CN116676267A - Non-tumorigenic MDCK gene engineering cell strain and preparation method and application thereof - Google Patents
Non-tumorigenic MDCK gene engineering cell strain and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a non-tumorigenic MDCK gene engineering cell strain, which is a miR-2779-x over-expression stable-transfer MDCK cell strain. miR-2779-x can be involved in regulating the MDCK cell tumorigenic phenotype by inhibiting Bak1 expression. The miR-2779-x over-expression stable transgenic cell strain is constructed, and a new idea is provided for establishing a low-tumorigenicity MDCK gene engineering cell strain.
Description
Technical Field
The invention relates to a non-tumorigenic MDCK genetically engineered cell strain, a preparation method and application thereof.
Background
Influenza virus is an acute respiratory infectious disease virus which has a high transmission speed and is very easy to change, and influenza vaccination is the best method for preventing influenza at present. MDCK cells have higher sensitivity to influenza viruses and shorter doubling time, and become the first cell strain for related researches on influenza viruses. However, the prior studies show that the injection of MDCK living cells into BALB/c nude mice can form tumors, namely MDCK cells have tumorigenicity, so that the application of MDCK cells is limited to a certain extent. In recent years, miRNA is discovered to be used as non-coding RNA, interacts with mRNA and is involved in the occurrence and development of various diseases such as tumors, but whether miRNA is involved in the regulation of the MDCK cell tumorigenic phenotype is not reported. Therefore, the regulatory effect of miRNA on MDCK cell tumorigenicity and the molecular mechanism thereof are explored, and the targeted intervention is beneficial to improving the application safety of MDCK cell matrix in producing influenza vaccine.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a non-tumorigenic MDCK gene engineering cell strain, and a preparation method and application thereof.
The invention provides a non-tumorigenic MDCK gene engineering cell strain, which is a miR-2779-x over-expression stable-transfer MDCK cell strain.
Preferably, the nucleotide sequence of miR-2779-x is as follows: UCCGGCUCGAAGGACCAU.
The invention also provides a construction method of the non-tumorigenic MDCK gene engineering cell strain, a miR-2779-x over-expression slow virus vector is designed and constructed, MDCK tumorigenic cells are transfected, and the miR-2779-x over-expression stable transgenic cell strain is obtained after puromycin screening.
Preferably, the miR-2779-x over-expression lentiviral vector is designed and constructed specifically by the following steps: and connecting the miR-2779-x and the GV369 vector through an AgeI/NheI enzyme cleavage site, transforming the connection product into an escherichia coli DH5 alpha competent cell, selecting a positive clone transformant for plasmid extraction to obtain a miR-2779 over-expression plasmid, co-transfecting the miR-2779 over-expression plasmid and a lentivirus packaging auxiliary plasmid into 293T cells, and collecting cell supernatant for purification to obtain the miR-2779-x over-expression lentivirus vector.
Preferably, the method comprisesWhen MDCK tumorigenic cells are transfected, the transfection MOI value is 100, and the temperature is 37 ℃ and the concentration of CO is 5% 2 Culturing under the condition.
The invention also provides application of the non-tumorigenic MDCK genetically engineered cell strain in influenza virus proliferation culture or preparation of influenza virus vaccines.
Preferably, the non-tumorigenic MDCK genetically engineered cell line is used as a cell matrix for influenza vaccine production.
Preferably, after the MDCK genetically engineered cell line is inoculated into an animal, the MDCK genetically engineered cell line does not form a tumor in the inoculated animal.
According to the invention, firstly, miRNA high-throughput sequencing and bioinformatics analysis are carried out on the introduced MDCK cell strain with the tumorigenicity and the non-tumorigenic monoclonal cell strain, so that 35 miRNAs with obvious differential expression are obtained. And predicting miRNA target genes by using RNAhybrid+svm-light, miranda and TargetScan software to obtain functional genes such as Bak1, PIK3R1, WNT9B, MTOR, BRAF, IL2 and the like related to tumorigenesis and development. Annotating the target gene by GO and KEGG, differentially expressed genes were significantly enriched in the first 20 pathways, with the pathways associated with neoplasia: the classical pathways related to tumors such as the Rap1 signaling pathway, notch pathway, MAPK pathway, VEGF signaling pathway, etc. And constructing an miRNA-mRNA interaction network participating in the MDCK cell tumorigenic phenotype, and obtaining 6 key miRNAs such as miR-2779-x, miR-424-x, miR-236-y and the like. Verification of the miRNA with differential expression by qRT-PCR shows that the miR-2779-x group has better parallelism and the group-to-group difference is most obvious. The Bak1 protein expression in different tumorigenic MDCK cells is detected by a Western Blot method, and as a result, compared with Gao Cheng tumor MDCK cells, the expression level of the Bak1 protein in (MDCK-CB 057) cells is obviously reduced, and the expression level and miR-2779-x expression are in negative regulation and control relation, so that miRNA-2779 and Bak1 are possibly key miRNA and target genes for regulating the tumorigenicity of the MDCK cells.
The binding relationship between Bak13' UTR and miR-2779-x is verified by using a dual-luciferase gene report vector, and the result shows that in Bak1 wild-type transfected cells, the luciferase activity of a miR-2779-x mimic treatment group is obviously reduced to 80% of that of a control group (P is less than 0.05); in Bak1 mutant transfected cells, the luciferase activity of the miR-2779-x mimetic treated group was significantly increased by 1.12 times (P > 0.05) that of the control group.
