shRNA and lentiviral vector for inhibiting human EDRADD gene expression as well as construction method and application thereof
阅读说明：本技术 一种抑制人EDARADD基因表达的shRNA、慢病毒载体及其构建方法和应用 (shRNA and lentiviral vector for inhibiting human EDRADD gene expression as well as construction method and application thereof ) 是由 孟箭 李梦 白玉婷 李晓东 管俊杰 王守鹏 于 2019-09-26 设计创作，主要内容包括：本发明公开了一种抑制人EDARADD基因表达的shRNA、慢病毒载体及其构建方法和应用,首先合成针对RNA干扰靶点序列的DNA oligo的正链和反链；正链和反链退火形成带粘性末端的双链DNA；通过酶切使慢病毒载体线性化,将线性化载体与双链DNA连接；将连接产物转化大肠杆菌感受态细胞,对得到的阳性克隆进行PCR鉴定；质粒抽提,用于下一步病毒包装。本发明以EDARADD基因为模板设计了RNA干扰靶点序列,根据选取的靶点序列设计shRNA干扰序列,包装构建RNA干扰慢病毒载体,对EDARADD基因进行敲减后,会显著影响舌鳞癌细胞CAL-27细胞的增殖,影响细胞克隆形成能力,并促进细胞的凋亡。(The invention discloses shRNA for inhibiting the expression of human EDRADD genes, a lentiviral vector, a construction method and application thereof, wherein the method comprises the steps of firstly synthesizing a positive strand and a negative strand of a DNA oligo aiming at an RNA interference target sequence; annealing the positive strand and the reverse strand to form double-stranded DNA with a sticky end; linearizing a lentivirus vector by enzyme digestion, and connecting the linearized vector with double-stranded DNA; transforming the ligation product into an escherichia coli competent cell, and performing PCR identification on the obtained positive clone; plasmid extraction is used for the next step of virus packaging. According to the invention, an RNA interference target sequence is designed by taking an EDRADD gene as a template, an shRNA interference sequence is designed according to the selected target sequence, an RNA interference lentiviral vector is constructed by packaging, and after the EDRADD gene is knocked down, the proliferation of a tongue squamous cell carcinoma CAL-27 cell is obviously influenced, the cell clone forming capability is influenced, and the apoptosis of the cell is promoted.)
1. A shRNA for inhibiting the expression of a human EDRADD gene is characterized in that: the shRNA comprises a positive strand and a reverse strand of a DNA oligo, wherein the positive strand is a base sequence shown in a sequence table SEQ ID NO.2, the reverse strand is a base sequence shown in a sequence table SEQ ID NO.3, and the positive strand and the reverse strand are annealed to form double-stranded DNA with a sticky end.
2. A lentiviral vector for the shRNA for inhibiting the expression of the human EDRADD gene according to claim 1, wherein the shRNA comprises: the lentiviral vector was made by cloning synthetic double-stranded DNA into lentiviral vector GV 115.
3. The lentiviral vector for inhibiting shRNA expressed by the human EDRADD gene according to claim 2, wherein the shRNA comprises a sequence selected from the group consisting of: the RNA interference target sequence aimed by the lentiviral vector GV 115-shEDRADD is as follows: 5'-GTACTTGTTCCTCCTGCTT-3' are provided.
4. The method for constructing the lentiviral vector for inhibiting shRNA expressed by the human EDRADD gene as claimed in claim 3, wherein the method comprises the following steps: the method comprises the following steps:
(1) synthesizing the positive strand and the reverse strand of the DNA oligo aiming at the RNA interference target sequence, dissolving the dry powder of the positive strand and the reverse strand of the synthesized DNA oligo in an annealing buffer solution, and carrying out water bath at 90 ℃ for 15 min; naturally cooling to room temperature to form a double chain with a sticky end; the annealing buffer solution contains Tris, EDTA and NaCl;
(2) carrying out double enzyme digestion on the GV115 vector by AgeI and EcoRI to linearize the vector, and connecting the linearized vector with the double-stranded DNA obtained in the step (1);
(3) transforming the ligation product into an escherichia coli competent cell, and performing PCR identification on the obtained positive clone;
(4) after the bacterial liquid qualified by PCR is transferred and cultured, plasmid extraction is carried out, and the plasmid qualified by quality inspection is transferred to a downstream platform for virus packaging.
