CN105063051B

CN105063051B - A kind of siRNA and its application for being used to suppress the hepatocellular carcinoma cells of high expression FAK genes

Info

Publication number: CN105063051B
Application number: CN201510540569.1A
Authority: CN
Inventors: 黄健
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-03-15
Filing date: 2013-03-15
Publication date: 2018-02-27
Anticipated expiration: 2033-03-15
Also published as: CN103184223A; CN105063050B; CN105063051A; CN103184223B; CN105063050A

Abstract

The invention discloses a kind of siRNA for being used to suppress SMMC7721, QGY7701 hepatocellular carcinoma cells of high expression FAK genes, the siRNA is the RNA sequence as shown in SEQ ID NO.11 12.The siRNA of the present invention can suppress human FAK gene expression, can be applied in the medicine for preparing anti-liver cancer and anti-tumour, new approach is provided to treat or preventing and treating the exploitation of liver cancer new drug.

Description

A kind of siRNA for being used to suppress the hepatocellular carcinoma cells of high expression FAK genes and its Using

This application is the divisional application of following patent application：Application No. 201310084396.8, the applying date are 2013 3 The moon 15, entitled " being used for antitumor siRNA and its application ".

Technical field

It is thin more particularly to a kind of liver for being used to suppress high expression FAK genes the present invention relates to a kind of siRNA and its application The siRNA of born of the same parents' cancer cell and its application.

Background technology

Focal adhesion kinase (focal adhesion kinase, FAK) is a kind of cytosolic non-receptor protein tyrosine kinase, It is in highly important position in cell signalling, mediates more signal paths, can adjust cell growth and and embryo Fetal hair is educated, tumour generation is relevant with migration.

The invasive growth of tumour cell is the complex process of a multi-step, has a variety of biochemical factors to participate in it In.Tumour cell must attach to extracellular matrix, by promoting the extracellular matrix signal dependent on PTK kinase activities to turn Lead, and then influence sticking, moving and migration for cell.Wherein, the signal transduction system of FAK mediations is wherein important cell letter One of number transduction pathway.

RNA interference (RNAi) refers to being highly conserved during evolution, being induced by double-stranded RNA (dsRNA), homologous The phenomenon of the efficient selective degradations of mRNA.Due to using RNAi technology can with specific depletion or close specific gene expression, So the technology has been widely used for exploring gene function and the field of gene of communicable disease and malignant tumour.

Thus, using RNA perturbation techniques, and FAK function is combined, good directive significance is provided for the treatment of tumour.

The content of the invention

The technical problem to be solved in the present invention is to provide a kind of hepatocellular carcinoma cells for being used to suppress high expression FAK genes SiRNA and its application.By using RNA perturbation techniques, it can effectively suppress the expression of FAK genes, thus, can more efficiently it enter Row oncotherapy.

In order to solve the above technical problems, a kind of SMMC7721 that FAK genes are expressed for suppressing height of present invention offer, The siRNA of QGY7701 hepatocellular carcinoma cells, the siRNA are the RNA sequence as shown in SEQ ID NO.11-12.

In addition, the present invention also provides a kind of DNA sequence dna for encoding siRNA as described above.

The present invention also provides a kind of expression vector, and the expression vector includes siRNA as described above DNA sequence dna.

The present invention provides a kind of host cell again, and the host cell is that one kind can express siRNA sequence as described above Cell, can such as include expression vector as described above.

In addition, the present invention, which also provides a kind of siRNA as described above, is preparing the high expression FAK genes of suppression Application in the medicine of SMMC7721, QGY7701 hepatocellular carcinoma cells.

In addition, the present invention also provides a kind of SMMC7721, QGY7701 hepatocellular carcinoma cells for suppressing high expression FAK genes Medicine, siRNA as described above and pharmaceutically acceptable carrier containing effective dose.

The medicine can be prepared into corresponding preparations using conventional method, can such as be made into injection form, such as use physiology salt Water or the aqueous solution containing glucose and other assistant agents are prepared by conventional method.Injection, solution etc. are preferably in aseptic condition Lower manufacture.

The medicine can be administered in a convenient way, such as by local, intravenous, intramuscular, subcutaneous, intracutaneous, intranasal give Medicine approach.

The dosage and the course for the treatment of of medicine of the present invention can appropriately adjust according to the light and heavy degree of formulation, the age of patient, disease, I.e. specific application dosage depends on many factors, such as administering mode, drug user's health status factor, and these are all skillfully to cure In the range of teacher's technical ability.

