CN108251455B - Construction method and application of RAGE stable low-expression cervical squamous carcinoma CaSki cells - Google Patents
Construction method and application of RAGE stable low-expression cervical squamous carcinoma CaSki cells Download PDFInfo
- Publication number
- CN108251455B CN108251455B CN201810107245.2A CN201810107245A CN108251455B CN 108251455 B CN108251455 B CN 108251455B CN 201810107245 A CN201810107245 A CN 201810107245A CN 108251455 B CN108251455 B CN 108251455B
- Authority
- CN
- China
- Prior art keywords
- rage
- cells
- squamous carcinoma
- cervical squamous
- lentivirus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Cell Biology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Virology (AREA)
- Plant Pathology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Oncology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a construction method and application of a RAGE stable low-expression cervical squamous carcinoma CaSki cell, wherein the construction method mainly comprises the following steps: constructing a lentivirus interference plasmid pLKO.1-TRC-shRAGE, packaging a lentivirus vector, and performing virus collection and amplification. The RAGE interference lentivirus can down-regulate the expression of RAGE protein in CaSki cells of cervical squamous carcinoma and inhibit the proliferation of the CaSki cells.
Description
Technical Field
The technology relates to construction and application of RAGE stable low-expression cervical squamous carcinoma CaSki cells
Background
Cervical cancer is one of the malignant tumors threatening the health of women, the third most common cancer in women worldwide, with about 52 million new cases each year. Persistent infection with high-risk HPV is considered to be the main cause of cervical cancer progression from precancerous lesion to cervical cancer, and its subtypes are mainly HPV16 type and HPV18 type, among which HPV16 is most common. The CaSki lines HPV16 and 18 are positive, and the cervical squamous carcinoma cells of the intestinal mesentery metastasis are cell lines commonly used for the research of cervical cancer.
The receptor for advanced-glycation end products (RAGE) gene is located on human chromosome 6p21.3, and RAGE protein is a transmembrane signal receptor protein consisting of 404 amino acids and having multiple ligands, and The complete structure of The RAGE protein comprises an intracellular domain, an extracellular domain and an extracellular domain 3 part. The extracellular domain contains 3 immunoglobulin structures, namely ligand binding sites, and can interact with ligands such as AGEs, high mobility group B1 (HMGB 1), S100/calgranulin, beta-amyloid protein (Abeta) and the like to activate related signal channels in cells, so that the cell function is disturbed, and the diseases are generated and developed. Recent researches show that RAGE is involved in the occurrence and development of various malignant tumors, such as esophageal cancer, oral squamous cell carcinoma, laryngeal cancer, liver cancer and the like.
In the preliminary study of the subject group, immunohistochemistry is adopted to detect the expression conditions of RAGE protein in cervical squamous carcinoma tissues, cervical intraepithelial neoplasias and chronic cervicitis tissues, and the result shows that the RAGE protein has the highest expression level in the squamous carcinoma tissues, the cervical intraepithelial neoplasias are the second highest, and the chronic cervicitis tissues are the lowest. RAGE is suggested to play a role in the development of cervical squamous cell carcinoma, but the specific mechanism is not yet clear.
At present, no existing RAGE low-expression model of the cervical squamous cell carcinoma CaSki cells is available for research, so a suitable RAGE low-expression model of the cervical squamous cell carcinoma CaSki cells is urgently needed to be established.
Disclosure of Invention
The invention aims to provide a cervical squamous carcinoma CaSki cell with stable and low expression of RAGE, which researches the influence of the RAGE on the proliferation of the cervical squamous carcinoma CaSki cell through the low expression of the RAGE mediated by lentivirus and provides a new idea and basis for the treatment of cervical cancer.
The invention is realized by the following technical scheme:
the invention discloses a construction method of a cervical squamous carcinoma CaSki cell with stable and low expression of RAGE, which comprises the steps of constructing a lentivirus interference plasmid pLKO.1-TRC-shRAGE, packaging a lentivirus vector, collecting and amplifying.
The specific construction method comprises the following steps:
1. vector and information of target gene
(1) Lentiviral vector pLKO.1-TRC map As shown in FIG. 1, the U6 promoter drives RNA polymerase III transcription to generate shRNA transcripts. EcoR I and Age I are the cleavage sites. The vector contains puromycin resistance and can be screened by puromycin.