5. The method for constructing the lentiviral vector for inhibiting shRNA expressed by the human EDRADD gene according to claim 4, wherein the lentiviral vector comprises: in the step (2), the reaction temperature of the enzyme digestion carrier is 37 ℃, and the reaction lasts for 1 h; the reaction temperature for connecting the carrier and the double-stranded DNA is 16 ℃, and the reaction lasts for 1-3 h.
6. The method for constructing the lentiviral vector for inhibiting shRNA expressed by the human EDRADD gene according to claim 4, wherein the lentiviral vector comprises: in the step (3), the specific steps of transforming the ligation product into the escherichia coli competent cells are as follows:
1) add 10. mu.l ligation product to 100. mu.l E.coli competent cells, ice-wash for 30 min;
2) heat shock at 42 deg.c for 90 sec in ice bath for 2 min;
3) adding 500 mul LB liquid culture medium without antibiotic, shaking and culturing for 1h at 200rpm and 37 ℃ in a shaking table;
4) 150 μ l of the bacterial solution was applied evenly to LB solid medium containing Amp and cultured overnight in an incubator at 37 ℃.
7. The method for constructing the lentiviral vector for inhibiting shRNA expressed by the human EDRADD gene according to claim 4, wherein the lentiviral vector comprises:
in the step (3), the identification primer adopted in the PCR identification process of the positive clone is an identification primer-F: 5'-CCTATTTCCCATGATTCCTTCATA-3' and identifying primer-R: 5'-GTAATACGGTTATCCACGCG-3', the specific process is as follows: selecting a single colony as a template, and carrying out PCR amplification under the reaction conditions: 3min at 94 ℃; 30s at 94 ℃, 30s at 55 ℃, 30s at 72 ℃ and 22 times of circulation; 5min at 72 ℃; after the PCR was completed, 5. mu.l of the product was subjected to 1% agarose gel electrophoresis to detect bands, and sequencing of positive clones was performed with the identifying primer-F.
8. The use of the shRNA for inhibiting the expression of the human EDRADD gene according to claim 1 in the preparation of a medicine for treating tongue squamous carcinoma.
9. The use of the lentiviral vector for inhibiting shRNA expressed by the human EDRADD gene of claim 2 in the preparation of a medicament for treating tongue squamous carcinoma.
Technical Field
The invention belongs to the technical field of molecular biology and biomedicine, and particularly relates to shRNA and a lentiviral vector for inhibiting the expression of human EDRADD genes, and a construction method and application thereof.
Background
Tongue Squamous Cell Carcinoma (TSCC), one of the most common malignant tumors of the oral and maxillofacial areas, is called tongue squamous cell carcinoma for short, and has high mortality and morbidity. Its development is a multistep process involving genetic alterations and epigenetic modifications. TSCC is a causative factor for many variables, a number of genetic alterations and accumulation of environmental factors such as tobacco use, alcohol consumption, chronic inflammation and Human Papillomavirus (HPV) infection, etc. some recent reports have shown that TSCC in young adults has increased morbidity, especially in women, with higher distant metastasis rates and poorer prognosis. Thus, TSCCs caused by different risk factors may have different molecular bases, involving loss of cell cycle control and aging control, dysregulation of apoptosis, and development of oral lichen planus and leukoplakia.
Current treatments for squamous cell carcinoma of the tongue include primarily traditional surgical treatment, chemotherapy, and radiation therapy. However, the trauma of operative treatment is large, the quality of life of a patient after the operation is reduced, and the traditional chemotherapy and radiotherapy lack specificity, so that the curative effect is obtained and the patient is often brought with larger toxic and side effects. In recent years, gene therapy and biotherapy play an increasingly important role in the comprehensive treatment of malignant tumors as new therapeutic means in addition to conventional therapies such as surgical therapy, radiotherapy and chemotherapy, and are receiving more and more attention. With the continuous and intensive research on the molecular biological behavior of tumors, a plurality of specific target sites which can be used for treatment are discovered. However, molecular targeted therapy for tongue squamous cell carcinoma is still in the experimental research stage, and it is crucial to correctly select specific molecular targets.
