CN116731976A - Construction method and application of lncRNA CPEB1-AS1 gene long fragment knockout cell strain - Google Patents
Construction method and application of lncRNA CPEB1-AS1 gene long fragment knockout cell strain Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a construction method and application of a cell strain knocked out by a long fragment of a human lncRNA CPEB1-AS1 gene. According to the invention, a CRISPR/Cas9 technology is adopted for the first time, the special sequence long fragment knockout of the gastric cancer cell lncRNA CPEB1-AS1 gene is carried out, the gastric cancer cell strain with the lncRNA CPEB1-AS1 gene knocked out is established, and an experimental material is provided for further elucidating the mechanism research of the lncRNA CPEB1-AS1 participating in gastric cancer.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a construction method and application of a lncRNA CPEB1-AS1 gene long fragment knockout cell strain.
Background
Long non-coding RNA (lncRNA) CPEB1-AS1 is lncRNA existing in the antisense strand of CPEB1 gene, only the sequence information of which is reported at present, and the research on the functions of which is not yet reported.
The CRISPR/Cas9 technology is a third generation gene editing technology, and the principle is that when sgRNA and Cas9 protein exist in a cell at the same time, the Cas9 protein can cut double-stranded DNA combined with the sgRNA, induce non-homologous recombination repair in the cell, cause insertion or deletion of a plurality of bases in the repair process, and lead the gene to generate frame shift mutation so as to generate nonfunctional protein. However, since lncRNA is not translated into protein and functions at RNA level, single-target cleavage using CRISPR/Cas9 technology, resulting in insertion or deletion of several bases of DNA sequence, may not result in functional changes of lncRNA.
Functional knockouts of lncRNA should be long fragment knockouts at the DNA level or interfering at the RNA level. The long fragment knockout of the gene is commonly used for double-target cutting by using a CRISPR/Cas9 technology at present, but the CRISPR/Cas9 technology has the problem of different cutting efficiency according to different target sequences, and has complex operation and low success rate.
Disclosure of Invention
Aiming at the problems, the invention provides a CRISPR/Cas9 multi-target cutting method for efficiently constructing a cell strain with the lncRNA CPEB1-AS1 gene long fragment knocked out.
The invention aims at providing a sgRNA sequence combination for knocking out CPEB1-AS1 gene long fragments.
The invention aims at providing a method for efficiently constructing a CPEB1-AS1 gene long fragment knockout cell strain based on a CRISPR-Cas9 technology.
The third object of the present invention is to provide the use of the cell line.
According to the invention, firstly, gastric cancer lncRNA chip data in a GEO database are analyzed, and the condition that the expression of lncRNA CPEB1-AS1 is up-regulated is found. Therefore, the invention preliminarily determines the lncRNA CPEB1-AS1 AS a molecular marker involved in the development of gastric cancer, knocks out the lncRNA CPEB1-AS1 and inspects the mechanism of inhibiting the occurrence and the development of gastric cancer.
Meanwhile, the inventor selects a unique sequence region of the gene according to the sequence position information of the lncRNA CPEB1-AS1 gene, designs multiple pairs of sgRNAs to perform multi-target knockout by using a CRISPR/Cas9 technology, improves the gene editing efficiency, and realizes the long-fragment knockout of the lncRNA CPEB1-AS1 gene.
Specifically, the invention designs sgRNA by using CRISPR/Cas9 technology, cuts CPEB1-AS1 genes in gastric cancer cells HGC-27 cells by multiple targets, completely cuts 2 and 3 exons of the genes, thereby realizing the loss of the function of the lncRNA CPEB1-AS 1.
According to the specific embodiment of the invention, the cell strain for knocking out the human CPEB1-AS1 gene is a gastric cancer cell strain HGC-27.
According to the specific embodiment of the invention, the cell strain for knocking out the human CPEB1-AS1 gene is obtained by transfection of a CRISPR/Cas9 vector targeting the CPEB1-AS1 gene.
Cell lines from which the human CPEB1-AS1 gene was knocked out according to specific embodiments of the present invention, the sequence of the sgRNA targeting the CPEB1-AS1 gene is shown in Table 1 below.
The invention provides application of the cell strain, which is used for researching the influence of CPEB1-AS1 gene long fragment knockout on cell behaviors such AS gastric cancer cell proliferation, migration and the like.