(2) The sequence of interest is as follows:
siRNA sequence: ggCTggTgTTCCCAATAAg are provided. The target shRAGE sequence is obtained by inserting a hairpin structure sequence TCAAGAG, an AgeI enzyme cutting site sequence CCGGT and an EcoRI enzyme cutting site sequence AATTC as follows:
RAGE-AgeI-F:CCGGTAAAAAggCTggTgTTCCCAATAAgTCAAGAGCTTATTgggAACACCAgCC(SEQ ID:NO.1)
RAGE-EcoRI-R:AATTCggCTggTgTTCCCAATAAgTCAAGAGCTTATTgggAACACCAgCCTTTTT(SEQ ID:NO.2)
2. the lentivirus vector pLKO.1-TRC is subjected to double enzyme digestion by EcoR I and Age I, and gel is recovered and amplified after the enzyme digestion is finished.
The RAGE single strand forms a double stranded fragment of interest with sticky ends via annealing.
4. And connecting the treated target fragment with a vector, transforming, sequencing and identifying to obtain the pLKO.1-TRC-shrAGE lentivirus interference plasmid.
5. And (3) taking a lentivirus interference plasmid pLKO.1-TRC-shRAGE and two lentivirus packaging plasmids psPAX2 and pMD2.G, transfecting HEK293 cells by using a Lipo2000 transfection reagent to obtain virus supernatant, and then measuring the titer.
6. Transfecting the interference lentivirus into the cervical squamous carcinoma CaSki cells to obtain RAGE stable low-expression cervical squamous carcinoma CaSki cells.
Further, the step of interfering the lentivirus transfection of the cervical squamous carcinoma CaSki cells in the step (6) comprises the following steps:
a. cell culture: inoculating the cervical squamous carcinoma CaSki cells in a culture dish; when the cell fusion degree reaches 40-60%, adding the coacervate amine with the final concentration of 8 mug/ml to act on the cells for 0.5 h;
b. infecting the cells according to the optimal lentivirus working concentration (1:10), namely infecting the cells after diluting the interfering lentivirus in the step (5) by 10 times; after 24h of infection, the medium was replaced with new complete medium;
c. and (3) screening by using puromycin with the final concentration of 2 mu g/ml, replacing culture solution containing puromycin every other day, observing the killing effect of puromycin on untransfected cells, screening 1-2 w, and obtaining the stable and low-expression RAGE cervical squamous carcinoma CaSki cells by using the screened survival cells.
The invention also provides a RAGE-stably low-expression cervical squamous carcinoma CaSki cell, which is obtained by the construction method of the RAGE-stably low-expression cervical squamous carcinoma CaSki cell.
In addition, the invention also provides application of the cervical squamous carcinoma CaSki cells with the stable and low expression of RAGE in researching the influence of RAGE on the proliferation of the cervical squamous carcinoma cells.
The invention successfully constructs the RAGE interference lentivirus and the cervical squamous cell carcinoma CaSki cell with RAGE stably low expression, provides good experimental basis and basis for researching the proliferation of the RAGE on the cervical squamous cell carcinoma cell and provides a more direct and convenient tool for the function of the RAGE in the cell. The hairpin structure of the invention has skillful design, can effectively reduce the possibility of base mismatching, and the pLKO.1-TRC vector, the restriction endonuclease, the ligase and other reagents are convenient to purchase, simple to operate, stable in interfering slow virus, and good in repeatability.
Drawings
FIG. 1 is a slow virus empty vector pLKO.1-TRC map.
FIG. 2 shows the sequencing result of pLKO.1-TRC-shRAGE lentivirus interference plasmid.
FIG. 3 is a schematic diagram of Western blot for detecting RAGE protein expression.
FIG. 4 is a graph showing the effect of pLKO.1-TRC-shRAGE lentivirus fluid on cell proliferation potency after transfection of CaSki cells.
Detailed Description
The invention successfully constructs pLKO.1-TRC-shRAGE interfering slow virus with stable and low expression of RAGE, and transfects cervical squamous cell carcinoma CaSki cells to ensure that the RAGE is low-expressed, and in vitro experiment detection results show that the RAGE can inhibit the proliferation of the CaSki cells after being down-regulated. The present invention will be described in further detail below.