Whether the human EDARADD gene is involved in apoptosis of tumor cells and its role in tumorigenesis and progression has not been reported in the literature. The role of the cadaveric edarad gene in tumors remains to be studied further.
Disclosure of Invention
One of the purposes of the invention is to provide shRNA for inhibiting the expression of the human EDRADD gene.
The invention also aims to provide the lentiviral vector of the shRNA for inhibiting the expression of the human EDRADD gene.
Still another object of the present invention is to provide a method for constructing the lentiviral vector for the shRNA that inhibits the expression of the human EDARADD gene.
The last purpose of the invention is to provide the application of the shRNA and the lentiviral vector for inhibiting the expression of the human EDRADD gene.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows: the shRNA for inhibiting the expression of the human EDRADD gene comprises a positive strand and a reverse strand of a DNA oligo, wherein the positive strand is a base sequence shown in a sequence table SEQ ID NO.2, the reverse strand is a base sequence shown in a sequence table SEQ ID NO.3, and the positive strand and the reverse strand are annealed to form double-stranded DNA with a sticky end.
The lentiviral vector of shRNA for inhibiting the expression of the human EDRADD gene is prepared by cloning synthetic double-stranded DNA into a lentiviral vector GV 115.
The RNA interference target sequence aimed by the lentiviral vector GV 115-shEDRADD is as follows: 5'-GTACTTGTTCCTCCTGCTT-3' (SEQ ID NO. 1).
The construction method of the lentiviral expression vector for inhibiting shRNA expressed by the human EDRADD gene comprises the following steps:
(1) synthesizing the positive strand and the reverse strand of the DNA oligo aiming at the RNA interference target sequence, dissolving the dry powder of the positive strand and the reverse strand of the synthesized DNA oligo in an annealing buffer solution, and carrying out water bath at 90 ℃ for 15 min; naturally cooling to room temperature to form a double chain with a sticky end; the annealing buffer solution contains Tris, EDTA and NaCl;
(2) carrying out double enzyme digestion on the GV115 vector by AgeI and EcoRI to linearize the vector, and connecting the linearized vector with the double-stranded DNA obtained in the step (1);
(3) transforming the ligation product into an escherichia coli competent cell, and performing PCR identification on the obtained positive clone;
(4) after the bacterial liquid qualified by PCR is transferred and cultured, plasmid extraction is carried out, and the plasmid qualified by quality inspection is transferred to a downstream platform for virus packaging.
In the step (2), the reaction temperature of the enzyme digestion carrier is 37 ℃, and the reaction lasts for 1 h; the reaction temperature for connecting the carrier and the double-stranded DNA is 16 ℃, and the reaction lasts for 1-3 h.
In the step (3), the specific steps of transforming the ligation product into the escherichia coli competent cells are as follows:
1) add 10. mu.l ligation product to 100. mu.l E.coli competent cells, ice-wash for 30 min;
2) heat shock at 42 deg.c for 90 sec in ice bath for 2 min;
3) adding 500 mul LB liquid culture medium without antibiotic, shaking and culturing for 1h at 200rpm and 37 ℃ in a shaking table;
4) 150 μ l of the bacterial solution was applied evenly to LB solid medium containing Amp and cultured overnight in an incubator at 37 ℃.
In the step (3), the identification primer adopted in the PCR identification process of the positive clone is an identification primer-F: 5'-CCTATTTCCCATGATTCCTTCATA-3' (SEQ ID NO.4) and identifying primer-R: 5'-GTAATACGGTTATCCACGCG-3' (SEQ ID NO.5), the specific process is as follows: selecting a single colony as a template, and carrying out PCR amplification under the reaction conditions: 3min at 94 ℃; 30s at 94 ℃, 30s at 55 ℃, 30s at 72 ℃ and 22 times of circulation; 5min at 72 ℃; after the PCR was completed, 5. mu.l of the product was subjected to 1% agarose gel electrophoresis to detect bands, and sequencing of positive clones was performed with the identifying primer-F.
The shRNA for inhibiting the expression of the human EDRADD gene is applied to preparing the medicine for treating the tongue squamous carcinoma.
