CN102643819B - ShRNA of Rab23 and ientiviral vector and applications thereof - Google Patents
ShRNA of Rab23 and ientiviral vector and applications thereof Download PDFInfo
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- CN102643819B CN102643819B CN201210123970.1A CN201210123970A CN102643819B CN 102643819 B CN102643819 B CN 102643819B CN 201210123970 A CN201210123970 A CN 201210123970A CN 102643819 B CN102643819 B CN 102643819B
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Abstract
The invention discloses an interference Rab23 gene of siRNA. The siRNA is hairpin RNA (ribonucleic acid) formed aiming to a target sequence of the Rab23 gene; the siRNA is double-strand siRNA, wherein one strand of nucleotide sequence is shown as SEQ. ID. NO.1 or SEQ. ID. NO.2; the expression of the Rab23 protein is positive correlation with the development of skin scale cancer and is especially related with invasion ability of the skin scale cancer; and the invasion ability of skin scale cancer cells is enabled to lowered by the expression of the interference Rab23 protein. The invention also discloses applications of interference shRNA of the Rab23 gene target sequence in preparing a skin scale cancer resisting drug.
Description
Technical field
The invention belongs to Skin Squamous Cell Carcinoma Prevention Technique field, relate to shRNA and lentiviral vectors and the application of a kind of Rab23.
Background technology
Rab23 is RAS proto-oncogene member, and the albumen of its coding is Small GTPases superfamily Rab family member, and its sudden change can cause that mouse opens brain syndrome (open brain syndrome), is therefore called again opb gene.Calendar year 2001, Eggenschwiler etc. study discovery, and Rab23 is the negative regulatory factor of Sonic Hedgehog signal path necessity, and in recent years, Rab23 is more and more paid attention to by scholar as the effect of proto-oncogene.Research discovery, Rab23 participates in developing of liver cancer, cancer of the stomach, lung cancer, mammary cancer and thyroid carcinoma, and in cancer of the stomach, finds with tumor invasion first closely related.
In prior art, there is scholar directly the siRNA fragment transfection mammalian cell energy specificity of chemosynthesis to be suppressed to homogenic expression in cell, but transfection efficiency is low, the interior siRNA fragment of cell is easy to be degraded, and therefore this method can not realize stable RNA interference.Lentiviral vectors efficiency of infection in mammalian body is high, immunogenicity is low and can infect division phase and non-division phase cell.These characteristics due to virus vector, lentivirus mediated RNA Recombinant Interferon α-2b is at the stable table of mammalian species cell long-period shRNA, inhibition of gene expression, has efficient, stable, high specificity, feature applied widely, has become the main research tool of RNA perturbation technique.The applied basic research that be applied as clinical tumor of RNA perturbation technique in biological medicine research increased strong means.
Summary of the invention
The problem that the present invention solves is to provide shRNA and lentiviral vectors and the application of a kind of Rab23, and the transfection of this lentiviral vectors can suppress the expression of the Rab23 gene in squamous cell carcinoma, suppresses the invasion and attack of squamous cell carcinoma.
The present invention is achieved through the following technical solutions:
Disturb a shRNA for Rab23 gene, this shRNA is that described target sequence is: gcgacaaatt caagttaat for the ShorthairpinRNA of the target sequence formation of Rab23 gene.
The shRNA of described Rab23, the nucleotide sequence of a chain is wherein as shown in SEQ.ID.NO.1 or SEQ.ID.NO.2.
Disturb a carrier for Rab23 gene, this carrier comprises the double-stranded shRNA consisting of the sequence shown in SEQ.ID.NO.1 and SEQ.ID.NO.2.
The carrier of described interference Rab23 gene, this carrier is GV248-Rab23-shRNA, is in GV248 carrier, to be cloned into the double-stranded shRNA consisting of the sequence shown in SEQ.ID.NO.1 and SEQ.ID.NO.2.