Reagents, materials and instruments used in the embodiment of the invention have no special requirements, and products purchased in the general market can repeatedly realize the invention.
Construction, amplification and identification of lentivirus interference plasmid pLKO.1-TRC-shrAGE
1. Enzyme digestion
FIG. 1 is a schematic representation of the empty lentiviral vector pLKO.1-TRC and key points therein. The empty lentiviral vector pLKO.1-TRC is commercially available.
Age I and EcoR I are insertion sites, pLKO.1-TRC vector is subjected to double enzyme digestion by Age I and EcoR I, and the enzyme digestion system is as follows:
RAGE double-stranded fragment retrieval
PCR procedure:
recovery of glue after completion of enzyme digestion
2. And (3) connecting the target fragment with the carrier:
the above ligation solution was incubated overnight at 16 deg.C
4. Lentivirus interference plasmid amplification
(1) Transformation of
1) 100 μ l of competent E.coli DH5 α stored at-80 ℃ was thawed on ice for 5-15 min.
2) When competent Escherichia coli just melted, 10ng of plasmid for transformation was added, and the mixture was allowed to stand on ice for 30 min.
3) And (3) thermally shocking for 90s in a water bath at 42 ℃, quickly transferring to ice, and standing for 2-3 min.
4) To competent E.coli DH5 α was added 900. mu.l of bacteriolysis broth (LB) medium (without antibiotics).
5) Shaking and culturing at 37 deg.C and 150rpm for 60min to recover thallus.
6) The E.coli was pelleted to the bottom of the tube by centrifugation at 3000rpm for 1min, and 800. mu.l of the supernatant was discarded.
7) And (3) blowing and beating the residual liquid uniformly by using a gun head, taking a proper amount of LB flat plate coated with ampicillin, and after the liquid is absorbed, inversely placing the flat plate in a bacterial incubator at 37 ℃ for culturing for 12-16 h.
(2) Liquid culture
1) The plate was removed from the incubator and a single colony was picked in a clean bench and inoculated into liquid LB medium. (15 ml of liquid LB medium was previously poured into a sterile 50ml centrifuge tube and ampicillin was added to the tube to a final concentration of 100. mu.g/ml)
2) Shaking the incubator at 37 ℃ overnight at 250 rpm.
3) When the liquid in the centrifugal tube becomes turbid, the oscillation is stopped.
(3) Plasmid extraction (performed according to the instruction of the endotoxin-free Plasmid Mini Kit II (OMEGA Co.) Ltd.)
1) 10ml of overnight-cultured bacterial liquid is taken, centrifuged at 8000rpm for 10min, and the supernatant is discarded.
2) Add 500. mu.l of solution I (RnaseA added in advance and stored at 4 ℃), shake up and down or blow repeatedly with a pipette tip to suspend the bacteria thoroughly and transfer into a clean sterile 2ml EP tube.
3) Adding 500 mu l of the solution II, mixing for 7-10 times to fully crack the thalli until a clear solution is formed, and the step is preferably gentle and rapid.
4) Add 250. mu.l of Buffer N3 precooled beforehand and invert the tube several times to neutralize it sufficiently until a white flocculent precipitate forms. 12000rpm, room temperature, centrifugal 10 min.
5) Transferring the supernatant obtained in the step 4 into a clean 1.5ml EP tube, adding 0.1 volume time of endotoxin removing solution according to volume, uniformly mixing, placing on ice for 10min, and shaking the centrifuge tube for several times.
6) Placing in 42 deg.C water bath for 5min, 12000rpm, centrifuging at room temperature for 3 min. The endotoxin-removing solution formed a blue precipitate at the bottom of the tube.
7) And (3) sucking the supernatant into a clean 2ml centrifugal tube, adding 0.5 time of absolute ethyl alcohol according to volume, reversing the centrifugal tube, uniformly mixing for several times, and standing for 1-2 min at room temperature.
8) The DNA micro-column is sleeved into a 2ml collecting tube, 700 mul of liquid is sucked into the collecting tube in times, the solution is centrifuged for 1min at the room temperature of 12000rpm, the DNA micro-column is taken out, and waste liquid in the collecting tube is poured off until all the liquid is filtered.