The construction method of the cell strain according to the specific embodiment of the invention comprises the following steps:
(1) The gRNA sequence of the CPEB1-AS1 gene is designed and targeted.
Designing a target point in a unique sequence area of the gene according to the position information of the CPEB1-AS1 gene sequence in the genome, wherein,
2 targets were designed for the intron 1 region (CPEB 1-AS1-sgRNA-1; CPEB1-AS 1-sgRNA-2);
the intron 3 region was designed for two targets (CPEB 1-AS1-sgRNA-3; CPEB1-AS 1-sgRNA-4).
(2) Construction of recombinant expression plasmids.
Annealing the sgRNA sequence designed in the step (1) and then connecting the annealed sgRNA sequence with a vector to construct 4 expression vectors containing the CPEB1-AS1 gene sgRNA sequence and the Cas9 gene;
(3) Performing cotransfection of a gastric cancer cell strain HGC-27 by using the vector obtained in the step (2), and screening by puromycin to obtain a CPEB1-AS1 gene knocked-out mixed cell clone; the method comprises the steps of carrying out a first treatment on the surface of the
(4) Cloning the mixed cells obtained in the step (3), and obtaining the CPEB1-AS1 gene long fragment knockout cell strain through monoclonal plating and PCR verification.
According to the construction method of the cell strain of the specific embodiment of the invention, in the step (1), a target sequence on the human CPEB1-AS1 gene is selected, wherein the sequence of the gRNA is shown AS SEQ ID NO.1-SEQ ID NO. 8.
In step (2), the expression vector is a pLenti-CRISPR-V2 vector (adedge: 52961).
In the step (3), three carriers are 1:1:1, selecting 2 targets upstream of the exon 2 and 1 target downstream of the exon 3 for combination, namely, the sgRNA1/2/3 or the sgRNA1/2/4 combination;
in the step (4), the monoclonal plating mode is a double dilution method, and 96-well plate plating is carried out to obtain the monoclonal.
The invention has the beneficial effects that:
according to the invention, by analyzing GEO data sets GSE95667 and GSE53137, the expression of lncRNA CPEB1-AS1 in gastric cancer tissues is obviously increased, and the average logFC is 3.3. The use of real-time PCR method further confirms that the expression of lncRNA CPEB1-AS1 in gastric cancer tissue is significantly higher than that in paracancerous tissue. Therefore, the invention judges that the lncRNA CPEB1-AS1 participates in the occurrence and development of gastric cancer. Therefore, researching the effect of the lncRNA CPEB1-AS1 in gastric cancer is expected to discover a new pathogenic mechanism of gastric cancer, and provides an important basis for researching the molecular mechanism of gastric cancer and discovering a new targeted treatment site.
The sgRNA provided by the invention is designed according to the position characteristics of the CPEB1-AS1 gene, so that the long fragment knockout of the special sequence of the CPEB1-AS1 gene is realized, and the knockout fragment does not influence the expression of the overlapped genes.
The invention uses CRISPR-Cas9 technology to knock out multiple targets, uses 3 knockout vectors to cotransfect, the proportion of the vectors is 1:1:1, the transfection efficiency is ensured, and meanwhile, the long fragment knockout efficiency is improved. When the monoclonal antibody is plated, a double dilution method is used for dilution plating, the operation is simple, and a large number of monoclonal antibodies can be obtained by one 96-well plate.
The invention establishes a gastric cancer cell strain with the CPEB1-AS1 gene knocked out by a long fragment, and provides a research platform for further elucidating the mechanism research of the CPEB1-AS1 participating in gastric cancer.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows an electrophoresis diagram of a polyclonal mixed cell PCR product, wherein no obvious band is found in the PCR band dispersion of the combined knocked-out cells of the sgRNA1/2/3 of lanes 2, the PCR product of the combined knocked-out cells of the sgRNA1/2/4 of lanes 3 contains smaller bands (about 1025bp and 1319 bp), and the PCR product of the control cells of lanes 4 is 10745bp;
FIG. 2 shows an electrophoresis diagram of PCR products of a CPEB1-AS1 gene long fragment knockout cell strain, wherein the left band is 1025bp of the PCR products after the CPEB1-AS1 gene knockout, and the right band is 10745bp of the PCR products of control cells;
FIG. 3 shows a sequence alignment of the PCR products of the CPEB1-AS1 gene of the control cells and the PCR products of the knockout cell line;
FIG. 4 shows the effect of CPEB1-AS1 gene long fragment knockout on cell proliferation;
FIG. 5 shows changes in cellular gene expression profile following CPEB1-AS1 gene knockout;
FIG. 6 shows the case of the differential gene after knockout of the CPEB1-AS1 gene long fragment.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.