The lentiviral vector of shRNA for inhibiting the expression of the human EDRADD gene is applied to the preparation of the medicine for treating the tongue squamous carcinoma.
The medicine for treating tongue squamous carcinoma has at least one of the following functions: inhibiting proliferation of tongue squamous carcinoma cells; promoting tongue squamous carcinoma cell apoptosis; inhibiting the clone of tongue squamous carcinoma cells; inhibiting tongue squamous carcinoma tumor formation.
Compared with the prior art, the invention has the following beneficial effects: according to the invention, an RNA interference target sequence is designed by taking an EDRADD gene as a template, an shRNA interference sequence is designed according to the selected target sequence, an RNA interference lentiviral vector is constructed by packaging, and after the EDRADD gene is knocked down, the proliferation of a tongue squamous cell carcinoma CAL-27 cell is obviously influenced, the cell clone forming capability is influenced, and the apoptosis of the cell is promoted. The gene is shown to be possible to regulate the occurrence and development of tumors, be possible to become a potential treatment target of tongue squamous cell carcinoma and provide reference for prognosis judgment and operation and targeted treatment schemes of tongue squamous cell carcinoma patients.
Drawings
FIG. 1 is a box plot of EDRADD overexpression in HNSCC tissue;
FIG. 2 is an agarose gel electrophoresis of a thread-granulated carrier, lane 1: 1kb Marker: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp and 250bp are sequentially arranged from top to bottom; lane 2: carrying out double enzyme digestion on the linearized vector plasmid by Age I and EcoR I; lane 3: vector plasmid without enzyme digestion;
FIG. 3 is an agarose gel electrophoresis of the PCR product, lane 1: negative control (ddH)2O), eliminating false positive results caused by exogenous nucleic acid pollution in the system; lane 2: self-ligation control (empty vector self-ligation control group); lane 3: 250bp Marker: 5kb, 3kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp and 100bp are sequentially arranged from top to bottom; lanes 4-8: monoclonal psc27052-1,2,3,4, 5;
FIG. 4 is a histogram of EDRADD gene expression in tongue squamous carcinoma cells;
FIG. 5 shows that EDRADD shRNA lentivirus inhibits EDRADD mRNA and protein expression: (A) fluorescent pictures of cells infected with
figure 6 shows that EDARADD knockdown inhibited TSCC cell proliferation: (A) immunofluorescence pictures of CAL27 cells infected with lentiviruses; (B) line graphs of cell counts for 5 consecutive days for the lentivirus suppression EDARADD gene and control; (C) fold line plot of cell count for 5 consecutive days for the lentivirus suppression EDARADD gene and control; (D) the line graphs of OD values at 490nm were measured for lentiviral suppressor edarad gene and control cells after MTT treatment; (E) OD490 fold line plot of lentivirus inhibition EDARADD gene and control cells after MTT treatment;
figure 7 shows that EDARADD knockdown induces apoptosis of TSCC cells: (A) the lentivirus inhibits the apoptosis result of the EDRADD gene and the control group; (B) histogram of apoptosis rate of lentivirus-inhibited EDARADD gene and control;
figure 8 is that EDARADD knockdown inhibited the clonogenic capacity of TSCC cells: (A) a comparison of cloning of the lentivirus-inhibited EDARADD gene and control; (B) the clones of lentivirus-inhibited EDARADD gene and control form a number histogram.
Detailed Description
The invention is described in further detail below with reference to the figures and specific examples.
The main indexes for evaluating the biological behavior of the tumor include tumor cell proliferation, colony forming ability, apoptosis and the like. According to the research, the influence of the gene on the biological behavior of tumor cells is observed by knocking down the expression of the endogenous EDADADD gene of the tongue squamous carcinoma cell line, so that reference is provided for prognosis judgment of a tongue squamous carcinoma patient and a surgical and targeted treatment scheme. The detection of the specific gene EDRADD in a noninvasive, safe and convenient mode is explored, and the diagnosis, treatment and prognosis prediction of tongue squamous carcinoma are facilitated.
1. Genetic information
Boxplots of the expression levels of EDARADD in head and neck tumor tissues as well as normal tissues were generated from the online source UALCAN database (http:// UALCAN. path. uab. edu. /).