A recombinant slow virus that suppresses squamous cell carcinoma is the genome that the protein encapsulation of being expressed by packaging plasmid Helper 1.0, Helper 2.0 contains GV248-Rab23-shRNA carrier;
This slow virus is by GV248-Rab23-shRNA expression plasmid and packaging plasmid Helper 1.0, Helper 2.0 polyinfection host cells, cracking after cultivating and producing.
Application for the interference shRNA of Rab23 gene target sequence as the preparation of anti-Skin Squamous Cell Carcinoma medicine.
Described target sequence is: gcgacaaatt caagttaat.
The ShorthairpinRNA of described interference shRNA for being formed by the sequence shown in SEQ.ID.NO.1 and SEQ.ID.NO.2.
Described interference shRNA is packaged as slow virus: the genome that the protein encapsulation of being expressed by packaging plasmid Helper 1.0, Helper 2.0 contains GV248-Rab23-shRNA carrier.
Described anti-Skin Squamous Cell Carcinoma medicine is the invasive medicine of anti-squamous cell carcinoma.
Compared with prior art, the present invention has following useful technique effect:
The shRNA of interference provided by the invention Rab23 gene and the application of anti-Skin Squamous Cell Carcinoma thereof are expression based on Rab23 albumen and the development positive correlation of Skin Squamous Cell Carcinoma, especially relevant to the invasive ability of squama cancer, disturb the expression of Rab23 albumen can make the invasive ability of squamous cell carcinoma decline.Described dependency shows as:
Use immunohistochemical method to detect the differential expression of Rab23 in precancerous lesion actinic keratosis, original position squama cancer and the aggressive squama cancerous tissue of excision descendant normal skin, Skin Squamous Cell Carcinoma, result shows not express in people's normal skin Rab23 albumen, the low expression of Rab23 albumen in actinic keratosis tissue, Rab23 albumen high expression level in original position squama cancer and aggressive squama cancerous tissue, in aggressive squama cancerous tissue, Rab23 expressing quantity is apparently higher than original position squama cancerous tissue.Development positive correlation prompting Rab23 gene or the albumen of the expression of Rab23 albumen and Skin Squamous Cell Carcinoma can be as the target spots of Skin Squamous Cell Carcinoma control.
Further, the invention provides specificity ShorthairpinRNA (the short hairpin RNA for Rab23 gene, shRNA), then build the lentiviral vectors that is loaded with this shRNA, use is packed slow viruss and infects squamous cell carcinoma for lentiviral vectors, helper plasmid Helper 1.0 and the Helper 2.0 of the shRNA of Rab23 gene, after importing in squamous cell carcinoma, can effectively suppress the expression of the Rab23 in squamous cell carcinoma.And after slow virus infection, the squama cancer that can continue to suppress surely turns cell, by Transwell Matrigel, find that the invasive ability of squamous cell carcinoma obviously declines.This shows that constructed slow virus can be applied to the control of squamous cell carcinoma, or the preparation of anti-squamous cell carcinoma medicine.
Accompanying drawing explanation
Fig. 1 is the differential expression result figure that immunohistochemical method detects Rab23 in precancerous lesion actinic keratosis, original position squama cancer and the aggressive squama cancerous tissue of normal skin, Skin Squamous Cell Carcinoma;
Fig. 2 is the plasmid map schematic diagram of GV248 plasmid;
Fig. 3 is that Western-blot detects GV248-Rab23-shRNA plastid rna interference effect knot figure;
Fig. 4 is the plasmid map schematic diagram of Helper1.0 plasmid;
Fig. 5 is the plasmid map schematic diagram of Helper2.0 plasmid;
Fig. 6-1～Fig. 6-2 surely turn cell result figure for HSQ-89NC and Rab23-shRNA;
Fig. 7-1～Fig. 7-3 reduce the invasive result figure of squamous cell carcinoma for Rab23 disturbs.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described, and the explanation of the invention is not limited.