And (3) designing and constructing a miR-2779-x over-expression slow virus vector, transfecting MDCK tumorigenic cells, screening for 10 generations by puromycin to obtain a miR-2779-x over-expression stable transgenic cell line, wherein compared with a control group, the miR-2779-x over-expression slow virus vector has the advantages that the miR-2779-x over-expression slow virus vector has the advantage that the miR-2779-x expression level of the MDCK cells is obviously up-regulated, and the Bak1 expression level is obviously down-regulated (P is less than 0.05). According to the results of the nude mice tumor-bearing test specified by the three pharmacopoeias of the people's republic of China (2020 edition), the tumor formation of the miR-2779-x over-expression cell strain is obviously reduced, and the weight growth speed of the nude mice is obviously higher than that of a control group (P < 0.05). After tumor-bearing test, MDCK cells, DNA of partial tissue and organ of nude mice formed by injection sites of the MDCK cells and tumor species sources are extracted, and 1% agarose gel electrophoresis is used for identifying tumor species sources, so that the nude mice tumor can be amplified to obtain COX1 fragments (canine genes), and canine genes are not expressed in other organs of the nude mice. Conclusion: miR-2779-x can participate in regulation of MDCK cell tumorigenic phenotype by inhibiting Bak1 expression; meanwhile, the miR-2779-x over-expression stable transgenic cell strain is experimentally constructed, and a new idea is provided for establishing the low-tumorigenicity MDCK gene engineering cell strain.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
in fig. 1, panel a is the number of differential mirnas up-and down-regulated, panel b is the thermogram analysis of differential mirnas in each group, panel c is the wien plot of the HT01 vs. nts 01 and HT02 vs. nts 02 comparison group, and miR-2779-x is the only intersection miRNA in both groups.
In fig. 2, panel a is the GO entry for the top 20 differentially expressed miRNA target gene and panel b is the KEGG analysis of the top 20 differentially expressed miRNA target gene.
In fig. 3, panel a is a network interaction diagram of mirnas and their target genes associated with neoplasia, in which dark grey is miRNA and light grey is mRNA. Panel b shows the total target genes for miR-2779-x, in which the target genes are off-white, the target genes associated with tumorigenicity are gray, the signal molecules and interactions associated with the target genes are dark gray, and the target genes associated with cell growth and death are light gray. Panel c shows the detection of miR-2779-x expression levels in MDCK-HT01, MDCK-NT01 and MDCK-NT02 cells by qPCR.
In FIG. 4, panel a shows the binding site between MiRanda predicted miR-2779-x and Bak13' UTR (CFA is abbreviated as Canine Canis lupus familiars); panel b shows the sequencing results of c-Bak1-WT and c-Bak1-MUT. Mutation sites are shown in the red region of the marker; panel c shows the fluorescence expression levels of c-Bak1-WT and c-Bak1-MUT co-transformed with miR-2779-x-mimic into 293T cells.
In FIG. 5, FIG. a shows that the fluorescence efficiency reaches more than 95% when MDCK cells are transfected with miR-2779-x lentiviral plasmid and control plasmid. FIG. b shows the relative expression levels of miR-2779-x in lv-miR-2779-x and lv-control. Panel c shows the relative expression levels of miR-2779-x target protein Bak 1.
FIG. 6, panel a shows the cloning efficiency of lv-miR-2779-x and lv-control; panel b shows the results of in vivo oncological examination of each group of cells; panel c is tumor volume and body weight growth rate for each group of nude mice.
In fig. 7, fig. a is a section of normal nude mouse subcutaneous tissue (40×). Panel b is a section of subcutaneous tumor tissue from nude mice (40X). Panel c is a local enlargement of the subcutaneous tissue of a normal nude mouse (400X). Panel d and e are local enlargement (400 x) of subcutaneous tumors in nude mice.
In FIG. 8, graphs A-C are respectively in sequence of lv-control, lv-miR-2779-x and myocardial structures of untreated MDCK cell groups (MDCK-M in the figure), graphs D-F are respectively in sequence of lv-control, lv-miR-2779-x and lung tissue structures of untreated MDCK cell groups, and graphs G-I are respectively in sequence of lv-control, lv-miR-2779-x and kidney tissue structures of untreated MDCK cell groups.
In fig. 9, M is DNAmarker, lanes 2, 3 are groups of nude mice tissue without MDCK cells injected; lanes 4, 5 are MDCK cell groups; lanes 6-13 are in turn groups of heart, liver, spleen, lung, kidney, brain, skin, subcutaneous lymph node tissue of nude mice after injection of MDCK cells; lanes 14-16 are MDCK cell-injected nude mouse tumor groups.
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional Biochemical reagents. The quantitative tests in the following examples were all set up in triplicate and the results averaged.
Example 1
1 method
1.1 cell culture and sequencing analysis
MDCK-adherent cell line was introduced from the American Type Culture Collection (ATCC) under the accession numberCCL-34 TM Lot number: 60245139, the introduction times are 55 generations. After introduction, three-level cell banks (an original bank, a main bank and a working bank) are established, and early experiments prove that the cell banks have the tumorigenicity. The clone cell strain is obtained by single cell clone self-making, and the MDCK monoclonal cell strain (MDCK-CA 005 and MDCK-CB 057) without tumorigenicity is obtained by multiple screening of karyotype identification, plate cloning test, soft agar cloning, nude mouse tumorigenicity and the like. In DMEM with 10% FBS (Cellmax, beijin China), 5% CO 2 Subculturing at 37 ℃ for every 36h, wherein the passaging ratio is 1:4. RNA of two main library cells MDCK-M01 (HT-01), MDCK-M02 (HT-02) and non-tumorigenic monoclonal cell strains MDCK-CA005 (NT-01) and MDCK-CB057 (NT-02) are extracted from the established MDCK three-level cell library. After detection of satisfactory RNA purity, sequencing was performed using IlluminaHiSeq TM 2500. small RNA-Seq was done by Dio Biotech Inc. of Guangzhou.