Boxplots of EDARADD expression show that EDARADD expression is significantly higher in head and neck tumor tissues than in normal tissues (p <0.01, as in fig. 1).
2. Cell lines and cell cultures
CAL-27 cell lines were purchased from Chinese academy of sciences (Shanghai, China), recovered by removal from liquid nitrogen tanks in RPMI 16 supplemented with 10% Fetal Bovine Serum (FBS), 100U/mL penicillin and 100. mu.g/100 mL streptomycin40 medium and at 37 ℃ in 5% CO2Is incubated in a humid atmosphere.
3. RNA interference target design and double-stranded DNA oligo preparation
According to the design principle of RNA interference sequences, EDRADD genes are used as templates, and a plurality of 19-21nt RNA interference target sequences are designed. After evaluation and determination by design software, the following sequences are selected as interference targets: 5'-GTACTTGTTCCTCCTGCTT-3' (SEQ ID No. 1).
And designing shRNA interference sequences according to the selected target sequences, and adding appropriate restriction enzyme cutting sites at two ends to complete vector construction. In addition, a TTTTT termination signal is added to the 3 '-end of the plus strand, and a termination signal complementary sequence is added to the 5' -end of the minus strand. After the design is completed, the DNA oligo is sent to the Czech company to synthesize the single-stranded DNA oligo.
CCGG: an AgeI enzyme cleavage site; AATTC: EcoRI enzyme cutting sites; g: EcoRI restriction site complementary sequence.
The synthesized single-stranded DNA oligo dry powder was dissolved in annealing buffer (Tris 10mM, NaCl50mM, EDTA1mM) (final concentration 20. mu.M) and water-bathed at 90 ℃ for 15 min. After naturally cooling to room temperature, a double strand with a cohesive end was formed.
A50. mu.l reaction was prepared according to the NEB protocol and the GV115 vector was linearized by double digestion with AgeI and EcoRI.
Reacting at 37 ℃ for 1h, carrying out agarose gel electrophoresis on the product of the enzyme digestion of the vector, and recovering a target band. The agarose gel electrophoresis picture is shown in FIG. 2.
4. RNA interference lentivirus vector construction
4.1A 20. mu.l reaction system was prepared according to Fermentas T4 DNA Ligase instructions and the double-stranded DNA oligo was ligated to the linearized vector.
After 1h-3h reaction at 16 ℃ the ligation product was named psc27052, after which the transformation experiment was performed.
4.2 the ligation products were transformed into E.coli competent cells, the detailed procedure was as follows:
1) add 10. mu.l ligation product psc27052 to 100. mu.l E.coli competent cells and ice-wash for 30 min;
2) heat shock at 42 deg.c for 90 sec in ice bath for 2 min;
3) adding 500 mul LB liquid culture medium without antibiotic, shaking and culturing for 1h at 200rpm and 37 ℃ in a shaking table;
4) 150 μ l of the bacterial solution was applied evenly to LB solid medium containing Amp and cultured overnight in an incubator at 37 ℃.
4.3 colony PCR identification
4.3.1 primers
4.3.2PCR amplification
Preparing a 20-mu-l PCR reaction system according to the following table, picking a single colony as a template by using a sterile gun head, and carrying out PCR amplification under the reaction conditions that: 3min at 94 ℃; 30s at 94 ℃, 30s at 55 ℃, 30s at 72 ℃ and 22 times of circulation; 5min at 72 ℃. After the PCR was completed, 5. mu.l of the product was subjected to 1% agarose gel electrophoresis to detect bands. The agarose gel electrophoresis picture is shown in FIG. 3.
PCR band size
The size of the positive clone PCR fragment ligated into the shRNA fragment was: 380 bp;
the size of the empty vector clone PCR fragment not ligated into the shRNA fragment was: 307 bp.
Therefore, the psc27052-1,2,3,4 and 5 is judged to be a positive clone, and the clone with the correct identification result is stored and sequenced.
4.4 analysis of sequencing results of Positive clones
And (3) carrying out positive clone sequencing by using the identification primer-F, and selecting a clone with a sequencing result completely consistent with a target sequence for the next experiment.
