CN116875558A - Lung cancer cell strain for stably expressing VGLL3 gene, construction method and application thereof - Google Patents
Lung cancer cell strain for stably expressing VGLL3 gene, construction method and application thereof Download PDFInfo
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Abstract
The application relates to the technical field of medical molecular biology, and particularly discloses a lung cancer cell strain for stably expressing VGLL3 gene, a construction method and application thereof. The application prepares a lentivirus solution by chemically synthesizing VGLL3 gene and transferring the VGLL3 gene into 293T cells to complete virus packaging after connecting lentivirus plasmid PCDH-CMV-MCS-EF 1-copGGFP-T2A-Puro by T4 ligase. Subsequently, RPMI1640 medium containing 10% FBS was used at 37℃with 5% CO 2 Culturing human lung cancer cell H460 in saturated humidity incubator, adding slow virus solution to infect for 24 hr when the cells grow to a certain amountAnd screening by puromycin to obtain the lung cancer cell strain stably expressing VGLL3 gene.
Description
Technical Field
The application relates to the technical field of medical molecular biology, in particular to a lung cancer cell strain for stably expressing VGLL3 gene, a construction method and application thereof.
Background
Lung cancer is one of the common malignant tumors worldwide, and its morbidity and mortality rate are high. The main reason for this research analysis is that the disease is difficult to diagnose early and its high metastatic nature. Among pathological forms of lung cancer, non-small cell lung cancer (non-small cell lung cancer, NSCLC) accounts for about 85% of lung cancer, and the disease has early stage concealment, and patients have advanced stage and middle stage at the time of diagnosis, miss the optimal treatment period and have low survival rate. For this reason, early screening for lung cancer is particularly important.
In recent years, molecular diagnosis technology has become a research hotspot for cancer screening due to its noninvasive and rapid diagnosis characteristics. The degenerated family member 3 (VGLL 3) is one of the homologous genes of drosophila degenerated gene products, and a significant increase in gene expression of VGLL3 is also observed in several tumors, and studies have also demonstrated that VGLL3 can affect tumor cell growth, but the molecular mechanism of action between VGLL3 and lung cancer is not known.
Disclosure of Invention
The application provides a lung cancer cell strain for stably expressing VGLL3 gene, a construction method and application thereof, and provides a basic and powerful research tool for further researching the VGLL3 gene in the occurrence, development, treatment and subsequent drug resistance processes of lung cancer.
In a first aspect, the present application provides a lung cancer cell line stably expressing the VGLL3 gene.
The application prepares a lentivirus solution by chemically synthesizing VGLL3 gene and transferring the VGLL3 gene into 293T cells to complete virus packaging after connecting lentivirus plasmid PCDH-CMV-MCS-EF 1-copGGFP-T2A-Puro by T4 ligase. Subsequently, RPMI1640 medium containing 10% FBS was used at 37℃with 5% CO 2 Culturing in saturated humidity incubatorAnd (3) when the human lung cancer cells H460 grow to a certain number, adding a slow virus solution to infect for 24 hours, and screening by puromycin to obtain a lung cancer cell strain stably expressing VGLL3 genes.
The lung cancer cell strain is preserved in 2023 and 4 months and 20 days, the preservation unit is China general microbiological culture Collection center (CGMCC), the preservation address is North Chenxi Lu No.1 and No.3 in the Korean area of Beijing city, and the preservation number is CGMCC No. 45548. The classification is named as target gene over-expression lung cancer cell strain.
The lung cancer cell strain can stably express VGLL3-GFP fusion gene.
In a second aspect, the present application provides the use of the above lung cancer cell line stably expressing the VGLL3 gene.
In a specific embodiment, the use of the above-described lung cancer cell line stably expressing the VGLL3 gene for studying lung cancer occurrence, development and drug resistance processes and mechanisms.
In a specific embodiment, the above-described lung cancer cell line stably expressing the VGLL3 gene is used as a cell model for early screening studies of lung cancer.
In a specific embodiment, the use of a lung cancer cell line stably expressing the VGLL3 gene as described above for screening or predicting molecular markers for early screening of lung cancer.
In a specific embodiment, the use of a lung cancer cell line stably expressing the VGLL3 gene as described above for the preparation of a medicament for preventing or treating early stage lung cancer.
In a specific embodiment, the use of a lung cancer cell line stably expressing the VGLL3 gene as described above for preparing a kit for early lung cancer risk assessment detection.
In a third aspect, the present application provides a method for constructing the above lung cancer cell line stably expressing the VGLL3 gene.