1, the expression of Rab23 albumen and the dependency of Skin Squamous Cell Carcinoma
The differential expression of Rab23 in the precancerous lesion actinic keratosis of normal skin, Skin Squamous Cell Carcinoma, original position squama cancer and aggressive squama cancerous tissue:
Normal skin tissue (Normal) 31 examples after collection excision, precancerous lesion actinic keratosis tissue (AK) 31 examples of Skin Squamous Cell Carcinoma, original position squama cancerous tissue (Bowen ' s) 42 examples, aggressive squama cancerous tissue (SCC) 45 examples.Through 15% formalin, fix, after row paraffin embedding, cut into slices.Paraffin section is after dimethylbenzene and gradient ethanol dewaxing rehydration, and citrate buffer solution boils does antigen retrieval, and 10%BSA seals 37 ℃ of 1h, and adding working concentration is the mouse anti human Rab23 primary antibodie of 1 μ g/ml, 4 ℃ of overnight incubation.Add anti-37 ℃ of the goat-anti mouse two of 1: 1000 HRP mark after dilution to hatch 2 hours.After PBS washing, with the colour developing of DAB solution, room temperature 3-5 minute, tap water is made phenodin lining and is dyed after rinsing, neutral gum mounting after dimethylbenzene transparence.Micro-Microscopic observation, judges that according to coloring degree Rab23 expresses degree.
As shown in the ImmunohistochemistryResults Results of Fig. 1, Rab23 does not express in normal skin, and low expression in the precancerous lesion actinic keratosis of Skin Squamous Cell Carcinoma obviously increases and express in squama cancer and aggressive squama cancer in position; And in aggressive squama cancerous tissue, Rab23 expressing quantity is apparently higher than original position squama cancerous tissue.Development positive correlation prompting Rab23 gene or the albumen of the expression of Rab23 albumen and Skin Squamous Cell Carcinoma can be as the target spots of Skin Squamous Cell Carcinoma control.
Further analyze and find, the expression of Rab23 and the differentiation degree of squamous cell carcinoma are negative correlation, and squama silver stain is lower, and the expression of Rab23 is stronger.And Rab23 expresses also and significantly increases in the squama cancerous tissue at exposure position, these all point out Rab23 to express may be relevant with the aggressive of squama cancer.
2, the design of shRNA sequence of Rab23 gene and the structure of carrier
2.1 design for people Rab23 gene shRNA sequence:
From genebank, transfer human Rab23 gene order (NM_016277), according to Rab23CDS sequence, use the online software design of BLOCK-iT RNAiDesigner for the Effective target site of Rab23 gene RNAi, the target sequence of screening is GCGACAAATTCAAGTTAAT.
The synthetic specificity ShorthairpinRNA of design subsequently, described ShorthairpinRNA is comprised of a positive-sense strand and an antisense strand:
Positive-sense strand: 5 '-ccggGAGCGACAAATTCAAGTTAATctcgagATTAACT TGAATTTGTCGCTCtttttg-3 '
Antisense strand: 5 '-aattcaaaaaGCGACAAATTCAAGTTAATCTCGAGATTAACTTGAATTTGTCGC-3 '
2.2 build GV248-Rab23-shRNA recombinant plasmid
Positive-sense strand and the antisense strand fragment of getting synthetic and purifying are dissolved in annealing buffer, 90 ℃ of water-bath 15min, and then natural condition is but to room temperature, and annealing forms complementary double-stranded.The complementation two strands of the GV248 plasmid (plasmid structure is as Fig. 2) of purifying after Age I and EcoR I double digestion and annealing formation is mixed with 1: 2 ratio, with T4 ligase enzyme, connect and spend the night, be transformed into DH5 α competent cell, with penbritin screening, contain and form GV248-Rab23-shRNA recombinant plasmid list bacterium colony, picking list bacterium colony is cultivated, extract plasmid and carry out sequencing, correct with confirmation composition sequence after synthetic fragment contrast, without transgenation.