1.2smallRNA-Seq differential expression analysis
Low quality data was FASTQ filtered and all Clean Tags were aligned with small RNAs in the GeneBank database (Release 209.0) using blastall 2.2.25 (blastn) software, identifying and removing rRNA, scRNA, snoRNA, snRNA and tRNA. Total miRNA comprises existing miRNA, known miRNA and new miRNA, the Total miRNA expression quantity in each sample is calculated, and the expression quantity is normalized by TPM. The conditions for screening for significantly differentially expressed miRNAs herein were set to miRNAs with fold differences greater than 2, p < 0.05.
1.3miRNA target Gene prediction and Signal pathway analysis
Based on the sequence information of existing miRNAs, known miRNAs and new miRNAs, RNAhybrid (Version 2.1.2) +svm-light (Version 6.01), miranda (Version 3.3 a) and TargetScan (Version 7.0) were used for joint analysis of candidate miRNAs target genes, and the intersection of the predictions was selected as predicted miRNAs target genes. After FDR correction, mapping candidate genes to a GO database by taking the corrected p value less than or equal to 0.05 as a threshold value, and counting a gene list and the number of genes with the GO function; the Pathway that is significantly enriched in candidate genes was found using KEGG and the hypergeometric test. And constructing an interaction relation network of the significant differential expression miRNAs and the tumorigenicity related target genes by using Cytoscape software.
1.4 double luciferase reporter assay
Adherent MDCK cells (No. MDCK-HT 02) were cultured in DMEM medium containing 10% fbs, resuspended as a cell suspension after 0.25% trypsin digestion using the original medium, centrifuged at 10000rpm for 1min, the supernatant removed, and DNA was extracted according to TIANamp Genomic DNAkit instructions. PCR amplification primers were designed and synthesized based on the 3' UTR sequence of the Bak1 gene in NCBI, and the primer sequences are shown as Nos. 1 and 2 in Table 1. Using MDCK cell DNA as template, PCR amplifying to synthesize 3' UTR sequence fragment, using restriction enzyme to make PCR product and carrier pmiR-RB-Report respectively TM Xhol and Notl cleavage site of Guangzhou Ruibo, guangdong, china are subjected to double cleavage, the primer sequences of the cleavage sites are shown as No.3 and No.4 in Table 1, the enzyme fragments are recovered by 2% agarose gel electrophoresis at 37 ℃ overnight, DH5 alpha competent cells are transformed after the ligation reaction of the products at 16 ℃, and positive colony PCR is selected and sequenced for identification. The wild type plasmid containing the sequence of interest was designated as c-Bak1-WT and the mutant plasmid was designated as c-Bak1-MUT.
Table 1 double luciferase assay plasmid sequences
DMEM medium containing 10% FBS at 37℃with 5% CO 2 293T cells were cultured under the conditions of (C). When the cells were cultured to logarithmic phase, 1.0X10 per well 4 Cells were seeded in 96-well plates in a total volume of 100. Mu.L per well and incubated at 37 ℃. Respectively diluting miRNA-2779-x micrometers (miRNA-2779-x enhancers, which can be synthesized by a company after providing miRNA-2779-x nucleic acid sequences and have the function of transiently overexpressing miRNA after transfection) with a 5 mu LOPTI-MEM culture medium, and then performing c-Bak1-WT and c-Bak1-MUT; mu.L of OPTI-MEM medium was diluted with 0.25. Mu.L of Lipo6000 TM And (3) a transfection reagent, mixing the transfection reagent and the transfection reagent, shaking the mixture uniformly, and standing the mixture at room temperature for 5min. mu.L of 293T cell-containing medium was added to each well, followed by 10. Mu.L of the above mixture, and finally 100. Mu.L of total volume per well. The experiments were divided into 4 groups, c-Bak1-WT+NC (wild-type plasmid+empty vector), c-Bak1-WT+miR-2779-x (wild-type plasmid+miR-2779-x enhancer), c-Bak1-MUT+NC (mutant plasmid+empty vector) and c-Bak1-MUT+miR-2779-x (mutant plasmid+miR-2779-x enhancer). Each set was set up with 3 replicates and the culture medium was changed after 6h of transfection. According toLuciferaseAssay System kit instructions, 48h post transfection, aspiration of culture broth, addition of PBS at 35. Mu.L/well, and luciferase reagent. Mu.L/well, shaking for 10min, transfer to LUMITRAC TM The firefly luciferase activity fluorescence values were measured in 20096 well white cell culture plates. Add 30. Mu.L Stop reagent, shake for 10min, and determine Renilla luciferase activity fluorescence.
1.5miR-2779-x over-expression vector construction
The miR-2779-x sequence is UCCGGCUCGAAGGACCAU, and a miR-2779-x primer sequence, miR-2779-x-F GAGGATCCCCGGGTACCGGTCTGGAGGCTTGCTGAAGGC and miR-2779-x-R CACACATTCCACAGGCTAGCGGGCCATTTGTTCCATGTG are designed. The microRNA is amplified by PCR reaction, the reaction system is shown in table 2, and the PCR reaction conditions are as follows: 98℃for 5min,98℃for 10s,55℃for 10s,72℃for 30s, 30 cycles in total; and at 72℃for 8min. GV369 vector (Ji Kai gene, shanghai, china) is selected. The vector element sequence is Ubi-MCS-SV40-EGFP-IRES-puromycin, and the cloning site is AgeI/NheI. The target fragment obtained by PCR was subjected to exchange reaction with the vector after cleavage, and the reaction system was as shown in Table 3.