Chemically synthesizing a sequence fragment of the VGLL3 gene, cloning the sequence fragment onto a vector PCDH, and transferring the sequence fragment into 293T cells to prepare a lentivirus solution; and (3) infecting the H460 cells for 24 hours by using the slow virus solution, and screening by using puromycin to obtain a lung cancer cell strain stably expressing the VGLL3 gene.
In a specific embodiment, the multiplicity of infection (MOI) with optimal infection effect is 10 when the H460 cells are infected with the lentiviral solution.
In a specific embodiment, the infection effect is 30% when the H460 cells are infected with the lentiviral solution for 24H.
In a specific embodiment, when the lentiviral solution is used to infect the H460 cells for 24 hours, the target gene VGLL3 in the lung cancer cell line stably expressing the VGLL3 gene is obtained in an amount of about 5 times the expression level of the control group.
In a fourth aspect, the present application also provides a primer set for amplifying the VGLL3 gene in the lung cancer cell strain.
Specifically, the nucleotide sequence of primer F is shown as SEQ ID NO. 3; the nucleotide sequence of primer R is shown as SEQ ID NO. 4.
In summary, the application has the following beneficial effects:
the application utilizes PCDH-CMV-MCS-EF 1-copGGFP-T2A-Puro vector to infect H460 cells through slow virus infection technology, and establishes lung cancer cell lines which stably express VGLL3 genes. The cell is detected by using the techniques of cell fluorescence and fluorescence quantitative PCR to prove that the cell strain is successfully constructed. The lung cancer cell strain constructed by the application can stably express the VGLL3 gene, and provides a basic and powerful research tool for further researching the VGLL3 gene in the occurrence, development, treatment and subsequent drug resistance processes of lung cancer.
Drawings
FIG. 1 is an identification chart of recombinant plasmids after double cleavage.
FIG. 2 is a block diagram of the lentiviral master plasmid PCDH-CMV-MCS-EF 1-copGGFP-T2A-Puro.
FIG. 3 is a graph showing the results of a cell infection experiment (wherein FIG. 3A is a cell map under normal light, and FIG. 3B is a cell fluorescence map after the same field of view of the cell map under normal light is irradiated with a laser having a wavelength of 450 to 490 nm).
Fig. 4 is a graph of the results of a fluorescent quantitative PCR reaction (relative mRNA expression level in ordinate; "indicates p < 0.0001).
Description of the embodiments
The application prepares a lentivirus solution by chemically synthesizing VGLL3 gene and transferring the VGLL3 gene into 293T cells to complete virus packaging after connecting lentivirus plasmid PCDH-CMV-MCS-EF 1-copGGFP-T2A-Puro by T4 ligase. Subsequently, RPMI1640 medium containing 10% FBS was used at 37℃with 5% CO 2 Culturing human lung cancer cells H460 in a saturated humidity incubator, adding a slow virus solution to infect for 24 hours when the cells grow to a certain quantity, and screening by puromycin to obtain a lung cancer cell strain stably expressing VGLL3 genes.
The lung cancer cell strain is preserved in 2023 and 4 months and 20 days, the preservation unit is China general microbiological culture Collection center (CGMCC), the preservation address is North Chenxi Lu No.1 and No.3 in the Korean area of Beijing city, and the preservation number is CGMCC No. 45548. The classification is named as target gene over-expression lung cancer cell strain.
In order to make the objects, technical solutions and advantages of the present application more apparent, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. All other embodiments, which can be made by a person skilled in the art without making any inventive effort, are intended to be within the scope of the present application.
The present application will be described in further detail with reference to examples and drawings.
Examples
This example provides a lung cancer cell line stably expressing the VGLL3 gene.
The construction method of the lung cancer cell strain specifically comprises the following steps:
(1) Synthesis of VGLL3 sequence fragments
In the database of national center for biological information gene (NCBI), the full-length sequence of VGLL3 gene was obtained as the target gene. In addition, restriction enzyme cleavage sites with NheI and NotI were designed at the 5-and 3-ends of the full-length sequence of the gene, respectively, and the gene synthesis was carried out by the company Shanghai, inc. of Biotechnology. The target gene was mounted on a plasmid (pUC 57) to obtain a recombinant plasmid.
The recombinant plasmid is transformed by using escherichia coli (competent cells), and the recombinant plasmid is plated and cultured on a solid medium (formula: 10g/L of tryptone, 5g/L of yeast powder, 10g/L of sodium chloride, 15g/L of agar powder and 1% of Ampicillin) containing Ampicillin to obtain a strain containing a target gene. After overnight culture, single colony is selected and inoculated into a liquid culture medium (formula: 10g/L of tryptone, 5g/L of yeast powder and 10g/L of sodium chloride) for culture, recombinant plasmid is extracted after overnight culture, restriction enzyme NheI and NotI are used for double enzyme digestion, target gene fragments are intercepted, and gel is cut and recovered.