Furthermore the present invention also provides a kind of medicine box, containing siRNA as described above or contains medicine as described above.Should Medicine box can be according to the indicative prompting given by the government authorities of manufacture, use or sale medicine or biological products, and this is carried Show that the government authorities for reflecting production, use or sale permit it to be applied on human body.

Present invention experiment proves expression of the FAK genes in liver cancer tissue apparently higher than cancer beside organism, therefore, FAK genes As the specific marker gene of diagnosing liver cancer diagnosing cancer of liver can be made more accurate, quick.Meanwhile siRNA of the invention can suppress Human FAK gene expression, liver cancer cells invasion and attack are reduced, therefore, siRNA of the invention can be applied to preparing the medicine of suppression liver cancer In, provide new approach to treat or preventing and treating the exploitation of liver cancer new drug.

Brief description of the drawings

The present invention is further detailed explanation with embodiment below in conjunction with the accompanying drawings：

Fig. 1 is expression signal of the RT-PCR checking FAK genes in 10 hepatocarcinoma patient cancerous tissues and cancer beside organism Figure, wherein, " N " refers to cancer beside organism, and " C " refers to liver cancer tissue；

Fig. 2 is expression figure of the fluorescence quantitative PCR detection FAK genes in 95 hepatocarcinoma patients；

Fig. 3 is expression figure of the Panomics analysis FAK genes in 146 hepatocarcinoma patients；

Fig. 4 is the FAK expression figures after siRNA transfection SMMC7721 cells；

Fig. 5 is the FAK expression figures after siRNA transfection WRL68 cells；

Fig. 6 is the FAK expression figures after siRNA transfection QGY7701 cells；

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

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

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

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

Embodiment

Following examples are only illustrative of the invention and is not intended to limit the scope of the invention.It is unreceipted specific in embodiment The experimental method of condition, generally according to normal condition, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or built according to manufacturer The condition of view.

Expression of the embodiment 1FAK genes in liver cancer tissue

1st, tissue separation

The tissue-derived operation patients in primary carcinoma of liver of experiment.The liver of surgery excision is cut rapidly once in vitro 5 centimeters of outer cancer beside organisms of focus and surrounding, are divided in cryopreservation tube, are put into liquid nitrogen (- 80 DEG C) preservations immediately.By cancer and cancer Diagnosis is using pathological diagnosis as final foundation.

2nd, Total RNAs extraction

Using Trizol (Invitrogen companies) reagent, the Total RNAs extraction of tissue is carried out according to its specification.Specific step It is rapid as follows：

1) Homogenize is homogenized：Tissue homogenate：Per 100mg, tissue adds 1ml TRIzol reagents, and even with homogenizer Slurry.

2) chloroform of about 1/5 volume is added, turns upside down and fully mixes 1min or so, be stored at room temperature 5min.

3) 4 DEG C, 12,000rpm centrifugation 15min.

4) supernatant is carefully taken out, avoids touching intermediate layer, supernatant is transferred to the body such as new 1.5ml centrifuge tubes, addition Long-pending isopropanol, gently overturn and mix, be stored at room temperature 5min.

5) 4 DEG C, 12000rpm centrifugations 15min.

6) supernatant is sucked, retains precipitation, and the ethanol of 1ml 75% is added into precipitation, 4 DEG C, 12000rpm centrifugations 15min Washing precipitation.

7) suck supernatant, precipitate the water that appropriate RNase-free is added after room temperature naturally dry, with Tip head pressure-vaccums, fully Dissolving precipitation.

8) 1-2 μ L are taken, determine RNA concentration and A260/A280 values.General A260/A280 ratios are in 1.8-2.1.RNA is deposited It is stand-by to be placed on -80 DEG C of refrigerators.

3rd, reverse transcription synthesis cDNA

With PrimeScript RT Master Mix Perfect Real Time (TAKARA companies) reverse transcription cDNA, Reaction system (total amount is 10 μ l) is as shown in table 1.

The reaction system of table 1

Reaction condition is as follows：

1) 37 DEG C 15 minutes；

2) 85 DEG C 5 seconds；

3) 4 DEG C preserve (- 20 DEG C long-term to preserve).

4、RT-PCR

It is poor using expression of the relative quantification method detection FAK genes in liver cancer sample.

Use instrument：Thermal Cycler Dice^TM Real Time System TP800,Takara。

Template：Above-mentioned reverse transcription product presses 1:Template after 10 dilution proportions as PCR reactions.