Sequencing results of psc27052
TTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGCTGTACTTGTTCCTCCTGCTTCTCGAGAAGCAGGAGGAACA AGTACAGTTTTTGAATTCTCGACCTCGAGACAAATGGCAGTATTCATCCACGAATTCGGATCCATTAGGCGGCCGCGTGGATAACCGTATTACCGCCATGCATTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTT(SEQ IDNO.6)
shRNA interfering sequence inserts are in bold, with the AgeI cleavage site disrupted.
4.5 plasmid extraction
Transferring the bacterial liquid with correct sequencing into 150ml LB liquid culture medium containing Amp antibiotics, and shaking and culturing overnight at 37 ℃ by a shaking table. Extracting plasmids according to the EndoFree Maxi Plasmid Kit instruction, and feeding the qualified plasmids into a downstream process.
The detailed operation steps are as follows:
1) centrifuging at 8000rpm for 4min, and collecting thallus;
2) adding 7ml of P1, shaking and mixing uniformly;
3) adding 7ml of P3, reversing and uniformly mixing for 6-8 times, and standing for 5 min;
4) adding 7ml of P4, reversing and uniformly mixing for 6-8 times, and carrying out ice bath for 10 min;
5) centrifuging at 9000rpm for 10min, transferring the supernatant to a filter CS, filtering, adding 10ml isopropanol, and mixing;
6) adding 2.5ml of balance liquid BL into the adsorption column, centrifuging at 8000rpm for 2min, pouring off waste liquid in the collecting pipe, and returning the column for standby;
7) pouring the supernatant into an adsorption column twice, centrifuging at 8000rpm for 2min, and discarding the waste liquid;
8) adding 10ml of rinsing liquid PW (added with absolute ethyl alcohol) into the adsorption column, centrifuging at the same rotation speed for 2min, discarding the waste liquid, and repeating the step once;
9) adding 3ml of absolute ethyl alcohol into the adsorption column, centrifuging at 8000rpm for 2min, and discarding the waste liquid;
10) air-throwing at 9500rpm for 5min to remove residual rinsing liquid;
11) transferring the adsorption column to a new white tube, dripping 800 μ l of elution buffer TB (preheated first) into the center of the column, standing at room temperature for 5min, and centrifuging at 9500rpm for 2 min;
12) the eluate from the tube was transferred to a clean 1.5ml EP tube and stored at-20 ℃;
13) the samples were electrophoresed, and the concentration of the plasmid was measured by a spectrophotometer (Thermo _ Nanodrop 2000) for quality control.
14) And (5) transferring the plasmids qualified in quality inspection to a downstream platform for virus packaging.
5. Virus package
(1) Digesting 293T cells in a logarithmic growth phase by using trypsin 24 hours before transfection, adjusting the cell density to about 5x 106 cells/15 ml by using a culture medium containing 10% serum, re-inoculating the cells in a 10cm cell culture dish, culturing the cells in a 5% CO2 culture box at 37 ℃ for 24 hours, and using the cells for transfection when the cell density reaches 70% -80%;
(2) replacing the medium with a serum-free medium 2h before transfection;
(3) adding each prepared DNA solution (20 μ g of GV115 vector plasmid, 15 μ g of pHelper1.0 vector plasmid, 10 μ g of pHelper 2.0 vector plasmid) into a sterilized centrifuge tube, mixing with corresponding volume of Gecky transfection reagent, adjusting the total volume to 1ml, and incubating at room temperature for 15 min;
(4) slowly adding the mixed solution dropwise into 293T cell culture solution, mixing, and culturing in a 5% CO2 cell culture box at 37 deg.C;
(5) culturing for 6h, discarding the culture medium containing the transfection mixture, adding 10ml of PBS (phosphate buffer solution) for washing once, gently shaking the culture dish to wash the residual transfection mixture, and then pouring and discarding;
(6) slowly adding 20ml of cell culture medium containing 10% serum, and culturing at 37 deg.C in 5% CO2 incubator for 48-72 hr.