The identification chart of the recombinant plasmid after double enzyme digestion is shown in figure 1. As shown in FIG. 1, the size of the target gene fragment is 963bp, and the target gene fragment is a nucleotide sequence shown as SEQ ID NO.1 through sequencing detection.
(2) Cloning of the Gene of interest into the vector PCDH
The target gene fragment is connected with a lentiviral main plasmid PCDH-CMV-MCS-EF 1-copGGFP-T2A-Puro (namely, the nucleotide sequence shown as SEQ ID NO.2 and the structure diagram shown as figure 2) by T4 ligase to obtain the vector with the connected structure.
Adding 10 μl of the carrier with the connection completion into 50 μl of competent cells (TOP 10) suspension on ice, standing on ice for 30min after mixing, activating competent cells in a water bath at 42 ℃ for 90s after transformation, plating on solid medium containing ampicillin for culturing to screen strains containing target genes, selecting single colony after overnight culture for inoculating to liquid medium for culturing, extracting plasmids thereof after overnight culture, and obtaining the carrier with target genes.
(3) Preparation of lentiviral solution with target Gene and cell infection experiment
To construct a cell over-expression gene model, 293T cells were first used to prepare lentiviral solutions with the target genes.
The method comprises the following steps: 293T cells were plated on a culture plate having a diameter of 10cm, and after the cells grew to about 8 confluence, vectors carrying the target gene and auxiliary vectors including envelope plasmid (envelop plasmid) and packaging plasmid (packaging plasmid) were co-transfected with lipid nanoparticle (Lipofectamine 3000) to obtain a cell culture solution. After 48h, fluorescence was observed with a fluorescence inversion microscope, and the transfection efficiency was analyzed.
Cell culture broth transfected for 48h was collected and 0.2. Mu.l nuclease was added for standing, reacted at 37℃for 30min, centrifuged at 4℃for 3,000 Xg to remove cells and protein fragments, and the supernatant was transferred to a new centrifuge tube to obtain a supernatant containing viruses. Preparing 15%, 25%, 40% and 60% iodixanol injection and a special ultra-high speed centrifuge tube, respectively adding iodixanol injection with different concentrations into the bottom of the ultra-high speed centrifuge tube by using a needle head (the flow rate is needed to be noted when adding, the mixing is avoided, the generation of bubbles is avoided), carefully adding the supernatant containing viruses slowly into the uppermost layers of the iodixanol injection with different gradients, balancing by using a microbalance, sealing the centrifuge tube by using a sealing tool, and centrifuging for 1h at 18 ℃ and 350,000Xg. After centrifugation, the cap of the tube was pierced with a needle to aspirate the virus solution at the layering site of the lentiviral particles, the aspirated virus solution was placed on ice, the virus was purified using an ultrafiltration tube, and PBS-MK (component: 137mM NaCl,5.2mM KCl,8 mM Na) 2 HPO 4 ，2mM KH 2 PO 4 , 1 mM MgCl 2 ) Recovering to obtain slow virus solution, and storing at-80 ℃.
Cell infection experiments: RPMI1640 medium containing 10% FBS was used at 37℃with 5% CO 2 Culturing H460 cells in saturated humidity incubator, spreading H460 cells into 24-well plate when cells grow to a certain amount, adding slow virus solution into each well with MOI gradient of 0, 10, 20, 40, 80, respectively, performing fluorescent cell count after 24 hr to find out infection complex (MOI) with optimal infection effect as 10, re-infecting with the virus concentration in 6cm diameter culture plate for 24 hr, screening with complete culture medium containing puromycin of 6 μg/ml for 24 hr, washing with PBS twice, and placing in 5% CO 2 And growing in an incubator to obtain the screened cells.
And carrying out cell infection experiments on the screened cells. The results of the cell infection experiment are shown in FIG. 3. Fig. 3A is a cell map under normal light, and fig. 3B is a cell fluorescence map after irradiation of the same field of view of the cell map under normal light with laser light having a wavelength of 450 to 490 nm.
As can be seen from FIG. 3, the cells in FIG. 3B emit green fluorescence, which represents that the cell line constructed according to the present application can stably express the fusion protein with the target gene VGLL3 and the green fluorescent protein.