Primer is as shown in table 2：

The primer sequence of table 2

The system of PCR reactions：

PCR reactions using TAKARA companies heat resistance Taq archaeal dna polymerases (DR001), during using human genome as template, The DNA fragmentation of 3.0kbp (p53 genes) can be expanded well.Wherein, the reagent of PCR reactions and its dosage are as shown in table 3.

The reagent and its dosage of the PCR of table 3 reactions

Reagent	Usage amount
		Takara Taq(5U/μl)	0.1μl

10 × PCR buffer solutions (Mg²⁺Plus)	2μl
		DNTP mixtures (every kind of 2.5mM)	1.6μl
Primer mixture (each 5 μM)	1.6μl
		Template	0.5-4μl
ddH₂O	Mend to 20 μ l

PCR reaction conditions：

Target gene：

As a result as shown in figure 1, expression of the FAK genes in liver cancer tissue is apparently higher than the expression water in cancer beside organism It is flat.

5th, quantitative fluorescent PCR

Using differential expression of the fluorescence quantitative PCR detection FAK genes in liver cancer sample.

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

Template：Above-mentioned reverse transcription product presses 1:Template after 5 dilution proportions as PCR reactions.

Primer sequence is as shown in table 4：

The primer sequence of table 4

The system of PCR reactions：PCR reactions use the SYBR Premix Ex Taq of TAKARA companies.Wherein, PCR reacts Reagent and its dosage it is as shown in table 5.

The reagent and its dosage of the PCR of table 5 reactions

Composition	Dosage
		SYBR Premix EX Taq	10.0μl
Sense primer (10 μM)	0.5μl

Anti-sense primer (10 μM)	0.5μl
		ROX Reference Dye(50*)	0.4μl
cDNA	1.0μl
		ddH₂O	7.6μl

PCR reaction conditions：

Fluorescence quantitative PCR detection shows, expression of the FAK genes in liver cancer tissue is apparently higher than in cancer beside organism Expression, and through using the statistical packages of Graphpad Prism 5 (GraphPad Software, San Diego, CA, USA) analyzed, the comparison of quantitative data is examined with t to be analyzed, and works as P<0.05 thinks that statistics is variant, as a result shows P <0.0001 (see Fig. 2).

6th, Panomics is analyzed

1) key instrument

Bio-plex suspension chip systems：Bio-plex 100system (Biorad companies)

Bio-plex board-washing machines：Bio-plex pro II wash station (Biorad companies)

Constant temperature oscillation shaking table：Vortemp 56shaking incubator(Labnet International)

2) operating procedure

The 30 nucleic acid factor panel of people suspension liquid chip inspecting reagent unit is Quantigene plex 2.0Kit (Panomics, Affymetrix corp).Applied sample amount is 400ng.

Proceeded as follows according to kit specification requirement：

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

2. being required to prepare microballon probe suspension according to kit specification, 80 μ l are taken to add in each hole of hybridization plate, then By in dummy or the μ l adding holes of RNA samples 20.After pad pasting closing, 600rpm in constant temperature oscillation shaking table, 54 DEG C of lucifuges are incubated 18-22h。

3. sample probe microbeads for periods liquid is transferred in the hole of Beads enrichment plate, washed using Bio-plex board-washing machines Wash, per hole 100ul, washing is three times.

4. adding pre-amplification liquid 100ul, 50 DEG C, 600rpm is incubated 1h.

After 5. board-washing machine washing is washed three times, 100 μ l amplification liquid is added, 50 DEG C, 600rpm is incubated 1h.

6. after washing, the μ l of label probe liquid 100 are added, 50 DEG C, 600rpm is incubated 1h.

7. after washing, add SAPE working solutions 100 μ l, room temperature 600rpm and be incubated 30min.

8. after SAPE cleaning solutions wash away unnecessary SAPE, adding 130 μ l SAPE cleaning solutions, Bio-plex suspension cores are sent into Piece system carries out readings, and through using the statistical packages of Graphpad Prism 5 (GraphPad Software, San Diego, CA, USA) to be analyzed, the comparison of quantitative data is examined with t to be analyzed, and works as P<0.05 thinks that statistics is variant.

As a result as shown in figure 3, expression of the FAK genes in liver cancer tissue is apparently higher than the expression water in cancer beside organism It is flat, P<0.0001 (see Fig. 3).