By GV248-Rab23-shRNA carrier transfection HSQ-89 squamous cell carcinoma, filter out successfully after the positive cell of transfection, at the cell with untransfected in contrast, Western-blot detects the expression of the Rab23 albumen in cell.The result detecting as shown in Figure 3, can see that the Rab23 protein normal in control cells is expressed, and after the cell of transfection carrier interferes Rab23 gene, Rab23 albumen is expressed hardly.This shows that constructed carrier can disturb the expression of Rab23 gene, thereby suppresses the expression of Rab23 albumen.
2.3 packing GV248-Rab23-shRNA recombinant virus particles
1. 24h before transfection, with the 293T cell of tryptic digestion logarithmic phase, take that containing the DMEM in high glucose substratum of 10% serum, to adjust cell density be 1.2 * 10
7individual/ml, is re-seeded into 15cm Tissue Culture Dish, 37 ℃, 5%CO
2in incubator, cultivate.24h can be used for transfection when cell density reaches 70%～80%.
2. before transfection, 2h is replaced by cell culture medium the DMEM in high glucose substratum of serum-free.
3. to adding each DNA solution in a sterilizing centrifuge tube: GV248-Rab23-shRNA plasmid 20 μ g, Helper 1.0 plasmids (are purchased, its plasmid structure is as shown in Figure 4) 15 μ g, Helper 2.0 plasmids (are purchased, its plasmid structure is as shown in Figure 5) 10 μ g, mix with the Opti-MEM of respective volume, adjustment cumulative volume is 2.5ml, and at room temperature incubation is 5 minutes.
4. Lipofectamine 2000 reagent are softly shaken up, get 100 μ l Lipofectamine 2000 reagent and mix at another Guan Zhongyu 2.4ml Opti-MEM, at room temperature incubation is 5 minutes.
5. the DNA after dilution is mixed with the Lipofectamine 2000 after dilution, put upside down and mix lightly.
6. after mixing, at room temperature incubation is 20 minutes, to form the transfection composite of DNA and Lipofectamine2000 diluent.
7. DNA and Lipofectamine 2000 mixed solutions are transferred in the nutrient solution of 293T cell, mix, in 37 ℃, 5%CO
2in cell culture incubator, cultivate.
8. cultivate and remove the substratum that contains transfection miscellany after 8h, every bottle of cell adds the PBS liquid of 20ml, gently double swerve once culturing bottle to wash remaining transfection miscellany, then go.
9. in every bottle of cell, add the DMEM in high glucose cell culture medium 25ml containing 10% serum, in 37 ℃, 5%CO2 incubator, continue to cultivate 48 hours.
10. collect after transfection the 293T cell conditioned medium liquid of 48 hours.
11. in 4 ℃, and the centrifugal 10min of 4000g, removes cell debris.
12. with 0.45 μ m filter filtering supernatant in 40ml ultracentrifugation pipe.
13. do 50000rpm ultracentrifugation by viral supernatant, draw viral supernatant layer.
14. adopt hole By Dilution poison titre, and with PBS, adjusting virus titer is 1 * 10
8tu/mL, after packing-80 ℃ frozen.
3, recombinant slow virus infects squamous cell carcinoma and Tanswell Matrigel
1. virus infection the day before yesterday, the good HSQ-89 cell of digestion growth conditions, is used 9 holes (NC contrast virus group 3 holes, GV248-Rab23-shRNA recombinant virus group 3 holes, Mock control group 3 holes) in 96 orifice plates, and each hole adds 1 * 10
4individual cell, volume is 90 μ l.
2.NC contrasts viral group, the every hole of Rab23-shRNA recombinant virus group and adds 10 μ l 1 * 10
8nC contrast virus and the GV248-Rab23-shRNA recombinant virus of Tu/mL, the every hole of Mock control group adds 10 μ l nutrient solutions.