10. Mu.L of the exchange reaction product was added to 100. Mu.L of DH 5. Alpha. Competent cells, and the walls of the flick tube were placed on ice for 30min. Heat shock at 42 ℃ for 90s and ice-water bath incubation for 2min. Adding into 500ul LB culture medium, shaking culture at 37deg.C for 1 hr, spreading appropriate amount of bacterial liquid on plate containing corresponding antibiotics, culturing in constant temperature incubator for 12-16 hr, selecting positive clone transformant, and sequencing and identifying. And comparing and analyzing the sequencing result with a target gene sequence, and then extracting plasmids to establish miR-2779-x overexpression plasmids. And co-transfecting miR-2779-x over-expression plasmid, lentiviral packaging auxiliary plasmid (Helper 1.0) and lentiviral packaging auxiliary plasmid (Helper 2.0) into 293T cells, collecting cell supernatant after transfection for 48 hours, concentrating and purifying to obtain miR-2779-x over-expression lentiviral solution, and entrusting Shanghai Ji Kai company to physical state detection, sterile detection and virus titer detection of the lentiviral solution.
TABLE 2PCR amplification microRNA reaction system
TABLE 3PCR products and Carrier exchange reaction System
1.6 cell transfection
Taking MDCK-M02 cells with good growth state and single layer fusion rate of 90%, and adjusting cell density to 1×10 4 cells/mL, inoculating to 12-well plate, respectively transfecting miR-2779-x lentiviral plasmid obtained in step 1.5 and control plasmid (empty plasmid) into MDCK-M02 cells, setting transfection MOI value as 100, calculating required virus liquid volume of each group according to lentiviral titer, 37 ℃ and 5% CO 2 Culturing for 12h, discarding the mixed solution, adding 500 μl of complete medium, 37deg.C, 5% CO 2 Culturing for 36h, and observing the transfection efficiency of the cells under a fluorescence inversion microscope. 0.4. Mu.g of anti-puromycin 10% FBS DMEM mediumAnd continuously screening for 10 generations to obtain a stably transfected miR-2779-x cell line.
1.7qRT-PCR
Extracting total RNA of cells by Trizol-isopropanol method, and collecting Bulge-Loop TM MiRNAPrimer (20 uM) was added 150uL RNase free water to prepare Bulge-Loop TM MiRNAPrimer (5 uM), 200uLPCR tube. Gently beating for 7-8 times, performing instantaneous centrifugation for 5-10 s, and setting a reverse transcription reaction program at 42 ℃ for 60min by a PCR instrument; 70 ℃ for 10min. Primers of 5s rRNA and miR-2779-x stem-Loop method are designed and synthesized by Guangzhou Ruibo biotechnology Co., ltd, and are prepared according to Bulge-Loop TM The mirnqRT-PCR Starter Kit protocol performs qPCR reactions, and each set of experiments was repeated three times. 1.8Western Blot
Cells were extracted with a cell scraper and added to RIPA & PMSF mix (1:100) to extract proteins. Protein quantification was performed by BCA method. Preparing 12% polyacrylamide separating gel, 5% polyacrylamide concentrated gel and glycine electrophoresis buffer solution for electrophoresis, wherein the loading amount is 10 μl,80V and 20min;120V,1h. After electrophoresis, transferring film by wet transfer method, sealing with 200mA,1h,5% skimmed milk powder for 1h, and cleaning with TBST for 3 times each for 5min. Primary antibody (1:1000) was incubated overnight, and TBST was washed 3 times for 5min each. Secondary antibody (1:2000) was incubated for 1h, and TBST was washed 3 times for 5min each. Mixing the ECL hypersensitive luminous liquid A and the ECL hypersensitive luminous liquid B in a dark place, blowing for 4-5 times, mixing, sucking a proper amount of mixed liquid drops onto a PVDF film, standing for 1min, setting the exposure time to be 2s, photographing and analyzing image data.
1.9 cloning experiments
MDCK cells which grow well and grow in logarithmic phase are taken, cell suspension is digested, inoculated into a 6-well plate at the concentration of 500 cells/well, and 4mL of complete culture medium is added for 6 days. After the cells form macroscopic colonies, discarding the culture medium, and washing twice with PBS; fixing with 100% methanol, standing at room temperature for 30min, and discarding methanol in each well; adding 2mL of crystal violet solution into each hole, and dyeing for 30min at room temperature; the crystal violet was discarded, rinsed with tap water, dried at room temperature, and then placed under an inverted microscope for observation and counting (five fields were randomly selected under a 10-fold objective lens, and counted separately). Only colonies containing more than 50 cells can be counted. This experiment was repeated at least three times.
