88)

FAK-siRNA41, positive-sense strand: 5 '-GUGAAGAACAAUGAUGUAAtt-3 ' (SEQIDNO.89)

FAK-siRNA41, antisense strand: 5 '-UUACAUCAUUGUUCUUCACtt-3 ' (SEQIDNO.90)

FAK-siRNA42, positive-sense strand: 5 '-GCCCAGGUUUACUGAACUUtt-3 ' (SEQIDNO.91)

FAK-siRNA42, antisense strand: 5 '-AAGUUCAGUAAACCUGGGCtt-3 ' (SEQIDNO.92)

FAK-siRNA43, positive-sense strand: 5 '-GCAGCAUCUAUCCAGGUCAtt-3 ' (SEQIDNO.93)

FAK-siRNA43, antisense strand: 5 '-UGACCUGGAUAGAUGCUGCtt-3 ' (SEQIDNO.94)

FAK-siRNA44, positive-sense strand: 5 '-GUGGAGGACUCUACAGUAUtt-3 ' (SEQIDNO.95)

FAK-siRNA44, antisense strand: 5 '-AUACUGUAGAGUCCUCCACtt-3 ' (SEQIDNO.96)

FAK-siRNA45, positive-sense strand: 5 '-GAGGGAUUGGGCAAGUGUUtt-3 ' (SEQIDNO.97)

FAK-siRNA45, antisense strand: 5 '-AACACUUGCCCAAUCCCUCtt-3 ' (SEQIDNO.98)

FAK-siRNA46, positive-sense strand: 5 '-GACUCUCUCGAGGCAGUAUtt-3 ' (SEQIDNO.99)

FAK-siRNA46, antisense strand: 5 '-AUACUGCCUCGAGAGAGUCtt-3 ' (SEQIDNO.100)

FAK-siRNA47, positive-sense strand: 5 '-GGUCGAAUGAUAAGGUGUAtt-3 ' (SEQIDNO.101)

FAK-siRNA47, antisense strand: 5 '-UACACCUUAUCAUUCGACCtt-3 ' (SEQIDNO.102)

FAK-siRNA48, positive-sense strand: 5 '-GCCUGGUGAAAGCUGUCAUtt-3 ' (SEQIDNO.103)

FAK-siRNA48, antisense strand: 5 '-AUGACAGCUUUCACCAGGCtt-3 ' (SEQIDNO.104)

FAK-siRNA49, positive-sense strand: 5 '-GGACAUUAUUGGCCACUGUtt-3 ' (SEQIDNO.105)

FAK-siRNA49, antisense strand: 5 '-ACAGUGGCCAAUAAUGUCCtt-3 ' (SEQIDNO.106)

FAK-siRNA50, positive-sense strand: 5 '-GCACAGAAGCUAUUGAACUtt-3 ' (SEQIDNO.107)

FAK-siRNA50, antisense strand: 5 '-AGUUCAAUAGCUUCUGUGCtt-3 ' (SEQIDNO.108)

FAK-siRNA51, positive-sense strand: 5 '-GCCCAGCAGUAUGUCAUGAtt-3 ' (SEQIDNO.109)

FAK-siRNA51, antisense strand: 5 '-UCAUGACAUACUGCUGGGCtt-3 ' (SEQIDNO.110)

FAK-siRNA52, positive-sense strand: 5 '-GCCUCCAGCAAGAGUACAAtt-3 ' (SEQIDNO.111)

FAK-siRNA52, antisense strand: 5 '-UUGUACUCUUGCUGGAGGCtt-3 ' (SEQIDNO.112)

FAK-siRNA53, positive-sense strand: 5 '-GCAUCAUGAAGAACAAUUUtt-3 ' (SEQIDNO.113)

FAK-siRNA53, antisense strand: 5 '-AAAUUGUUCUUCAUGAUGCtt-3 ' (SEQIDNO.114)

FAK-siRNA54, positive-sense strand: 5 '-GCUGCAUAGUGGAAGAGGAtt-3 ' (SEQIDNO.115)

FAK-siRNA54, antisense strand: 5 '-UCCUCUUCCACUAUGCAGCtt-3 ' (SEQIDNO.116)

FAK-siRNA55, positive-sense strand: 5 '-GAGCAUGAAGCAAAGAAUUtt-3 ' (SEQIDNO.117)

FAK-siRNA55, antisense strand: 5 '-AAUUCUUUGCUUCAUGCUCtt-3 ' (SEQIDNO.118)