6. Purification of viruses
(1) Collecting 293T cell supernatant 48h after transfection (which can be counted as 0h after transfection) according to cell states;
(2) centrifuging at 4000g for 10min at 4 deg.C to remove cell debris;
(3) filtering the supernatant with a 0.45 μm filter in a 40ml ultracentrifuge tube;
(4) respectively balancing samples, putting ultracentrifuge tubes with virus supernatant into a Beckman ultracentrifuge one by one, setting the centrifugation parameters to be 25000rpm, setting the centrifugation time to be 2h, and controlling the centrifugation temperature to be 4 ℃;
(5) after centrifugation is finished, removing supernatant, removing liquid remained on the tube wall as much as possible, adding virus preservation solution (which can be replaced by PBS or cell culture medium), and lightly and repeatedly blowing and resuspending;
(6) after full dissolution, centrifuging at high speed of 10000rpm for 5min, and taking the supernatant to subpackage according to the requirement;
(7) preparing a sample to be detected.
7. RNA extraction and qPCR
Total RNA was extracted from TSCC cell lines using Trizol as per the manufacturer's protocol. And cDNA was synthesized using M-MLV RT according to the manufacturer's instructions, with reverse transcription primers from Ruibo Biotech, Guangzhou. Then, real-time quantitative PCR was performed in two steps to detect the mRNA expression abundance of EDRADD in CAL-27 cells, and the MicroRNAPCR primers were obtained from Ribo Biotech, Guangzhou. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an internal reference gene, and the control and comparative threshold cycle (2- Δ Ct) methods were used to calculate the relative expression levels of mRNA. qPCR was performed on Stratagene Mx3000P using a SYBR Green master mix.
(1) The reaction system (12. mu.L system) was prepared in the following proportions:
①MicroRNA PCR
primers were from Ribo Biotech, Inc., Guangzhou. Primer information:
②RNA PCR
(2) performing Real-Time PCR by a two-step method, and making a melting curve, wherein the procedure is as follows: 30s at 95 ℃; 5s at 95 ℃, 30s at 60 ℃ and 40 times of circulation; 95 ℃ for 15s, 60 ℃ for 30s, 95 ℃ for 15 s.
The results are shown in FIG. 4, indicating that the EDRADD gene is highly expressed in the mRNA level in CAL-27 cells.
8. Lentiviral transfection of cells and measurement of the interference efficiency of EDRADD shRNA by qPCR and Western blot
Using 5'-TTCTCCGAACGTGTCACGT-3' as a negative control (shCtrl), the interference sequence shrrnapscc 3741 was synthesized and lentiviruses were constructed. Lentivirus titers were determined by real-time PCR according to the manufacturer's instructions and were transfected into CAL-27 cells with a lentiviral vector with a multiplicity of infection (MOI) of 20. Seeding cells (2X 10)5Individual cells/mL) were placed in a 6-well plate and incubated, and successfully infected cells were positive for green fluorescent protein and observed under a fluorescent microscope after 72 h.
As shown in FIG. 5A, CAL-27 cells were successfully infected with a lentivirus expressing EDRADD shRNA or the control shrNAPsc 3741. As shown in fig. 5B, the EDARADD shrna (shedaradd) cell line was inhibited in mRNA expression (P <0.01) compared to the negative control cell (shCtrl) with a knockdown efficiency of 60.4%.
9. Western blot
Cells were washed twice with ice-cold PBS and lysed with RIPA buffer. Aliquots of cell lysates containing 30. mu.g of protein were separated on 10% SDS-PAGE gels and transferred to PVDF membrane by electroporation at a constant current of 300mA for 150 min. Membranes were blocked in TBST buffer containing 5% skim milk and incubated with Anti-EDARADD: 1:100, Anti-GAPDH: 1:2000 dilution overnight, then mixed with Anti-rabbitigg: 1:2000, Anti-Mouse IgG: 1:2000 incubation. Bands were visualized using ECL kit in conjunction with X-ray film.
The Western blot result shows that the protein level of EDRADD is remarkably reduced after shRNA mediated knockdown in CAL-27 cells (FIG. 5C), and the target has remarkable knockdown effect on the endogenous expression of the EDRADD gene at the protein level and is an effective target.