EXAMPLE 1LncRNA CPEB1-AS1 Gene knockout gastric cancer cell line
1. Construction of knockout vectors
1) Selection of target sequences
And screening target sequences according to the genome sequence of the human CPEB1-AS1 gene, and finally selecting two targets in the regions of the intron 1 and the intron 3 of the CPEB1-AS1 gene, wherein the targets can effectively avoid overlapping parts of the CPEB1-AS1 gene and the CPEB1 gene on the complementary strand and overlapping with the AP3B2 gene.
2) Design two pairs of sgRNAs targeting CPEB1-AS1 genes
The sgRNA-1, the sgRNA-2 is located in the region of the gene intron 1, and the sgRNA-3, the sgRNA-4 is located in the region of the gene intron 3.
sgRNA-1F:5'-CACCGTAAGAGTAGAAGTCCACGAG-3' ；
sgRNA-1R:5'-AAACCTCGTGGACTTCTACTCTTAC-3';
sgRNA-2F:5'-CACCGGGCCCAAGGCACACCCAAAA-3'；
sgRNA-2R:5'-AAACTTTTGGGTGTGCCTTGGGCCC-3'；
sgRNA-3F:5'-CACCGCAAGGACTTGCCCACACCTG-3'；
sgRNA-3R:5'-AAACCAGGTGTGGGCAAGTCCTTGC-3'；
sgRNA-4F:5'-CACCGTGGCTGGGGTACTGACCCAC-3';
sgRNA-4R:5'-AAACGTGGGTCAGTACCCCAGCCAC-3'。
Exon 2 and exon 3, which do not overlap with other genes, were excised using the sgrnas described above.
3) The sgrnas were dissolved to 100uM with water. 75pmol of sgRNA-F and sgRNA-R were phosphorylated using T4 PNK enzyme and ATP (25 nmol) at 37℃for 30min;95 ℃ for 5min. And then the temperature is reduced to 25 ℃ at the speed of 5 ℃/min for annealing, and double-chain sgRNA is obtained.
4) The lentiCRISPR v2 plasmid (addgene, 52961) was digested with BsmBI-v2 enzyme and the large fragment linear vector was recovered by excision.
5) The double-stranded sgRNA was ligated with the linear vector using T4 ligase (Takara, 6022) in an amount of 2pmol for the sgRNA and 0.03pmol for the linear vector.
6) Mu.l of ligation product was added to 50. Mu.l of DH 5. Alpha. Competent cells thawed on ice, transformed by heat shock, and left on ice for 2min after 45 seconds in a water bath at 42 ℃.
To the transformation product was added 500. Mu.l of SOC medium and shaken at 200rpm at 37℃for 1h. Mu.l of the bacterial liquid was streaked on LB agar plates (containing ampicillin) and incubated overnight at 37 ℃.
7) The following day the monoclonal was picked into round bottom centrifuge tubes containing 5ml of ampicillin LB medium and shaken overnight at 37℃at 200 rpm.
8) The bacterial cells were collected, plasmid extraction was performed using a plasmid extraction kit (a biotechnological organism), and the plasmids obtained by the extraction were sequenced and identified, and pRNA-U6Forward was used as a sequencing primer, TACGATACAAGGCTGTTAGAGAG.
Positive clones inserted into the target sequence are found through sequence alignment, and knock-out vectors, namely, the lentiCRISPR v2-sgRNA1, the lentiCRISPR v2-sgRNA2, the lentiCRISPR v2-sgRNA3 and the lentiCRISPR v2-sgRNA4 are successfully constructed.
2. Transfecting cells
1) HGC-27 cells were digested one day in advance, plated at 20% density into 6-well plates, and the cell density reached 40-50% on the day of transfection.