FAK-siRNA56, positive-sense strand: 5 '-GCUAAUCCCACUUUACAAAtt-3 ' (SEQIDNO.119)

FAK-siRNA56, antisense strand: 5 '-UUUGUAAAGUGGGAUUAGCtt-3 ' (SEQIDNO.120)

FAK-siRNA57, positive-sense strand: 5 '-GUCGGGAACUAGCUGUAGAtt-3 ' (SEQIDNO.121)

FAK-siRNA57, antisense strand: 5 '-UCUACAGCUAGUUCCCGACtt-3 ' (SEQIDNO.122)

FAK-siRNA58, positive-sense strand: 5 '-GGGAACUAGCUGUAGAACAtt-3 ' (SEQIDNO.123)

FAK-siRNA58, antisense strand: 5 '-UGUUCUACAGCUAGUUCCCtt-3 ' (SEQIDNO.124)

FAK-siRNA59, positive-sense strand: 5 '-GUAGCAAUGUUAUUUCUCUtt-3 ' (SEQIDNO.125)

FAK-siRNA59, antisense strand: 5 '-AGAGAAAUAACAUUGCUACtt-3 ' (SEQIDNO.126)

3, siRNA transient transfection

Select SMMC7721, WRL68, QGY7701 cell strain of the higher FAK gene of endogenous expression.Synthetic often kind siRNA (being respectively FAK-siRNA1, FAK-siRNA2, FAK-siRNA3 and negative control siRNA) is used DEPC water dissolution, obtains the solution of 20 μMs of concentration.With 1 μ lLipofectamine ^tM2000 (Invitrogen) and 1 μ l concentration are after often kind of siRNA of 20 μMs mixes respectively, mixed solution is forwarded to and puies forward passage cell density the day before yesterday (24 porocyte culture dish) in SMMC7721, WRL68 and QGY7701 cell of about 30-40%, change complete culture solution after 4 hours, continue to cultivate.Turn collecting cell after 48h wink, then carry out follow-up Total RNAs extraction.

4, the extraction of cell total rna

Adopt Trizol (TAKARA company) reagent, carry out according to its specification sheets the total serum IgE extracting cell, step is as follows:

1) attached cell (24 hole culture dish) cultivated, after adopting above-mentioned " 3, siRNA transient transfection " method to carry out transfection, cultivates 48h, sucks nutrient solution, add 500 μ lTRIzol in culture dish, with the piping and druming of rifle head several times;

2) be transferred in the centrifuge tube of 1.5ml by lysate, room temperature places 5min;

3) add chloroform by the amount of 0.2ml chloroform/mlTRIzol, build pipe lid, violent jolting 15s, room temperature places 5min, 4 DEG C of centrifugal 15min of 12000g;

4) upper phase be transferred in a clean centrifuge tube, add Virahol (0.5ml/mlTRIzol), put upside down mixing gently for several times, room temperature places 10min, 4 DEG C of centrifugal 15min of 12000g;

5) abandon supernatant, the ethanol (1ml/mlTRIzol) adding 75% fully vibrates mixing, 4 DEG C of centrifugal 10min of 12000g;

6) remove supernatant, be deposited in room temperature and place 5 ~ 10min, make its natural airing (not complete drying); Add the DEPC water dissolution RNA of 30-50 μ l; 260/280 ratio is measured also quantitatively with ultraviolet spectrophotometer;

7) DNA is removed: adopt DNaseI (RNaseFree, TaKaRa company) to remove DNA pollution possible in total serum IgE, operation steps is as follows:

1. in Eppendorf tube, prepare reaction solution (total amount 50 μ l) as shown in table 6, then, 37 DEG C of reaction 20min.

Table 6 reaction solution

Composition	Consumption
		Total serum IgE	20～50μg
10×DNase I Buffer	5μl
		DNase I(RNase-free，5U/μl)	2μl
RNase Inhibitor(40U/μl)	0.5μl
		The H of DEPC process ₂O	Until reaction solution total amount is 50 μ l

2. the H of the DEPC process of 50 μ l is added ₂o.

3. add the phenol/chloroform/primary isoamyl alcohol (volume ratio is respectively 25:24:1) of 100 μ l (equivalent), fully mix.

4. centrifugal 12000rpm, 15min, gets upper strata (water layer) and moves in another Eppendorf tube.

5. add the chloroform/primary isoamyl alcohol (volume ratio is 24:1) of 100 μ l (equivalent), fully mix.