1.10 nude mouse nodulation test
30 female BALB/c nude mice of 4-6 weeks old are purchased from Beijing Veitz Lihua, the ambient temperature is 23+/-2 ℃, and the illumination condition is 12h illumination: and the people are dark for 12 hours and drink water freely. It was randomly divided into positive control group (Hela cells injected) and negative control group (MRC-5 cells injected) before the experiment, empty vector control group (lv-control cells injected), and miR-2779-x group (lv-miR-2779-x cells injected) were overexpressed, 10 in each group. And (3) preparing a cell suspension by taking MDCK cells transfected by slow viruses with the monolayer cell fusion rate reaching 90%. Subcutaneously inoculating 0.2mL of cell suspension, 1×10, to scapula of nude mice 7 Every five days after injection, the body weight change of the nude mice and the tumor volume change formed at the injection position are recorded, the mice are sacrificed and dissected after 30 days, and the nude mice tumor and tissues and organs such as heart, lung, liver, spleen, kidney and the like are separated and stored in 4% formaldehyde.
1.11 species identification
The canine COX1 primer, COX1-F, was designed and synthesized: ATCCGAGCCGAACTAGGTCAGC, COX1-F: AGGATGGAGGAAGGAGTCAGAAGC. MDCK-HT02 cells, subcutaneous tumors and normal tissues of nude mice stored at the temperature of minus 80 ℃ are taken, and tissues and cell genome DNA are respectively extracted. After detecting the concentration and purity, carrying out PCR reaction, taking 2 mu L of PCR product, carrying out 0.8% agarose gel electrophoresis under 120V for 45min, observing the electrophoresis result under an ultraviolet detector and photographing.
1.12 HE staining
Taking tissue blocks fixed in paraformaldehyde, and flushing the tissue blocks for 6h by running water; setting dehydration and transparency procedures: 70% (24 h) →80% (24 h) →95% (2 h) →95% (12 h) →100% (1 h) →benzene alcohol (1:1, 5 min) →xylene I (5 s) →xylene II (5 s) →benzene wax (1:1 m,1 h) →paraffin I (52-56 ℃ for 30 min) →paraffin II (56-58 ℃ for 1 h) →paraffin III (58-60 ℃ for 1 h), an automatic embedding machine embeds tissues, wax blocks are continuously sliced, thickness slices are 4 μm, and after a fishing piece is fixed on a glass slide, a piece is baked at 58 ℃. Dewaxing in paraffin section xylene for 2 times each for 5-10min. Serial ethanol (100%, 95%, 85%, 75%) was rehydrated, 3min per gradient. Distilled water for 2min. Hematoxylin dye liquor is used for dyeing for 2-20min, and the floating color is removed by washing with distilled water. Differentiation liquid is differentiated for 3min, and is washed for 2 times with tap water for 2min each time. Placing eosin dye liquor for 30s-2min, slightly washing with distilled water for 2-3s, and rapidly dehydrating. Each of 75% ethanol, 85% ethanol, 95% ethanol and 100% ethanol (I) was immersed for 2-3s. Soaking in 100% ethanol (II) for 1min, and observing with a mirror after xylene is transparent twice for 1min each time.
1.13 influenza Virus susceptibility test
Spreading lv-miR-2779-x and lv-control cells into a 6-well plate, and growing the cells to 80-90% after about 12h, wherein Ca-free cells are used 2+ 、Mg 2+ Is inoculated with influenza A/California/7/2009X-179A (H1N 1) virus strain at a multiplicity of viral infection (MOI) of 0.001 in DMEM medium (without serum) and 2.5mg/mL TPCK trypsin is added at a ratio of 1:1000 (v: v) at 34℃at 5% CO 2 Culturing in incubator, collecting virus solution 24h, 48h, 72h after virus inoculation, and storing in 1.5ml centrifuge tube in-80 refrigerator.
TCID 50 Is detected: observing MDCK main library cells under an inverted microscope, placing the MDCK main library cells in a 200 mu L/Kong Jiazhi 96-well plate after the adherent cells in a T75 bottle reach 80% -90% of density degree, and placing the MDCK main library cells in a CO at 37 DEG C 2 Culturing in an incubator. After observing that MDCK adherent cells in the wells grew into a single 80% -90%, the medium in the cell plates was discarded and washed 2 times with PBS buffer solution. After removing the residual serum from the medium, 100. Mu.L/well (200. Mu.L/well around the perimeter) of a cell-retaining solution (DMEM containing TPCK pancreatin) was added.
The samples were diluted in a gradient with serum-free cell-maintenance solution (DMEM containing TPCK pancreatin 2.5. Mu.g/ml). The final concentration of the virus sample after serial dilution reaches 10 respectively -1 lgTCID 50 /mL、10 -2 lgTCID 50 /mL、10 -3 lgTCID 50 /mL、10 - 4 lgTCID 50 /mL、10 -5 lgTCID 50 /mL、10 -6 lgTCID 50 /mL、10 -7 lgTCID 50 /mL、10 -8 lgTCID 50 /mL、10 - 9 lgTCID 50 /mL. The inoculation is performed by selecting the appropriate concentration of the virus solution according to the experiment. To eliminate edge effects, experimental junctionsWith interference of the fruits, the edge wells served as blank controls without virus sample. 100 μl of serial diluted virus samples were pipetted into 96 well plates, 6 multiplex wells were made per virus sample gradient. Samples were inoculated from low dilution to high dilution, 100. Mu.L/well, 6 wells/sample, and a column of maintenance solution alone (DMEM containing TPCK pancreatin) was left for each plate as negative control (200. Mu.L/well). The 96 plates were placed at 34℃constant CO 2 Culturing in an incubator for 1-2h, adding 100 mu L of virus maintenance solution after virus is completely adsorbed, and culturing in the incubator for 72h to observe cytopathic effect.
1.14 data statistics
All statistical evaluations were performed using GraphPad Prism 8.0.2 for group-to-group variance analysis, with P <0.05 indicating significant variance, and each group experiment was repeated three times, comparing group-to-group variance using T-test.