Embodiment 2 utilizes the expression of RNAi technology silence FAK genes

1st, cell culture

Using common hepatoma cell strain, i.e. SMMC7721, WRL68, QGY7701 (laboratory preservation) carries out FAK genes Expression study.Wherein, the condition of culture of cell line is to contain 10% (percentage by volume) hyclone (Life Technologies DMEM (dulbecco's modified eagle medium) nutrient solution), at 37 DEG C, containing 5%CO₂Training Support and cultivated in case.

2nd, siRNA design

The free service device provided according to some companies on network, such as the interface http of Clontech companies:// Bioinfo2.clontech.com/rnaidesigner/sirnaSequenceDesign.d o, the boundary of Invitrogen companies Face https://rnaidesigner.invitrogen.com/sirna/ and according to InvivoGene server (network address http://www.sirnawizard.com) design corresponding siRNA.

Wherein, the siRNA sequence difference for FAK genes of design is as follows：

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)

3rd, siRNA transient transfections

Select SMMC7721, WRL68, QGY7701 cell line of the higher FAK genes of endogenous expression.Will be synthetic every Kind siRNA (being respectively FAK-siRNA1, FAK-siRNA2, FAK-siRNA3 and negative control siRNA) is dissolved with DEPC water, is obtained To the solution of 20 μM of concentration.With 1 μ l Lipofectamine^TM2000 (Invitrogen) and 1 μ l concentration are 20 μM every kind of After siRNA is mixed respectively, mixed liquor is gone to SMMC7721, the WRL68 for putting forward the previous day passage cell density in 30-40% or so With (24 hole Tissue Culture Dish) in QGY7701 cells, complete culture solution is changed after 4 hours, continues to cultivate.Collected carefully after turning 48h winks Born of the same parents, then carry out follow-up Total RNAs extraction.

4th, the extraction of cell total rna

Using Trizol (TAKARA companies) reagent, according to its specification extract the total serum IgE of cell, step is as follows：

1) attached cell (24 hole culture dish) of culture, after being transfected using above-mentioned " 3, siRNA transient transfections " method, 48h is cultivated, sucks nutrient solution, adds 500 μ l TRIzol into culture dish, with pipette tips piping and druming several times；

2) lysate is transferred in 1.5ml centrifuge tube, room temperature places 5min；

3) chloroform is added by 0.2ml chloroforms/ml TRIzol amount, covers lid, acutely shake 15s, room temperature is placed 5min, 4 DEG C of 12000g centrifuge 15min；

4) upper phase is transferred in a clean centrifuge tube, adds isopropanol (0.5ml/mlTRIzol), gently run Mix for several times, room temperature places 10min, and 4 DEG C of 12000g centrifuge 15min；

5) supernatant is abandoned, adds 75% ethanol (1ml/ml TRIzol) fully vibration mixing, 4 DEG C of 12000g centrifugations 10min；

6) remove supernatant, be deposited in room temperature and place 5~10min, make its natural airing (not be completely dried)；Add 30- 50 μ l DEPC water dissolving RNA；260/280 ratio is determined with ultraviolet specrophotometer and is quantified；

7) DNA is removed：Possible DNA pollution in total serum IgE is removed using DNase I (RNase Free, TaKaRa companies), Operating procedure is as follows：

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

The reaction solution of table 6

Composition	Dosage
		Total serum IgE	20~50 μ g
10×DNase I Buffer	5μl
		DNase I (RNase-free, 5U/ μ l)	2μl
RNase Inhibitor(40U/μl)	0.5μl
		The H of DEPC processing₂O	Until reaction solution total amount is 50 μ l

2. add the H of 50 μ l DEPC processing₂O。

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

4. centrifuging 12000rpm, 15min, upper strata (water layer) is taken to move in another microcentrifugal tube.

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

6. centrifuging 12000rpm, 15min, upper strata (water layer) is taken to move in another microcentrifugal tube.

7. add 10 μ l (1/10 amount) 3M sodium acetates (pH5.2).

8. add the cold absolute ethyl alcohol of 250 μ l (2.5 times of amounts), -20 DEG C of placement 30-60 minutes.

9. centrifugation recovery precipitation 12000rpm, 10min, clean precipitation with 70% cold ethanol, dry.

10. the H handled with appropriate DEPC₂O dissolves, quantitative.

5th, reverse transcription synthesis cDNA

With PrimeScript RT Master Mix Perfect Real Time (TAKARA companies) reverse transcription cDNA, Reaction system (total amount is 10 μ l) is as shown in table 7.