6. centrifugal 12000rpm, 15min, gets upper strata (water layer) and moves in another Eppendorf tube.

7. the 3M sodium acetate (pH5.2) of 10 μ l (1/10 amount) is added.

8. add the cold dehydrated alcohol of 250 μ l (2.5 times amount), place 30-60 minute for-20 DEG C.

9. centrifugal recovery precipitation 12000rpm, 10min is by the cold ethanol purge precipitation of 70%, dry.

10. with the H of appropriate DEPC process ₂o dissolves, quantitatively.

5, reverse transcription synthesis cDNA

With PrimeScriptRTMasterMixPerfectRealTime (TAKARA company) reverse transcription cDNA, reaction system (total amount is 10 μ l) is as shown in table 7.

Table 7 reaction system

Composition	Consumption
		5*PrimeScript RT Master Mix(for Real Time)	2μl
Total serum IgE	500ng
		Remove the dH of RNase ₂O	Complement to 10 μ l

Reaction conditions is as follows:

1) 37 DEG C 15 minutes;

2) 85 DEG C of 5 second;

3) 4 DEG C of preservations (preserving for a long time for-20 DEG C).

6, quantitative fluorescent PCR

ABI7300 real-time fluorescence quantitative PCR instrument and SYBRPremixExTaq test kit is used to carry out, using β-actin (ACTB) as internal reference, analyze the impact that transfection siRNA expresses hepatoma cell strain FAK, 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

PCR reaction conditions is: 94 DEG C of denaturation 30s, afterwards 94 DEG C of 10s, 60 DEG C of 30s, after carrying out 40 circulations altogether, carries out the detection of solubility curve.

Wherein, autofluorescent background signal and threshold value generally adopt instrument default number, and automatically generate after each run terminates, the cycle number experienced when the fluorescent signal in each reaction tubes reaches the threshold value of setting is defined as Ct value; Data analysis is carried out as follows: often pair of primer (gene) is done 3 and repeated pipe in each template, and the Ct value obtained is averaged; The Ct mean value of each goal gene deducts the Ct mean value of the reference 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 proportion 2-Δ Δ Ct of the testing gene in control group and experimental group.

Experimental result shows, these three siRNA fragment of FAK-siRNA1, FAK-siRNA2 and FAK-siRNA3 all can the expression of reticent FAK gene effectively, and the institute that prompting may be used for next step uses.Wherein, the experimental result in SMMC7721, WRL68, QGY7701 cell as Figure 4-Figure 6.

7, the mensuration of cell growth curve

1) day before transfection inoculates appropriate cell, and when making transfection, cell density controls between 30% ~ 50%;

2) liposome Lipofectamine is adopted ^tM2000 (Invitrogen) transfection siRNA

Preparation siRNA and Lipofectamine ^tMthe mixed solution of 2000, the formula of mixed solution is with 1 μ lLipofectamine ^tM2000 and 1 μ l concentration is after often kind of siRNA (FAK-siRNA1, FAK-siRNA2, FAK-siRNA3 and negative control siRNA use DEPC water dissolution respectively) of 20 μMs mixes respectively, and room temperature is placed about 20 minutes, forms siRNA-Lipofectamine ^tM2000 mixtures; Then, by siRNA-Lipofectamine ^tM2000 mixtures (2 μ l) add in cell, mixing; Normal nutrient solution is changed after 4 ~ 6 hours;

3), after 24 hours, SMMC7721, WRL68, QGY7701 cell after digestion transfection siRNAs, according to its growth characteristics by 1 × 10 ³/ 100 μ l/ holes calculate cell total amount, and after fully diluting, plant in 96 orifice plates.Often organize multiple hole every day six, generally press 7-8 days inoculating cells;

4) treat about half an hour, the substantially adherent rear observation of cell state of cell and number.With chromogenic reagent reaction, to determine the initial density of cell reality, as growth relative zero.The usage of developer Cellcountingkit-8 (CCK-8, Dojindo, Japan): every 100 μ l train liquid and add 10 μ lCCK-8,37 DEG C, 5%CO ₂place 1h, microplate reader measures the absorbancy at 450nm place;

5) observation of cell form under light microscopic.Timing measurement on request, record upgrowth situation;

6) survey 4 to 7 days are generally needed.After data gathering, then process, draw chart with Excel.

Found that, these three siRNA fragment 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, QGY7701 cell as Figure 7-9.