2 results
2.1 expression of different tumorigenic MDCK cell miRNAs and target Gene prediction
A total of 14597969 ～ 16523140 clean Tags were obtained by miRNA-seq, and in total 256 dogs miRNAs were found to be present in the 4 samples, which belong to the 204 families of miRNAs; 602 known miRNAs and 991 new miRNAs. HT vs.NT found 35 significant differentially expressed miRNAs in total, including 21 up-regulated and 14 down-regulated miRNAs (FIG. 1a and FIG. 1 b). Inter-group differential expression miRNAs venn figure as shown in figure 1c, miR-2779-x is the only inter-group differential expression miRNA.
In fig. 1, panel a is the number of differential mirnas up-and down-regulated, panel b is the thermogram analysis of differential mirnas in each group, panel c is the wien plot of the HT01 vs. nts 01 and HT02 vs. nts 02 comparison group, and miR-2779-x is the only intersection miRNA in both groups.
Target gene prediction was performed on 1849 existing miRNAs and new miRNAs in the sequencing data by three methods of RNAhybrid (v2.1.2) +svm_light (v 6.01), miranda (v 3.3 a), targetScan (Version: 7.0), and a total of 26641 target genes were obtained, wherein the number of target genes corresponding to DE miRNAs was 18331. Analysis has resulted in tumor progression-related functions based on, for example, PIK3R1, WNT9B, MTOR, BRAF, IL, BAK1, etc., which may be key molecules for modulating neoplasia.
2.2 target Gene analysis of differentially expressed miRNAs
miRNAs are involved in biological processes by binding to a target gene to regulate factors associated with the signaling pathway in which the target gene is located. Analysis of target Gene function using the Gene on log (GO) database, as shown in fig. 2, found that binding, metabolic processes, biological regulation, catalytic activity and protein binding were enriched by Biological Process (BP), suggesting that we these biological processes might be associated with the neoplastic processes of cells, most of the target genes were enriched at cell surface receptor signaling pathway, localization, regulation ofbiological quality, establishment oflocalization. Molecular Function (MF) is enriched in binding and adhesion, and cytoplasmic membranes and cell circumferences are enriched with Cellular Component (CC), suggesting that the target gene may function biologically in these regions.
In fig. 2, panel a is the GO entry for the top 20 differentially expressed miRNA target gene and panel b is the KEGG analysis of the top 20 differentially expressed miRNA target gene.
Pathway analysis the pathways associated with neoplasia enriched to the top 20 were: rap1 signaling pathway, microRNA in cancer, PI3K-Akt signaling pathway, signaling pathway in cancer, and the like. The tissue characteristics of the nodulation histochemical staining formed after MDCK is inoculated into a newborn nude mouse are similar to that of adenocarcinoma, the nodulation histochemical staining has a certain tissue migration capacity, lung metastasis and spinal cord metastasis can be formed, and tumor cells have high proliferation, so that the passages related to continuous angiogenesis, tissue invasion, metastasis and proliferation are considered when searching for enrichment passages related to the tumorigenicity. The pathways involved in persistent angiogenesis are VEGF signaling pathway, MAPK signaling pathway, jak-STAT signaling pathway, PI3K-Akt signaling pathway, m-TOR signaling pathway, notch signaling pathway, HIF1 signaling pathway; the signaling pathway associated with tissue invasion and migration is the Wnt signaling pathway.
2.3 establishment of an oncologic related miRNA-mRNA interaction network
Constructing a regulation network by using Cytoscape software according to the interaction relation between the differential expression miRNA and the target gene; taking data related to neoplasia according to gene functions and the located pathways, establishing a neoplasia related miRNA-mRNA interaction network (FIG. 3a & FIG. 3 b). Wherein miR-2779-x is the only inter-group differential expression miRNA, and target genes thereof comprise a plurality of genes related to tumor progress, such as: BAK1 (a Bcl2 family protein member) is one of its target genes, bcl2 family proteins play an important role in apoptosis, tumorigenesis and anticancer therapy. The qPCR method detects the expression level of miR-2779-x in each sequencing cell, and the result shows that the miR-2779-x has better parallelism in groups and the difference between groups is most obvious; the expression level of miR-2779-x was significantly higher in the non-neoplastic monoclonal cells (NT 01 and NT 02) than in the master pool cells HT-01 and HT-02 (FIG. 3 c). Thus, we selected miR-2779-x for subsequent study.
In fig. 3, panel a is a network interaction diagram of mirnas and their target genes associated with neoplasia, in which dark grey is miRNA and light grey is mRNA. Panel b shows the total target genes for miR-2779-x, in which the target genes are off-white, the target genes associated with tumorigenicity are gray, the signal molecules and interactions associated with the target genes are dark gray, and the target genes associated with cell growth and death are light gray. Panel c shows the detection of miR-2779-x expression levels in MDCK-HT01, MDCK-NT01 and MDCK-NT02 cells by qPCR.