2) Transfection day: (1) Cells were prepared, cell densities were observed at 40-50%, medium was discarded, and serum-free 1640 medium was added. (2) The transfection complex was prepared by adding 3000. Mu.l lipo to 125. Mu.l Opti-MEM medium, mixing well, adding 3000. Mu.l P and 850ng of each of the three knockdown vectors (sgRNA 1/2/3 or sgRNA 1/2/4) to 125. Mu.l Opti-MEM medium, mixing well. 125. Mu.l lipo3000 was mixed with 125. Mu.l vector DNA and incubated for 15min at room temperature. (3) Transfection, the transfection complex is added drop-wise to the cell culture medium. (4) Changing the liquid, changing the liquid 6h after transfection, and continuously culturing by using a serum-containing culture medium.
3) Puromycin screening: transfected cells were cultured until full growth on day three, post-digestion 1:2 plates were plated to 2 wells/six well plate, and one well of cells was screened the next day using 1.5ug/ml puromycin. The selection of untransfected cells was performed simultaneously as a control, and when the control cells all died, the selection was stopped and the culture expansion was continued using complete medium.
3. Polyclonal validation
1) Collecting cells: the screened cells are continuously cultured in a six-hole plate until the cells are full, the cells are digested, half of the cells are continuously cultured, and half of the cells are used for DNA extraction.
2) Extracting DNA: after cell collection, the cells were centrifuged, washed once with PBS, 200. Mu.l DNAiso was added, and the mixture was blown and mixed well for 5 minutes at room temperature. 10000g of supernatant were transferred to a fresh tube at 4℃for 10Min. Adding 1/2 volume (100 μl) of absolute ethanol, and mixing for 1-3min to obtain cloud DNA. The DNA was wound into a new tube with a gun head. Washing was performed using 1ml of 75% ethanol, 12000g of 4℃for 5min, the supernatant was discarded, and the DNA was dried. The solubilization was performed using 40 μl EB. The DNA concentration was measured using a multifunctional reader.
3) And (2) PCR: PCR was designed to verify that the upstream primer was designed upstream of the sgRNA1 in the intron 1 region and the downstream primer was designed downstream of the sgRNA4 in the intron 3 region. The primer sequences were as follows:
verify-primer-F:5'-GCGCACTCACTTTAGACCCT-3';
verify-primer-R:5'-GTGGAAGAGACATCTGGGCTG-3'。
using the knocked-out cell DNA and the negative cell DNA as templates, amplification was performed using LA taq enzyme (Takara, RR02 MA), verify-primer-F, verify-primer-R, and PCR procedures were 94℃1min,98℃10s,62℃30s,72℃15min, (2-4) 30 cycles,72℃10min.
The result of agarose gel electrophoresis detection of the PCR product is shown in figure 1, lane 4 is a negative control cell, a 10745bp band is obtained by PCR, no obvious band is found in the PCR band dispersion of the sgRNA1/2/3 combined knockdown cell of lane 2, the PCR product of the sgRNA1/2/4 combined knockdown cell of lane 3 contains smaller bands (about 1025bp and 1319 bp), and the cells are proved to contain the CPEB1-AS1 gene long fragment knockdown cells.
4. Monoclonal screening
1) And (5) paving. 100 μl of complete medium was added to 96-well plates, 100 μl of medium containing 100 sgRNA1/2/4 of the combined knocked-out cells was added to each of the eight wells in column 1, 100 μl was aspirated after air-beating and mixing into the second well, dilution to the last column was continued by fold ratio, and 100 μl of medium was aspirated for discarding.
2) Counting. Cells were observed 2-3 days after plating and labeled on wells containing the monoclonal. Culturing is continued until the tenth day, and liquid is changed every 2-3 days.
3) And (5) passage. Cells in 96-well plates grew to day ten and were digested and passaged into 24-well plates for continued culture.
4) And (5) verification. After the cells in the 24-well plate are full, a small amount of cells are remained at the bottom of the hole after digestion for continuous culture, and the rest cells are subjected to DNA extraction, and PCR verification is carried out by using CPEB1-AS1 gene primers, wherein the verification method is AS described above. Clones with the PCR product electrophoresis band of single 1025bp band were selected as candidate monoclonal cell lines (see FIG. 2).