The reaction system of table 7

Composition	Dosage
		5*PrimeScript RT Master Mix(for Real Time)	2μl
Total serum IgE	500ng
		Remove RNase dH₂O	Complement to 10 μ l

Reaction condition is as follows：

1) 37 DEG C 15 minutes；

2) 85 DEG C 5 seconds；

3) 4 DEG C preserve (- 20 DEG C long-term to preserve).

6th, quantitative fluorescent PCR

Carried out using ABI7300 real-time fluorescence quantitative PCRs instrument and SYBR Premix Ex Taq kits, with β-actin (ACTB) internal reference is used as, the influence that analysis transfection siRNA is expressed hepatoma cell strain FAK, the primer is as shown in table 4, instead Answer system (total amount is 20 μ l) as shown in table 8.

The PCR reaction systems of table 8

PCR reaction conditions are：94 DEG C of pre-degeneration 30s, 94 DEG C of 10s, 60 DEG C of 30s, after carrying out 40 circulations altogether, are carried out afterwards The detection of solubility curve.

Wherein, autofluorescent background signal and threshold value typically use instrument default number, and each run automatically generates after terminating, Fluorescence signal in each reaction tube reaches the period undergone during the threshold value of setting and is defined as Ct values；Data analysis is according to such as Lower method is carried out：Each pair primer (gene) does 3 repeating pipes in each template, and obtained Ct values are averaged；Each purpose The Ct average values of gene subtract the Ct average values of the reference gene (ACTB) of corresponding templates, obtain Δ Ct.The Δ Ct of experimental group subtracts The Δ Ct of control group is removed, obtains Δ Δ Ct values, the multiple proportion 2- Δ Δs Ct of the testing gene in control group and experimental group.

Test result indicates that these three siRNA fragments of FAK-siRNA1, FAK-siRNA2 and FAK-siRNA3 can be effective The expression of ground silence FAK genes, the research institute that can be used for next step is prompted to use.Wherein, SMMC7721, WRL68, QGY7701 Experimental result in cell is as Figure 4-Figure 6.

7th, the measure of cell growth curve

1) day before transfection is inoculated with appropriate cell, cell density during transfection is controlled between 30%~50%；

2) liposome Lipofectamine is used^TM2000 (Invitrogen) transfect siRNA

Prepare siRNA and Lipofectamine^TM2000 mixed liquor, the formula of mixed liquor is with 1 μ l Lipofectamine^TM2000 and 1 μ l concentration is 20 μM every kind of siRNA (FAK-siRNA1, FAK-siRNA2, FAK-siRNA3 With negative control siRNA respectively with DEPC water dissolve) respectively mix after, room temperature place about 20 minutes, formed siRNA- Lipofectamine^TM2000 compounds；Then, by siRNA-Lipofectamine^TM2000 compounds (2 μ l) add cell In, mix；Normal nutrient solution is changed after 4~6 hours；

3) after 24 hours, SMMC7721, WRL68, QGY7701 cell after digestion transfection siRNAs are special according to its growth Property press 1 × 10³/ 100 μ l/ holes calculate cell total amount, and are fully planted after dilution in 96 orifice plates.Every group of daily six multiple holes, typically By 7-8 days inoculating cells；

4) treat or so half an hour, the substantially adherent rear observation cell state of cell and number.Reacted with chromogenic reagent, with true The actual initial density of cell is determined, as growth relative zero.Developer Cell counting kit-8 (CCK-8, Dojindo, Japan) usage：Every 100 μ l trainings liquid adds 10 μ l CCK-8,37 DEG C, 5%CO₂1h is placed, is determined on ELIASA Absorbance at 450nm；

5) light Microscopic observation cellular morphology.Timing measurement on request, record upgrowth situation；

6) survey 4 to 7 days is generally required.After Data Collection, then handled, chart is drawn with Excel.

As a result find, these three siRNA fragments of FAK-siRNA1, FAK-siRNA2 and FAK-siRNA3 can effectively press down The growth of liver cancer cells processed.Wherein, the experimental result in SMMC7721, WRL68, QGY7701 cell is as Figure 7-9.

8th, cell scratch experiment

SMMC7721 cells are spread in 24 hole Tissue Culture Dish with 80%-90% density.With 1 μ l Lipofectamine^TM2000 and 1 μ l concentration is 20 μM every kind of siRNA (FAK-siRNA1, FAK-siRNA2, FAK-siRNA3 With negative control siRNA respectively with DEPC water dissolve) respectively mix after, respectively transfectional cell, carry out silence FAK.After 24 hours, Cell surface, 37 DEG C, 5%CO are streaked using plastics pipette tips₂Cell culture incubator in cultivate 4 days, adopted respectively at 0,1,2,3 day Collect the image of same position.Experiment is in triplicate.The cell or cell without any processing transfected by the use of negative control as Control.