(4) Detection of expression of target gene by fluorescent quantitative PCR method
And taking out the screened cells, sucking the complete culture medium, and dissolving the cells by using a Lysis Buffer containing 1% of 2-mercaptoethanol, wherein the obtained cell lysate is used for extracting total RNA according to the standard operation flow of Biospin Total RNA Extraction Kit (Biospin total RNA extraction kit, bori technology Co., hangzhou).
Removing gDNA: mu.g of total RNA was mixed with 2. Mu.l of 5 XgDNA Buffer and the volume was homogenized with ddH 2 O was added to 10. Mu.l, and the mixture was incubated at 42℃for 3 minutes to obtain a reaction solution from which gDNA was removed, and the reaction solution was placed on ice.
Reverse transcription: mu.l of 10 XKing RT Buffer, 1. Mu. l FastKing RT Enzyme Mix, 2. Mu.l of FQ-RQ Primer Mix were mixed and the volume was mixed with ddH 2 Supplementing O to 10ul, mixing with the reaction solution after gDNA removal, incubating at 42 ℃ for 15min, incubating at 95 ℃ for 3min, and placing on ice to obtain cDNA product.
Fluorescent quantitative PCR reaction: according to the instruction manual (instruction manual of qPCR premix reagent, bai Meng medical science, product number: BM 60304S), 10. Mu.l LUNA qPCR Mix, 1. Mu.l cDNA, 0.5. Mu.l primer F, 0.5. Mu.l primer R, volume ddH 2 O was fed to 20ul, the reaction conditions were pre-denatured at 95℃for 5min, denatured at 95℃for 20s in cycles, annealed at 60℃for 15s, 40 cycles were set, and fluorescence signals were collected at the end of extension of each cycle.
The results of the fluorescent quantitative PCR reaction are shown in FIG. 4. From FIG. 4, it is clear that the target gene VGLL3 is expressed in an amount 5 times that of the control group, and that the obtained lung cancer cell line can stably express the VGLL3 gene, indicating that the construction of the desired lung cancer cell line was successful.
The lung cancer cell strain is preserved in 2023 and 4 months for 20 days, wherein the preservation unit is China general microbiological culture Collection center (CGMCC), the preservation address is North Chengxi Lu No.1 and No.3 in the Korean region of Beijing city, and the preservation number is CGMCC No. 45548. The classification is named as target gene over-expression lung cancer cell strain.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and are not limiting; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present application.
Claims (10)
1. A lung cancer cell strain for stably expressing VGLL3 gene is characterized in that the preservation number of the lung cancer cell strain is CGMCC NO. 45548.
2. The lung cancer cell line of claim 1, wherein the lung cancer cell line stably expresses a VGLL3-GFP fusion gene.
3. Use of the lung cancer cell line of claim 1 for stably expressing VGLL3 gene for studying the occurrence, development and drug resistance processes and mechanisms of lung cancer.
4. Use of the lung cancer cell line stably expressing the VGLL3 gene of claim 1 as a cell model for early screening studies of lung cancer.
5. Use of a lung cancer cell line stably expressing the VGLL3 gene of claim 1 in screening or predicting molecular markers for early screening of lung cancer.
6. Use of a lung cancer cell line stably expressing the VGLL3 gene according to claim 1 in the manufacture of a medicament for the prevention or treatment of early stage lung cancer.
7. Use of the lung cancer cell line stably expressing VGLL3 gene of claim 1 in the preparation of a kit for early lung cancer risk assessment detection.
8. A method of constructing a lung cancer cell line stably expressing the VGLL3 gene according to claim 1, wherein:
chemically synthesizing a sequence fragment of the VGLL3 gene, cloning the sequence fragment onto a vector PCDH, and transferring the sequence fragment into 293T cells to prepare a lentivirus solution; and (3) infecting the H460 cells for 24 hours by using the slow virus solution, and screening by using puromycin to obtain a lung cancer cell strain stably expressing the VGLL3 gene.
9. The method according to claim 8, wherein when the H460 cells are infected with the lentiviral solution, the optimal multiplicity of infection (MOI) is 10;
preferably, when the H460 cells are infected with the lentiviral solution for 24 hours, the infection effect is 30%;
preferably, when the H460 cells are infected with the lentiviral solution for 24 hours, the target gene VGLL3 in the lung cancer cell line stably expressing the VGLL3 gene is obtained in an amount of about 5 times the expression level of the control group.
10. A primer group for amplifying the VGLL3 gene in the lung cancer cell line of claim 1, wherein the nucleotide sequence of primer F is shown in SEQ ID No. 3; the nucleotide sequence of primer R is shown as SEQ ID NO. 4.
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