3. infect after 8h, abandon nutrient solution, add 100 μ l fresh mediums, in 37 ℃, 5%CO
2continue in incubator to cultivate 72 hours, under inverted fluorescence microscope, observe egfp expression, with the cell of green fluorescence, be the successful cell of virus infection.
4.NC contrasts viral group, the every hole of Rab23-shRNA recombinant virus group and adds the tetracycline of 2.5 μ g/ml concentration to kill the squamous cell carcinoma HSQ-89 not infecting, in 37 ℃, 5%CO
2in incubator, continue to cultivate 48 hours, under inverted fluorescence microscope, observe cell egfp expression under normal visual field and the fluorescence visual field, HSQ-89NC (as in Figure 6-1) and HSQ-89Rab23-shRNA that the cell under normal visual field is all acquisition with green fluorescence under the fluorescence visual field surely turn cell (as shown in Fig. 6-2).
5. because tumour cell is by the matrix adhesion in chamber face on film surface special receptor and Transwell cell, then can discharges proteolytic ferment or activate already present proenzyme in matrix, matrix degradation, and then cell movement is to the matrix gap being hydrolyzed.This process constantly repeats, and tumour cell finally enters chamber face under Transwell cell through matrix, and more the speak more invasive ability of bright tumour cell of the tumour cell passing is stronger.Select aperture is the Transwell cell of 8 μ m for this reason, uses matrigel and the coated matrigel of serum-free DMEM in high glucose substratum dilution in 1: 8 of BD company, and 37 ℃ air-dry, uses front with DMEM in high glucose aquation basilar membrane.
6. routine digests HSQ-89NC cell and HSQ-89Rab23-shRNA cell respectively, and PBS washes 2 times, resuspended with serum-free DMEM in high glucose substratum, adjusts cell concn to 5 * 10
5individual/ml, then joins respectively in different Transwell cells:
Upper chamber adds cell suspension 200 μ l, and lower chamber adds the DMEM in high glucose substratum 900 μ l containing 10%FBS, guarantees between lower chamber nutrient solution and cell without bubble cellar culture.
7. every 4h, take out a sample view, there is appropriate cell lower chamber and stops cultivating during without propagation.Take out cell, cotton swab is wiped face cell in chamber on cell gently, and 100% methyl alcohol is 30min fixedly, 0.4% violet staining liquid dyeing 15min, clean, dry and under rear inverted microscope, take pictures and count, adopt Prism 5.0 statistical softwares map and use two groups of data differences of independent sample t check analysis.
As shown in Fig. 7 result, HSQ-89Rab23-shRNA surely turns cell (Fig. 7-2) and HSQ-89NC and surely turns cell (Fig. 7-1) and compare in Transwell cell by upper chamber face and pass to the cell count of lower chamber face and obviously reduce compared with control group, choosing at random 10 visuals field takes pictures and counts, adopt Prism 5.0 statistical softwares map and use two groups of data differences of independent sample t check analysis, result shows that HSQ-89Rab23-shRNA surely turns cell and compared with HSQ-89NC, surely turns the cell ability of attacking and decline, two groups of data just have significant difference, and P < 0.05 represents (Fig. 7-3) with *.Above experimental result shows to lower the invasive ability of HSQ-89 squamous cell carcinoma after Rab23 genetic expression and obviously reduces, and Rab23 gene can be used as the target molecule that suppresses squama cancerous invasion.
In view of the development positive correlation of Rab23 gene and Skin Squamous Cell Carcinoma, for Rab23 gene, carry out the development of Recombinant Interferon α-2b impact, blocking-up Skin Squamous Cell Carcinoma; And for aggressive squamous cell carcinoma, disturb Rab23 gene can reduce the aggressive of squamous cell carcinoma, so this technology can carry out with chemotherapeutics coupling the treatment of squama cancer, also can be used as the auxiliary treatment means that prevents squamous cell carcinoma invasion and attack after operation.Therefore, can be applied to the control of Skin Squamous Cell Carcinoma for the shRNA of Rab23 gene target sequence, be potential novel anti-Skin Squamous Cell Carcinoma medicine.