As a result find, these three siRNA fragments of FAK-siRNA1, FAK-siRNA2 and FAK-siRNA3 can suppress The growth of SMMC7721 cells.Wherein, FAK-siRNA1 and FAK-siRNA3 experimental result is as shown in Figure 10.

9th, cell invasion is tested

Appropriate cell (SMMC7721, SK-hep1) is inoculated with, cell density during transfection is controlled between 30%~50%； Liposome Lipofectamine is used in 24 hours^TM2000 (Invitrogen) transfect siRNA：Prepare siRNA with Lipofectamine^TM2000 mixed liquor, i.e., with 1 μ l Lipofectamine^TM2000 and 1 μ l concentration is 20 μM every kind of After siRNA (FAK-siRNA1 and negative control siRNA are dissolved with DEPC water respectively) is mixed respectively, room temperature is placed about 20 minutes, Form siRNA-Lipofectamine^TM2000 compounds；By siRNA-Lipofectamine^TM2000 compounds (every kind of 2 μ l) Add in cell, mix；Normal nutrient solution is changed after 4~6 hours.

Cell invasion experiment uses 24 hole Transwell cells (8- μm of aperture, BD Biosciences companies), scribbles Artificial basement membrane (Falcon354480；BD Biosciences companies).The liver cancer cells in serum free medium (SMMC7721, SK-hep1) overnight starvation, is handled with Trypsin Induced, and washs 3 in the DMEM nutrient solutions containing 1%FBS It is secondary.1 × 10 will be contained⁵In DMEM nutrient solutions of the 500 μ l containing 1%FBS of individual cell, upper chamber is added to.Simultaneously by 750 μ l (catalog number 356008, BD Biosciences are public for the fibronectin of DMEM culture mediums and 10 μ g/ml containing 10%FBS Department) it is placed at lower chamber.As control, lower chamber will be added in the culture medium containing 1%FBS.It is latent by 48 hours Fu Qi, remaining artificial basement membrane and cell are removed with cotton swab in upper chamber.4% paraformaldehyde of cell on film lower surface It is fixed, and with 0.5% violet staining.Cell is counted and taken pictures in the random field of microscope of at least six (multiplying power × 100). All experiments are repeating, and are repeated 3 times, and using the statistical package (GraphPad of Graphpad Prism 5 Software, San Diego, CA, USA) to be analyzed, the comparison of quantitative data is examined with t to be analyzed, and works as P<0.05 thinks Statistics is variant.

As a result find, FAK-siRNA1 can substantially suppress the invasion and attack of liver cancer cells SMMC7721, SK-hep1.

In summary, the present invention can be suppressed the cell growth of liver cancer, be moved and invaded using RNAi technology silence FAK expression Attack.Thus, siRNA of the invention can be applied to prepare treatment or anti-curing oncoma (such as liver cancer) medicine, be the treatment of tumour, there is provided New approach.

Claims

1. a kind of siRNA for being used to suppress SMMC7721, QGY7701 hepatocellular carcinoma cells of high expression FAK genes, its feature exist In the siRNA is the RNA sequence as shown in SEQ ID NO.11-12.

A kind of 2. DNA sequence dna for encoding siRNA as claimed in claim 1.

A kind of 3. expression vector, it is characterised in that：The expression vector includes the DNA sequence dna described in claim 2.

A kind of 4. host cell, it is characterised in that：The host cell is that one kind can express siRNA as claimed in claim 1 The cell of sequence.

5. host cell as claimed in claim 4, it is characterised in that：The host cell includes described in claim 3 Expression vector.

6. a kind of siRNA as claimed in claim 1 is thin in SMMC7721, QGY7701 liver for preparing the high expression FAK genes of suppression Application in the medicine of born of the same parents' cancer cell.

A kind of 7. medicine for SMMC7721, QGY7701 hepatocellular carcinoma cells for suppressing high expression FAK genes, it is characterised in that：Contain There are the siRNA as claimed in claim 1 and pharmaceutically acceptable carrier of effective dose.

A kind of 8. medicine box, it is characterised in that：Containing siRNA as claimed in claim 1 or contain medicine as claimed in claim 7 Thing.