9) The DNA microcolumn was returned to the collection tube again, 500. mu.l of a weak acid buffer was added thereto, 12,000rpm was applied thereto, and the tube was centrifuged at room temperature for 1min to remove waste liquid from the collection tube.
10) The DNA microcolumn was replaced into the collection tube again, 700. mu.l of DNA washing buffer was added, 12,000rpm was applied, centrifugation was carried out at room temperature for 1min, and the waste liquid in the collection tube was discarded.
11) Step 10 is repeated once.
12) The waste liquid in the collection tube was decanted, centrifuged at 12000rpm for 3min at room temperature, and the DNA wash buffer was removed completely.
13) Putting the DNA microcolumn into a clean 1.5ml centrifuge tube, adding 80-100 mul of endotoxin-free elution buffer solution into the center of the microcolumn membrane, and standing at room temperature for 2 min. 12000rpm, room temperature centrifugation for 1min, collecting the liquid in the EP tube, measuring the plasmid concentration, and storing at-20 ℃ for later use.
5. Identification
Sequencing the cloned bacterial liquid. The results are as follows: ACTTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGAGTATCTGTGAAGGAACATTCAAGAGATGTTCCTTCACAGATACTTTTTTGAATTCTCGACCTCGAGACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCACTTTGGCCGCGGCTCGAGGGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGCGGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGGGAAGCTTCCTGGTCCGCCCCTAAATCGGGAAAGTTCCTTTGGGTTCGCGGCGTGCCGGAAGTGACAAACGGAAACCGCACGTCTCACTTATACCCTCCCAGAAGGACAGCGCCAGGGAACAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCCATAACGGCTGCTCAGCAAGGGCGCGCCCAGAGCAACGGCCCGGAAGGGGCGGTGCGGGAAGCGGGGTGTGGGGCCGGTAATGTGGGCCTGGTTTCCTGGCCCGCGCGGTGTTCCGCAATTCTGCAAGCCTCGGAGCGCACGTCGGCAGTCGCTTCCTCGTTGACCGATTCACGAACTCTCTCCCAGGGATTCACGGAGCTTACATGACCGAGTACAAGCCCACGGGTGGGCTTCGCA (SEQ ID: NO. 3). As a result of sequencing, the underlined region is the sequence of shRAGE linked to pLKO.1-TRC vector, and is completely matched with the sequence of shRAGE designed by us, which indicates that shRAGE has been successfully linked to pLKO.1-TRC vector and that the construction of lentiviral interference plasmid pLKO.1-TRC-shRAGE has been successful. As shown in figure 2.
Second, packaging and reclaiming poison of slow virus interference plasmid pLKO.1-TRC-shRAGE
1. Transfection of 293T cells:
(1) one day before transfection, 293T cells in logarithmic growth phase are taken, after conventional digestion and centrifugation, 2.5x10^6 cells are inoculated into a T25 culture bottle, the culture medium is DMEM + 10% fetal calf serum, and the cells are cultured in a cell culture box with the temperature of 37 ℃ and the concentration of 5% CO 2. Let 3 groups be blank control group, negative control group (empty vector group) and shRAGE group.
(2) Transfection
When the cells grow until the confluence reaches 70% -80% and the cells are in good state, 2ml of complete culture medium without double antibody is replaced 2h before transfection, and transfection is carried out according to the following table.
Adding the mixed solution into a T25 culture flask, gently shaking the mixed solution, and culturing in an incubator at 37 ℃ and 5% CO 2.
(3) Liquid changing device
And after 6-8 h of transfection, replacing the medium with a fresh complete medium.
2. Collecting the viral supernatant
After 48h of transfection, the cell status was observed, and all the liquid in the T25 flask was collected by a 10ml sterile syringe, and the viral supernatant was collected by filtration through a 0.45 μm sterile PVDF needle filter and stored at-80 ℃ for a long period of time.
Thirdly, RAGE interference lentivirus transfection CaSki cell
(1) Cell culture: a6 cm culture dish was added with 3ml of a cell culture medium containing 3X 10^5 CaSki cells.