Claims (6)
1. a shRNA who disturbs Rab23 gene, is characterized in that, this shRNA is that described target sequence is: gcgacaaatt caagttaat for the ShorthairpinRNA of the target sequence formation of Rab23 gene;
Described shRNA is ShorthairpinRNA, and its corresponding deoxynucleoside acid sequence is as shown in SEQ.ID.NO.1.
2. a carrier that disturbs Rab23 gene, is characterized in that, this carrier comprises the shRNA consisting of the corresponding Nucleotide of sequence shown in SEQ.ID.NO.1 and SEQ.ID.NO.2;
This carrier is GV248-Rab23-shRNA, is in GV248 carrier, to be cloned into the shRNA consisting of the corresponding Nucleotide of sequence shown in SEQ.ID.NO.1 and SEQ.ID.NO.2.
3. a recombinant slow virus that suppresses squamous cell carcinoma, is characterized in that, is the genome that the protein encapsulation of being expressed by packaging plasmid Helper1.0 and Helper2.0 contains GV248-Rab23-shRNA carrier;
Described GV248-Rab23-shRNA carrier is in GV248 carrier, to be cloned into the shRNA consisting of the corresponding Nucleotide of sequence shown in SEQ.ID.NO.1 and SEQ.ID.NO.2;
This slow virus is by GV248-Rab23-shRNA expression plasmid and packaging plasmid Helper1.0 and Helper2.0 polyinfection host cell, cracking after cultivating and producing.
4. the application in the anti-Skin Squamous Cell Carcinoma medicine of preparation for the shRNA of Rab23 gene target sequence, described target sequence is: gcgacaaatt caagttaat;
The ShorthairpinRNA of described shRNA for being formed by the corresponding Nucleotide of the sequence shown in SEQ.ID.NO.1.
5. application as claimed in claim 4, is characterized in that, described shRNA is packaged as slow virus, the genome that the protein encapsulation that this slow virus packaging plasmid Helper1.0 and Helper2.0 express contains GV248-Rab23-shRNA carrier.
6. application as claimed in claim 5, is characterized in that, described anti-Skin Squamous Cell Carcinoma medicine is the invasive medicine of anti-squamous cell carcinoma.
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| CN101363027A (en) * | 2007-08-06 | 2009-02-11 | 武汉大学 | Method for preparing recombination plasmid carrying short hairpin RNA and use |
| TW201030340A (en) * | 2009-02-10 | 2010-08-16 | Nat Defense Medical Ct | Method for renal disease diagnosis and prognosis using annexin a1 and rab23 as markers |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101363027A (en) * | 2007-08-06 | 2009-02-11 | 武汉大学 | Method for preparing recombination plasmid carrying short hairpin RNA and use |
| TW201030340A (en) * | 2009-02-10 | 2010-08-16 | Nat Defense Medical Ct | Method for renal disease diagnosis and prognosis using annexin a1 and rab23 as markers |
Non-Patent Citations (4)
| Title |
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| MMP-9小干扰RNA重组慢病毒抑制喉癌体内生长的实验研究;孙亚男等;《临床耳鼻咽喉头颈外科杂志》;20081220;第22卷(第24期);1129-1131 * |
| Rab23在皮肤鳞状细胞癌中的表达;刘博强等;《现代生物医学进展》;20110731;第11卷(第13期);2474-2476 * |
| 刘博强等.Rab23在皮肤鳞状细胞癌中的表达.《现代生物医学进展》.2011,第11卷(第13期),2474-2476. |
| 孙亚男等.MMP-9小干扰RNA重组慢病毒抑制喉癌体内生长的实验研究.《临床耳鼻咽喉头颈外科杂志》.2008,第22卷(第24期),1129-1131. |
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