2.4miR-2779-x has targeting binding relation with BAK13' UTR
MiRanda software predicts the binding relationship between the miR-2799-x seed region and the Bak13 '-non-coding region, and as a result, a binding region exists in theory between the miR-2779-x seed region and the Bak13' non-coding region, the binding region is located at 1006-1012bp, 7 bases are added, and the binding free energy is-17.25 kCal/Mol, as shown in FIG. 4 a. A reverse primer is designed aiming at the predicted binding site (5 ' -3 ') mutation site end of miR-2779 and Bak1, the sequence of the reverse primer is 5'-AATGCGGCCGCGCCGGCCTCTACGTAAAACTTCGAGCCAGTATCGAGGGGGCAAG TGGGCCGAGCTGCCCCTTCTACC-3', and the c-Bak1-WT recombinant plasmid is successfully introduced into a Bak13' UTR region with the length of 695bp, so that a mutant recombinant plasmid c-Bak1-Mut is obtained. Sequencing results of the c-Bak1-Mut recombinant plasmid show that the GAGCCGG of the binding region of the Bak13' UTR region and miR-2779-x is successfully mutated into CTCGGCC, and the c-Bak1-Mut recombinant plasmid is successfully constructed (figure 4 b).
The fluorescence intensity is detected after c-Bak1-WT recombinant plasmid and c-Bak1-MUT recombinant plasmid are respectively co-transformed with miR-2779mimics into 293T cells, and analysis data after correction of the fluorescence intensity shows that compared with NC, the fluorescence expression level of c-Bak1-WT is significantly reduced after transfection of miR-2779mimics (P is less than 0.05, as shown in FIG. 4 c); the c-Bak1-Mut fluorescence expression levels were slightly up-regulated, but there was no significant difference (P > 0.05, as shown in FIG. 4 c). The result shows that miR-2779-x can be combined with the 3'UTR of Bak1 in a targeted way, so that the transcriptional activity of Bak1 is inhibited, and the mutation of the 3' UTR site of Bak1 inhibits miR-2779-x combination.
In FIG. 4, panel a shows the binding site between MiRanda predicted miR-2779-x and Bak13' UTR (CFA is abbreviated as Canine Canis lupus familiars); panel b shows the sequencing results of c-Bak1-WT and c-Bak1-MUT. Mutation sites are shown in the red region of the marker; panel c shows the fluorescence expression levels of c-Bak1-WT and c-Bak1-MUT co-transformed with miR-2779-x-mimic into 293T cells.
2.5 establishment of MDCK cell lines with stable overexpression of miR-2779-x
The miR-2779-x sequence is UCCGGCUCGAAGGACCAU, and the designed primer sequence designs miR-2779-x-F GAGGATCCCCGGGTACCGGTCTGGAGGCTTGCTGAAGGC and miR-2779-x-R CACACAT TCCACAGGCTAGCGGGCCATTTGTTCCATGTG. Double digestion experiments miR-2779-x was ligated onto lentiviral plasmids, and positive clones were selected for sequencing. The sequencing result of the bacterial liquid is as follows:
wherein the bold portion is the inserted target sequence and the underlined portion is the reverse complement of the primer.
We succeeded in constructing lentiviral plasmid carrying miR-2779-x sequence, and can carry out subsequent experiments. Plasmid carrying miR-2779-x and transformationAfter the transfection auxiliary plasmid is used for co-transfecting 293T cells, the infection efficiency can reach more than 90 percent by observation of an inverted fluorescence microscope. Mycoplasma, chlamydia, bacteria and fungi detection are negative, endotoxin content is less than 50EU/mL, and lentivirus titer is 3×10 8 TU/mL。
And respectively transfecting miR-2779-x lentiviral plasmid and control plasmid into MDCK cells, and carrying out puromycin passage screening for 10 generations. The fluorescence efficiency of the inverted fluorescence microscope can reach more than 95 percent, as shown in figure 5 a. After passage 10, the expression level of miR-2779-x in the transfected group was significantly increased by 2.9 times (P <0.05, FIG. 5 b) and the expression level of the miR-2779-x target gene Bak1 mRNA and protein was significantly decreased (P <0.05, FIG. 5 c) compared with the control plasmid transfected cells. Thus, this MDCK cell was designated as lv-miR-2779-x, and the MDCK cell transfected with the control plasmid was designated as lv-control.
In FIG. 5, FIG. a shows that the fluorescence efficiency reaches more than 95% when MDCK cells are transfected with miR-2779-x lentiviral plasmid and control plasmid. FIG. b shows the relative expression levels of miR-2779-x in lv-miR-2779-x and lv-control. Panel c shows the relative expression levels of miR-2779-x target protein Bak 1.
2.6miR-2779-x can inhibit MDCK cell tumorigenicity
The plate cloning test facilitates in vitro determination of the neoplastic properties of cells. In this study, 500 seed cells were routinely cultured for 6 days for each group. The results showed that the colony formation rate was lower in the lv-miR-2779-x group than in the control group (P < 0.05), FIG. 6a.
After miR-2779-x over-expressed MDCK cells (lv-miR-2779-x), miR-2779-x over-expressed control cells (lv-control), hela cells (positive control) and MRC-5 cells (negative control) are injected into nude mice, tumors formed by the Hela cells show solid tumors with more surface nodules, the tumor formation rate is 100%, the boundaries of the tumors formed by the MDCK cells are clear, most of the tumors contain liquid hollow tumors, the morphology is shown in figure 6b, and the statistical results are shown in Table 4.
FIG. 6, panel a shows the cloning efficiency of lv-miR-2779-x and lv-control; panel b shows the results of in vivo oncological examination of each group of cells; panel c is tumor volume and body weight growth rate for each group of nude mice.