(2) When the cell fusion degree reaches 40-60%, adding the coagulant cells with the final concentration of 8 mug/ml into the culture medium for 0.5 h.
(3) Cells were infected at the optimal lentivirus working concentration (1: 10).
(4) After 24h of infection, the medium was replaced with fresh complete medium.
And (3) screening by using puromycin with the final concentration of 2 mu g/ml, replacing culture solution containing puromycin every other day, observing the killing effect of puromycin on untransfected cells, screening by 1-2 w, and continuously culturing the screened survival cells for subsequent experiments.
Fourth, application
Schematic diagram for detecting RAGE protein expression by Western blot
As shown in the attached figure 3, pLKO.1-TRC-shRAGE interfering slow virus is transferred into cervical squamous cell carcinoma CsSki cells, protein is extracted after 48h, the expression condition of the RAGE protein is detected, a single-factor variance analysis is adopted, the difference between a transfected group and an empty vector group and an untransfected group is statistically significant (P is less than 0.05), the difference between the empty vector group and the untransfected group is not statistically significant (P is more than 0.05), the expression quantity of the RAGE protein after transfection is obviously reduced compared with that before transfection, the transfection is successful, and the construction of the cervical squamous cell carcinoma CaSki cells with the RAGE stably and low in expression is successful.
CCK-8 detection of cell proliferation Capacity
(1) Grouping experiments: untransfected, empty vector, shRAGE.
(2) Taking each group of cells in logarithmic phase, digesting and centrifuging to prepare cell suspension, adjusting the cell density to 5 multiplied by 10^3/100 mu l, adding 100 mu l of cell suspension into each hole of a 96-hole plate, arranging 5 compound holes in each group of cells, and adding sterile PBS into the edge hole.
(3) After 48h of culture, discarding the original culture medium, adding 10 mul of CCK-8 reagent and 90 mul of 1640 basic culture medium into each hole, and incubating for 1-4 h in a dark place.
(4) And taking out the 96-well plate, and placing the 96-well plate on a microplate reader to detect the absorbance value at the wavelength of 450 nm.
CCK-8 results are shown in FIG. 4, and it can be seen that the absorbance values of the cells in the transfected group at a wavelength of 450nm are significantly reduced (OD value of 0.85. + -. 0.06) compared with those in the untransfected group (OD value of 1.11. + -. 0.08) and the empty vector group (OD value of 1.13. + -. 0.11), and the difference is statistically significant (P < 0.05). Suggesting that the expression quantity of RAGE in the cervical squamous carcinoma CaSki cells can be reduced to obviously inhibit the proliferation capacity of the cells.
In view of CCK-8 experimental results, the expression of the down-regulated RAGE gene can inhibit the growth of cervical squamous cell carcinoma, provide a basis for developing compounds related to the down-regulated RAGE gene, and provide a new idea and experimental basis for the treatment of clinical cervical cancer.
SEQUENCE LISTING
<110> second Hospital affiliated to Wenzhou medical university and English child care Hospital affiliated to Wenzhou medical university
Construction method and application of low-expression stable-expression cervical squamous carcinoma CaSki cells of RAGE
<130> 2018
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 55
<212> DNA
<213> Artificial sequence
<400> 1
ccggtaaaaa ggctggtgtt cccaataagt caagagctta ttgggaacac cagcc 55
<210> 2
<211> 55
<212> DNA
<213> Artificial sequence
<400> 2