Further carrying out sequencing verification on PCR products of the gene knockout monoclonal cell strain, and after sequence comparison, finding that the upper and lower sections of the PCR product sequence (Query) of the CPEB1-AS1 gene of the knockout cell strain and the PCR product sequence (Sbjct) of the CPEB1-AS1 gene of the control cell can be completely compared, and 9720bp deletion exists in the middle (see figure 3), so AS to prove that the monoclonal cell strain is a monoclonal cell strain with the CPEB1-AS1 gene long fragment knocked out.
Example 2 verification of CPEB1-AS1 Gene knockout monoclonal cell lines
In this example, a verification test was performed on the function of the cell line obtained in example 1.
1. CPEB1-AS1 gene long fragment knockout results in slower cell proliferation
After a baseline is detected by adding 50 mu l of cell culture medium into each hole of an E-plate (Agilent), inoculating control cells (transfected control plasmid) and HGC-27 cell strain knocking out CPEB1-AS1 gene long fragments into the E-plate holes according to 30% of cell density, inoculating four holes of two groups of cells respectively, placing the cells into a multifunctional real-time label-free cell analyzer (Agilent) for detection, setting the detection cell index once every 30 minutes, and reacting the cell proliferation condition according to the change of the cell index.
The results are shown in FIG. 4, and the results show that the proliferation of cells is obviously slowed down after the CPEB1-AS1 gene is knocked out.
2. CPEB1-AS1 gene long fragment knockout resulting in cell gene expression changes
RNA was extracted from control cells and cells from which long fragments of CPEB1-AS1 gene were knocked out, and then transcriptome sequencing was performed.
As a result, AS shown in FIG. 5, the gene expression profile of the cell was changed after the CPEB1-AS1 gene was knocked out, and some genes were up-regulated and some genes were down-regulated.
The differential genes are mainly enriched on tumor-associated signal pathways, such as TRKA activation, WNT signal pathway, FGFR2b signal pathway, etc., and the results are shown in fig. 6.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (7)
1. A cell strain for knocking out the human lncRNA CPEB1-AS1 gene, which is characterized in that: the cell strain is a gastric cancer cell strain HGC-27.
2. The cell line for knocking out the human lncRNA CPEB1-AS1 gene according to claim 1, wherein: the cell strain is obtained by cotransfection of three targeting lncRNA CPEB1-AS1 genes CRISPR/Cas9 vectors; the ratio of co-transfection of three of the vectors was 1:1:1.
3. the cell line knocked out of human lncRNA CPEB1-AS1 gene according to claim 2, wherein: the sequence of the gRNA of the targeting lncRNA CPEB1-AS1 gene is shown AS SEQ ID NO. 1-8.
4. Use of a cell line according to any one of claims 1-3.
5. The method for constructing a cell line according to claim 1, comprising the steps of:
(1) Constructing a vector for knocking out the human lncRNA CPEB1-AS1 gene;
(2) And (3) transfecting a gastric cancer cell strain HGC-27 by using the vector obtained in the step (1) to obtain a cell strain for knocking out the human lncRNA CPEB1-AS1 gene.
6. The method of constructing a cell line according to claim 5, wherein in the step (1), a target sequence on the human CPEB1-AS1 gene is selected, wherein the sequence of the gRNA is shown in SEQ ID NO. 1-8.
7. The method of constructing a cell line according to claim 6, wherein the step (1) is to design sgrnas targeting the CPEB1-AS1 gene according to a target sequence, and the nucleotide sequences thereof are AS follows:
SEQ ID NO.1: 5'-CACCGTAAGAGTAGAAGTCCACGAG-3' ；
SEQ ID NO.2: 5'-AAACCTCGTGGACTTCTACTCTTAC-3'；
SEQ ID NO.3: 5'-CACCGGGCCCAAGGCACACCCAAAA-3'；
SEQ ID NO.4: 5'-AAACTTTTGGGTGTGCCTTGGGCCC-3'；
SEQ ID NO.5: 5'-CACCGCAAGGACTTGCCCACACCTG-3' ；
SEQ ID NO.6: 5'-AAACCAGGTGTGGGCAAGTCCTTGC-3' ；
SEQ ID NO.7: 5'-CACCGTGGCTGGGGTACTGACCCAC-3' ；
SEQ ID NO.8: 5'-AAACGTGGGTCAGTACCCCAGCCAC-3' 。
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