TABLE 4 statistics of tumor formation rate in nude mice tumor-bearing test
2.7 observation of tissue structure of tumor formed by MDCK cells
The tumor body formed by injecting the lv-miR-2779-x MDCK cells into the scapula part of the nude mouse is subjected to HE staining, and the tumor body tissue is compared with the normal skin tissue structure of the nude mouse (shown in figure 7). Obvious glandular tissues are visible in the tumor body tissues, the boundary with surrounding tissues is clear, and obvious infiltration or adhesion is not found. The lv-miR-2779-x group myocardial fibers are tightly connected, coarse long-strip myocardial cells can be seen, and no obvious abnormality is found compared with the control group tissue; lung tissue with intact alveoli, inflammatory cell infiltration around alveoli, thickening of alveoli walls; kidney tissue was seen as glomeruli that were oval and clearly bordered by the renal interstitium, and no apparent inflammatory cell infiltration was observed. In conclusion, tumor bodies formed by injecting the lv-miR-2779-x MDCK cells into the nude mice do not obviously transfer in the heart, lung and kidney of the nude mice. The heart, lung and kidney tissue structures of the nude mice are shown in figure 8.
In fig. 7, fig. a is a section of normal nude mouse subcutaneous tissue (40×). Panel b is a section of subcutaneous tumor tissue from nude mice (40X). Panel c is a local enlargement of the subcutaneous tissue of a normal nude mouse (400X). Panel d and e are local enlargement (400 x) of subcutaneous tumors in nude mice.
In FIG. 8, graphs A-C are respectively in sequence of lv-control, lv-miR-2779-x and myocardial structures of untreated MDCK cell groups (MDCK-M in the figure), graphs D-F are respectively in sequence of lv-control, lv-miR-2779-x and lung tissue structures of untreated MDCK cell groups, and graphs G-I are respectively in sequence of lv-control, lv-miR-2779-x and kidney tissue structures of untreated MDCK cell groups.
2.8 species identification
The specific primers of canine COX1 are designed, the PCR technology is utilized to amplify COX1 fragments in MDCK-HT02 cells, subcutaneous tumors and normal tissues of nude mice, the agarose gel is used for detecting the expression condition of COX1, the MDCK cells are injected into nude mice to form tumor bodies and the expression condition of tissues is shown in figure 9, the higher expression of COX1 is detected in the MDCK cells and the genomic DNA of the tumor bodies, but the expression of COX1 is not detected in the normal tissues of the nude mice.
In fig. 9, M is DNAmarker, lanes 2, 3 are groups of nude mice tissue without MDCK cells injected; lanes 4, 5 are MDCK cell groups; lanes 6-13 are in turn groups of heart, liver, spleen, lung, kidney, brain, skin, subcutaneous lymph node tissue of nude mice after injection of MDCK cells; lanes 14-16 are MDCK cell-injected nude mouse tumor groups.
2.9 testing of susceptibility of non-tumorigenic genetically engineered cell lines to influenza Virus
Reed-Muench two-way method: distance ratio = (percentage of disease rate above 50%)/(percentage of disease rate above 50% below 50% percentage of disease rate), lgTCID 50 Log of dilution that = difference between distance ratio x log of dilution + greater than 50% disease rate. The results showed that TCID for 48H of A/California/7/2009X-179A (H1N 1) influenza virus strain was inoculated using lv-control, lv-miR-2779 50 Respectively 10 -5 /0.1ml、10 -5.334 0.1ml, (meaning: diluting the virus 10) 5 、10 -5.334 Inoculation of 100 μl resulted in 50% of the cells being diseased). TCID of MDCK adherent cells with original strain 50 The non-tumorigenic genetically engineered cell line has high sensitivity to influenza virus, and is suitable for replication of influenza virus and production of influenza vaccine.
TABLE 5 TCID 48h after the detoxification of lv-control and lv-miR-2779 cells 50
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. The non-tumorigenic MDCK genetically engineered cell strain is characterized in that: the MDCK gene engineering cell strain is a miR-2779-x over-expression stable-transformation MDCK cell strain.
2. The non-tumorigenic MDCK genetically engineered cell line of claim 1, wherein: the nucleotide sequence of miR-2779-x is as follows: UCCGGCUCGAAGGACCAU.
3. The method for constructing the non-tumorigenic MDCK genetically engineered cell line according to claim 1 or 2, wherein: and (3) designing and constructing a miR-2779-x over-expression slow virus vector, transfecting MDCK tumorigenic cells, and screening by puromycin to obtain a miR-2779-x over-expression stable transgenic cell strain.
4. A method of construction according to claim 3, wherein: the miR-2779-x over-expression lentiviral vector is designed and constructed specifically by the following steps: and connecting the miR-2779-x and the GV369 vector through an AgeI/NheI enzyme cleavage site, transforming the connection product into an escherichia coli DH5 alpha competent cell, selecting a positive clone transformant for plasmid extraction to obtain a miR-2779 over-expression plasmid, co-transfecting the miR-2779 over-expression plasmid and a lentivirus packaging auxiliary plasmid into 293T cells, and collecting cell supernatant for purification to obtain the miR-2779-x over-expression lentivirus vector.
5. A method of construction according to claim 3, wherein: when MDCK tumorigenic cells are transfected, the transfection MOI value is 100, and the temperature is 37 ℃ and the concentration of CO is 5 percent 2 Culturing under the condition.
6. Use of the non-tumorigenic MDCK genetically engineered cell line of claim 1 or 2 in the proliferation culture of influenza virus or in the preparation of influenza virus vaccines.
7. The use according to claim 6, characterized in that: the non-tumorigenic MDCK genetically engineered cell strain is used as a cell matrix for influenza vaccine production.
8. The use according to claim 7, characterized in that: after the MDCK genetically engineered cell strain is inoculated into animals, the MDCK genetically engineered cell strain does not form tumors on the inoculated animals.
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