aattcggctg gtgttcccaa taagtcaaga gcttattggg aacaccagcc ttttt 55
<210> 3
<211> 870
<212> DNA
<213> Artificial sequence
<400> 3
actttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgg agtatctgtg 60
aaggaacatt caagagatgt tccttcacag atactttttt gaattctcga cctcgagaca 120
aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg 180
ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat 240
tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccac tttggccgcg 300
gctcgagggg gttggggttg cgccttttcc aaggcagccc tgggtttgcg cagggacgcg 360
gctgctctgg gcgtggttcc gggaaacgca gcggcgccga ccctgggtct cgcacattct 420
tcacgtccgt tcgcagcgtc acccggatct tcgccgctac ccttgtgggc cccccgggga 480
agcttcctgg tccgccccta aatcgggaaa gttcctttgg gttcgcggcg tgccggaagt 540
gacaaacgga aaccgcacgt ctcacttata ccctcccaga aggacagcgc cagggaacaa 600
tggcagcgcg ccgaccgcga tgggctgtgg cccataacgg ctgctcagca agggcgcgcc 660
cagagcaacg gcccggaagg ggcggtgcgg gaagcggggt gtggggccgg taatgtgggc 720
ctggtttcct ggcccgcgcg gtgttccgca attctgcaag cctcggagcg cacgtcggca 780
gtcgcttcct cgttgaccga ttcacgaact ctctcccagg gattcacgga gcttacatga 840
ccgagtacaa gcccacgggt gggcttcgca 870
Claims (3)
1. A method for constructing a cervical squamous carcinoma CaSki cell with stable and low expression of RAGE is characterized by comprising the following specific construction steps of constructing a lentivirus interference plasmid pLKO.1-TRC-shRAGE, packaging a lentivirus vector, and performing virus harvesting and amplification:
(1) the sequence of interest is as follows:
siRNA sequence: ggCTggTgTTCCCAATAAg, the target shRAGE sequence is obtained by inserting hairpin structure sequence TCAAGAG, AgeI restriction site sequence CCGGT and EcoRI restriction site sequence AATTC as follows:
RAGE-AgeI-F:CCGGTAAAAAggCTggTgTTCCCAATAAgTCAAGAGCTTATTgggAACACCAgCC
RAGE-EcoRI-R:AATTCggCTggTgTTCCCAATAAgTCAAGAGCTTATTgggAACACCAgCCTTTTT ；
(2) annealing the target single-chain fragment into a double-chain fragment with a sticky end by a PCR instrument;
(3) double digestion of lentivirus vector pLKO.1-TRC: double enzyme digestion is carried out on a lentiviral vector pLKO.1-TRC by Age I and EcoR I, and glue is recovered and purified after the enzyme digestion is finished;
(4) connecting, transforming and identifying the product obtained in the step (2) and the vector subjected to double enzyme digestion in the step (3) to obtain a lentivirus interference plasmid pLKO.1-TRC-shrAGE;
(5) co-transfecting HEK293 cells with a lentivirus interference plasmid pLKO.1-TRC-shRAGE and two lentivirus packaging plasmids psPAX2 and pMD2.G to obtain interference lentiviruses;
(6) and transfecting the interference lentivirus into the cervical squamous carcinoma CaSki cells to obtain the cervical squamous carcinoma CaSki cells with stable and low expression of RAGE.
2. The method for constructing the RAGE-stabilized underexpressed cervical squamous carcinoma cells, CaSki, as claimed in claim 1, wherein the step of (6) interfering with lentiviral transfection of the cervical squamous carcinoma cells comprises:
a. cell culture: inoculating the cervical squamous carcinoma CaSki cells in a culture dish; when the cell fusion degree reaches 40-60%, adding the coacervate amine with the final concentration of 8 mug/ml to act on the cells for 0.5 h;
b. infecting cells after diluting the interfering lentivirus in the step (5) by 10 times; after 24h of infection, the medium was replaced with new complete medium;
c. and (3) screening by using puromycin with the final concentration of 2 mu g/ml, replacing culture solution containing puromycin every other day, observing the killing effect of puromycin on untransfected cells, screening 1-2 w, and obtaining the stable and low-expression RAGE cervical squamous carcinoma CaSki cells by using the screened survival cells.