8, cell scratch experiment

SMMC7721 cell is spread in 24 porocyte culture dish with the density of 80%-90%.With 1 μ lLipofectamine ^tM2000 and 1 μ l concentration is after often kind of siRNA (FAK-siRNA1, FAK-siRNA2, FAK-siRNA3 and negative control siRNA use DEPC water dissolution respectively) of 20 μMs mixes respectively, respectively transfectional cell, carrys out reticent FAK.After 24 hours, plastics rifle head is used to streak cell surface, 37 DEG C, 5%CO ₂cell culture incubator in cultivate 4 days, 0,1,2,3 day time, gather the image of same position respectively.Experiment in triplicate.Cell with negative control transfection or the cell without any process are in contrast.

Found that, these three siRNA fragment 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 as shown in Figure 10.

9, cell invasion experiment

Inoculate appropriate cell (SMMC7721, SK-hep1), when making transfection, cell density controls between 30% ~ 50%; Liposome Lipofectamine is adopted in 24 hours ^tM2000 (Invitrogen) transfection siRNA: preparation siRNA and Lipofectamine ^tMthe mixed solution of 2000, namely uses 1 μ lLipofectamine ^tM2000 and 1 μ l concentration is after often kind of siRNA (FAK-siRNA1 and negative control siRNA uses DEPC water dissolution respectively) of 20 μMs mixes respectively, and room temperature is placed about 20 minutes, forms siRNA-Lipofectamine ^tM2000 mixtures; By siRNA-Lipofectamine ^tM2000 mixtures (often kind of 2 μ l) add in cell, mixing; Normal nutrient solution is changed after 4 ~ 6 hours.

Cell invasion experiment all adopts 24 hole Transwell cells (8-μm of aperture, BDBiosciences company), scribbles artificial basement membrane (Falcon354480; BDBiosciences company).In serum free medium, liver cancer cell (SMMC7721, SK-hep1) overnight starvation, uses tryptic digestion process, and washs 3 times in containing the DMEM nutrient solution of 1%FBS.Will containing 1 × 10 ⁵500 μ l of individual cell, containing in the DMEM nutrient solution of 1%FBS, join upper chamber.750 μ l are placed at lower chamber containing the DMEM substratum of 10%FBS and the fibronectin (catalog number 356008, BDBiosciences company) of 10 μ g/ml simultaneously.In contrast, lower chamber is added by containing in the substratum of 1%FBS.Through the latent period of 48 hours, in upper chamber, remaining artificial basement membrane and cell cotton swab were removed.Cell on film lower surface 4% paraformaldehyde is fixed, and uses 0.5% violet staining.Cell counts and takes pictures at least 6 random fields of microscope (multiplying power × 100).All experiments are all repeating, and repeat 3 times, and adopt GraphpadPrism5 statistical package (GraphPadSoftware, SanDiego, CA, USA) analyze, the comparison t inspection of quantitative data is analyzed, when P<0.05 thinks that statistics is variant.

Found that, FAK-siRNA1 obviously can suppress the invasion and attack of liver cancer cell SMMC7721, SK-hep1.

In sum, the present invention utilizes the expression of the reticent FAK of RNAi technology can suppress Growth of Cells, the mobility and invasiveness of liver cancer.Thus, siRNA of the present invention can be applicable to preparation treatment or anti-curing oncoma (as liver cancer) medicine, is the treatment of tumour, provides new approach.

Claims

1. for suppressing a siRNA for SMMC7721, QGY7701 liver cancer cell of high expression level FAK gene, it is characterized in that, described siRNA is the RNA sequence such as shown in SEQIDNO.7-8.

2. the DNA sequence dna of a coding siRNA as claimed in claim 1.

3. an expression vector, is characterized in that: described expression vector comprises DNA sequence dna according to claim 2.

4. a host cell, is characterized in that: described host cell is a kind of cell of expressing siRNA sequence as claimed in claim 1.

5. host cell as claimed in claim 4, is characterized in that: described host cell includes expression vector according to claim 3.

6. the application of siRNA as claimed in claim 1 in the medicine of SMMC7721, QGY7701 liver cancer cell of preparation suppression high expression level FAK gene.

7. suppress a medicine for SMMC7721, QGY7701 liver cancer cell of high expression level FAK gene, it is characterized in that: the siRNA as claimed in claim 1 containing significant quantity and pharmaceutically acceptable carrier.

8. a medicine box, is characterized in that: containing, for example siRNA according to claim 1 or containing, for example medicine according to claim 7.