10. Celigo cell counting method for detecting cell growth
TSCC cells were digested with 0.25% trypsin to prepare single cell suspensions, and then the cells were counted using a hemocytometer. Mixing cells (2X 10)3Individual cells/well) were seeded onto 96-well plates and incubated at 37 ℃ with 5% CO2The culture was carried out in an incubator for 24 hours. Starting from the next day after plating, the reading of the plate is performed once per day by the Celigo cell counter, the reading of the plate is performed for 5 consecutive days, and the number of cells having green fluorescence is accurately calculated and statistically analyzed by adjusting input parameters set for analysis. Fold cell counts represent cell counts per time point relative to the mean on
The results of Celigo cell counts showed that the proliferation rate of the hyosquamous carcinoma cells was significantly lower in the sheddadd group than in the negative control group, particularly on
11. MTT detection of cell viability
Cells from lentivirus-transfected TSCC cell line (1.5X 10)3Individual cells/well) were seeded into 96-well plates and incubated for 24 hours. Then, 20 μ L of 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl-tetrazolium bromide in PBS was added to each well plate. After 20. mu.l of MTT was incubated at 37 ℃ for 4 hours, the supernatant was discarded, and the precipitate was dissolved in 100. mu.l of dimethyl sulfoxide. The absorbance of each well was measured using a microplate reader at 490nm, and OD490 times represent the OD value at each time point relative to the average value on
MTT results showed that OD490 fold values for shEDARADD group were significantly lower than shCtrl group at 4 and 5 days (fig. 6D and E), with results consistent with Celigo cell counter detection.
12. Detection of apoptosis by Annexin V-APC single staining method
Cells were seeded 72 hours after transfection (2X 10)5Individual cells/mL) onto 6-well plates and incubated to about 85% confluence. The supernatant and adherent cells were harvested, centrifuged, washed with 4 ℃ pre-cooled D-Hanks solution and washed at
The results showed that the apoptosis rate of shEDRADD group cells was significantly higher than that of shCtr group cells (P <0.01, FIG. 7), indicating that EDRADD gene was significantly related to the apoptosis of CAL-27 cells.
13. Cell clonogenic assay
Cells (1.5X 10) were transfected 72 hours later3Cells/well) were seeded on 6-well plates and cultured for 10 days, the medium was changed every 3 days and the cell status was observed. Before the end of the culture, cell clones were photographed using a fluorescence microscope. Cells were fixed with 4% paraformaldehyde for 30min, washed once with Phosphate Buffered Saline (PBS), and then stained with Giemsa. After washing with distilled deionized water and completely drying, cell clones were photographed with a digital camera and then counted.
The results showed (fig. 8) that the number of clones of shedarad tongue squamous carcinoma cells was significantly lower than that of the negative control group (. times.p <0.01), suggesting that EDARADD gene was significantly correlated with the clonogenic capacity of CAL-27 cells.
Sequence listing
<110> Xuzhou city central hospital
<120> shRNA and lentiviral vector for inhibiting human EDRADD gene expression, and construction method and application thereof
<160>10
<170>SIPOSequenceListing 1.0
<210>1
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
gtacttgttc ctcctgctt 19
<210>2
<211>58
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
ccggctgtac ttgttcctcc tgcttctcga gaagcaggag gaacaagtac agtttttg 58
<210>3
<211>58
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
aattcaaaaa ctgtacttgt tcctcctgct tctcgagaag caggaggaac aagtacag 58
<210>4
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
cctatttccc atgattcctt cata 24
<210>5
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
gtaatacggt tatccacgcg 20
<210>6
<211>442
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg 60
atttcttggc tttatatatc ttgtggaaag gacgaaacac cggctgtact tgttcctcct 120
gcttctcgag aagcaggagg aacaagtaca gtttttgaat tctcgacctc gagacaaatg 180
gcagtattca tccacgaatt cggatccatt aggcggccgc gtggataacc gtattaccgc 240
catgcattag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg 300
agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc 360
gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt 420
gacgtcaatg ggtggagtat tt 442
<210>7
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
gaccaaccca aagaggacag 20
<210>8
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
ccagaatgat gaggcaccat 20
<210>9
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
tgacttcaac agcgacaccc a 21
<210>10
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
caccctgttg ctgtagccaa a 21