The CaSki cell of the cervical squamous carcinoma with the RAGE stably low expression, which is obtained by the method for constructing the CaSki cell of the cervical squamous carcinoma with the RAGE stably low expression as claimed in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810107245.2A CN108251455B (en) | 2018-02-02 | 2018-02-02 | Construction method and application of RAGE stable low-expression cervical squamous carcinoma CaSki cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810107245.2A CN108251455B (en) | 2018-02-02 | 2018-02-02 | Construction method and application of RAGE stable low-expression cervical squamous carcinoma CaSki cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108251455A CN108251455A (en) | 2018-07-06 |
| CN108251455B true CN108251455B (en) | 2021-02-26 |
Family
ID=62743605
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810107245.2A Active CN108251455B (en) | 2018-02-02 | 2018-02-02 | Construction method and application of RAGE stable low-expression cervical squamous carcinoma CaSki cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108251455B (en) |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2650013A1 (en) * | 2012-04-10 | 2013-10-16 | Deutsches Krebsforschungszentrum | Inhibitors of Receptor for Advanced Glycation-End products (RAGE) for use in treating and/or preventing inflammation- and/or damage-induced cancer |
| RU2735141C2 (en) * | 2013-03-15 | 2020-10-28 | Иликсиэджен Терапьютикс, Инк. | Methods of using zscan4 for human cell rejuvenation |
| CN107254470B (en) * | 2017-06-26 | 2019-11-26 | 浙江大学 | The siRNA and its recombinant vector of specificity inhibition PADI2 gene expression and application |
-
2018
- 2018-02-02 CN CN201810107245.2A patent/CN108251455B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| "RAGE在宫颈鳞癌癌变过程中的表达及意义";朱雪洁等;《浙江医学》;20100526;第32卷(第3期);第318页右栏第2段，第319页左栏第1段 * |
| "特异性靶向晚期糖基化终产物受体（RAGE）的siRNA降低人类乳腺癌增殖研究";热娣;《中国博士学位论文全文数据库 医药卫生科技辑》;20140215(第2期);第5页第4段,第24页第3-4段,第32页第3段，第33页第1段，图3-4 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108251455A (en) | 2018-07-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107586777A (en) | People's PDCD1 genes sgRNA purposes and its related drugs | |
| CN109097402B (en) | Preparation method of recombinant vector CAR-CD244-anti CD19 | |
| CN105924533B (en) | ROR1 specific chimeric antigen receptor and application thereof | |
| CN111172114B (en) | Humanized pre-intestinal cancer lesion immortalized epithelial cell line, construction method and application thereof | |
| CN110684120B (en) | Chimeric antigen receptor targeting GPC3 and application thereof | |
| CN105368859A (en) | Chimeric antigen receptor hCD87-CAR, lentivirus carrying hCD87-CAR gene structure, plasmid and application of chimeric antigen receptor hCD87-CAR | |
| CN107604005A (en) | The dual-gene construction methods for being overexpressed slow virus carrier of VEGF and Smad7 | |
| CN106191121A (en) | A kind of T1R2 gene overexpression slow virus carrier, slow virus and construction method thereof | |
| CN104694576A (en) | Method for silencing IFNARI gene in DF-1 cell line | |
| CN108251455B (en) | Construction method and application of RAGE stable low-expression cervical squamous carcinoma CaSki cells | |
| CN105624162B (en) | For the siRNA of mammal R-Spondin2 gene targets, ShorthairpinRNA and carrier and application | |
| CN105039342A (en) | siRNA capable of inhibiting MAT2A genetic expression and application of siRNA | |
| CN114672460B (en) | Preparation method and application of CD 44-targeted heterogeneous CIC cell model | |
| CN113736742B (en) | Application of PRTN3 gene as target for activating cytotoxic immune cells in tumor immunotherapy | |
| CN111849917B (en) | Fourth generation CAR-T cell for fusion expression of type I IL-10 receptor and preparation method and application thereof | |
| CN115505600A (en) | Method for efficiently infecting human NK cells by lentiviruses | |
| CN112980888B (en) | Mesenchymal stem cell secreting IL-6 antibody/CD 20 antibody, construction method and application thereof | |
| CN108465108A (en) | A kind of specific gene target spot prevented or treat glioma | |
| CN114917325A (en) | Application of human soluble TIM-4 protein in preparation of antitumor drugs | |
| CN104630221B (en) | Suppress shRNA and its recombinant vector and the application of growth of tumour cell | |
| CN111378621B (en) | B lymphoma cell strain stably transfected by EB virus latent membrane protein 1, construction method and application thereof | |
| CN112779262A (en) | Application of pig RagC gene | |
| CN117024605B (en) | Chimeric antigen receptor, microglia expressing chimeric antigen receptor and application thereof | |
| CN104745635B (en) | A kind of method of OASL genes in silence DF-1 cell lines | |
| CN110642953B (en) | BCMA-targeted T cell receptor fusion protein and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |