CN116814552A - Esophageal cancer metastasis and invasion cell model and application thereof - Google Patents
Esophageal cancer metastasis and invasion cell model and application thereof Download PDFInfo
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- CN116814552A CN116814552A CN202211637615.6A CN202211637615A CN116814552A CN 116814552 A CN116814552 A CN 116814552A CN 202211637615 A CN202211637615 A CN 202211637615A CN 116814552 A CN116814552 A CN 116814552A
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Abstract
The invention discloses an esophageal cancer metastasis and invasion cell model and application thereof. In vitro screening and analysis are carried out on a plurality of esophageal cancer cell lines, the transfer capacity and invasion capacity of the esophageal cancer cell lines in vivo are researched, and the expression of miR150 in the esophageal cancer cell lines with high transfer capacity and invasion capacity is often expressed in a low mode, and the esophageal cancer cell lines can be used as model cells which are directly opposite to the transfer of esophageal cancer cells and the in vitro screening of invasion medicines, so that the medicine screening efficiency is greatly improved.
Description
Technical Field
The invention relates to the technical field of esophageal cancer metastasis and invasion, in particular to an esophageal cancer metastasis and invasion cell model and application thereof.
Background
Esophageal Cancer (EC) is one of the common cancers of the upper digestive tract, with poor prognosis and high mortality. Esophageal cancer can be classified into squamous cell carcinoma, adenocarcinoma, and adenosquamous carcinoma according to histopathological classification, wherein Esophageal Squamous Cell Carcinoma (ESCC) accounts for more than 90% of esophageal cancers. Despite the great progress currently made in diagnosis and treatment of esophageal cancer, prognosis of patients with esophageal cancer remains poor. Esophageal cancer cell metastasis is a multi-step complex process, and the growth, invasion, anti-apoptosis and other abilities of tumor cells are closely related to the remote metastasis ability of tumor cells. In order to fully study the relevant mechanisms of esophageal cancer cell metastasis, especially lung metastasis, and to explore targeted drugs for the metastasis, model cells related to esophageal cancer cell metastasis cassette invasion need to be constructed in vitro first to provide assistance for drug screening and improving the screening efficiency thereof.
Disclosure of Invention
Therefore, the invention carries out in vitro screening and analysis and research on the metastasis capability and invasion capability of a plurality of esophageal cancer cell lines in vivo, and discovers that the expression of miR150 in the esophageal cancer cell lines with high metastasis and invasion capability is often expressed in low, and the esophageal cancer cell lines can be used as model cells which are directly opposite to the metastasis and invasion medicaments in vitro screening, so that the medicament screening efficiency is greatly improved. The invention also discovers a molecular marker of miR150 which can be related to the metastatic invasion capacity of the esophageal cancer cells, and can inhibit the metastasis and invasion of the esophageal cancer cells through the intervention of over-expressing miR150, thereby providing another treatment prospect for the drug intervention. Therefore, the embodiment of the invention at least shares the following technical scheme:
in a first aspect, the embodiment of the invention discloses an esophageal cancer model cell line with strong metastatic invasion capability, wherein the esophageal cancer model cell line is one of miR150 knockdown EC-109-1 cells, miR150 knockdown EC-109-2 cells or miR150 knockdown EC-109 cells.
In a second aspect, a method for constructing a strong invasive esophageal cancer model cell line, the method comprising:
in situ lung metastasis tissue cells EC-109-1 isolated from the nodulation of EC-109 cell esophageal carcinoma nude mice were placed in a Transwell six-well plate;
the cells were co-cultured in a Transwell chamber at 37℃with 5% CO2 for 3 weeks with 1 exchange of liquid every 3 days.
In a third aspect, a kit for miR150 knockdown in a esophageal cancer model cell line, the kit comprising a lentiviral vector carrying a miR150 knockdown sequence and a reagent for transfecting cells, wherein the miR150 knockdown sequence comprises a pair of most complementary oligonucleotide fragments, a sense strand of the oligonucleotide fragments is shown in SEQ ID NO.4, and an antisense strand of the oligonucleotide fragments is shown in SEQ ID NO. 5.
Further, the expression cassette of the lentiviral vector carrying the miR150 knockdown sequence comprises a CMVPromoter, emGFP reporter gene, a 5 'miRNAflavangregion, an inserted knockdown sequence, a 3' miRNAflavangregion and a transcription termination sequence.
In a fourth aspect, a method for preparing a kit for knockdown of miR150 in an esophageal cancer model cell line, which comprises the steps of preparing a lentiviral vector carrying a miR150 knockdown sequence and preparing a reagent for transfected cells; the preparation method of the lentiviral vector carrying the miR150 knockdown sequence comprises the following steps:
obtaining a miR150 knockdown sequence, wherein the miR150 knockdown sequence comprises a sense strand shown as SEQ ID NO.4 and an antisense strand shown as SEQ ID NO. 5;
connecting the miR150 knockdown sequence to a pcDNA6.2-GW/EmGFP-miR carrier, transforming DH5 alpha competent cells, selecting positive clones, sequencing and identifying to obtain plasmid pGW-miR150;
the small fragment recovered by ClaI/SalI double digestion of pGW-miR150 is reacted with
The large fragment recovered by double digestion of pCDH-CMV-MCS-EF 1-copGGFP is connected with T4ligase at 16 ℃ overnight to transform Stabl3 competent cells, select positive clone, sequence and identify to obtain the lentiviral vector carrying miR150 knockdown sequence.
In a fifth aspect, a weakly invasive esophageal cancer model cell line is a miR150 over-expressed EC-109-1 cell and/or miR150 over-expressed EC-109-2 cell.
In a sixth aspect, a kit for over-expressing miR150 in an esophageal cancer model cell line comprises a miR150 over-expression vector and a reagent for transfecting cells, wherein the miR150 over-expression vector comprises a miR150 precursor sequence shown in SEQ ID NO. 6.
In a seventh aspect, an agent or medicament for inhibiting invasion of an esophageal cancer cell metastasis cassette comprises a miR150 over-expression vector, wherein the miR150 over-expression vector comprises a miR150 precursor sequence, and the precursor sequence is shown in SEQ ID NO. 6.
In the eighth aspect, the marker related to metastasis and invasion of esophageal cancer cells is miR150, and the nucleotide sequence of miR150 is shown in SEQ ID NO. 3.
In a ninth aspect, the kit for detecting the metastatic capacity of esophageal epithelial cells and/or esophageal tumor cells comprises a miRNA extraction reagent, a PCR reaction reagent and a PCR product electrophoresis reagent, wherein the miR150 overexpression vector comprises a precursor sequence of miR150, and the precursor sequence is shown as SEQ ID No. 6.
Compared with the prior art, the invention has at least one of the following beneficial effects:
the invention prepares an esophageal cancer tumor-bearing mouse by transplanting human esophageal cancer cells EC-109, separates a cell strain EC-109-1 with strong metastasis and invasion capability from lung metastasis thereof, further establishes a cell line EC-109-2 with more metastasis and invasion capability by culturing the cell strain EC-109-2, and discovers that miRNA expression has obvious difference by analyzing miRNA of the EC-109 cells, the EC-109-1 cells and the EC-109-2 cells.
Therefore, the invention further proves that miR150 can inhibit metastasis and clarity of esophageal cancer through over-expression or knocking down miRNAs in the cell lines, and the targeting effect of miR150 on CYR61 is verified through double luciferase report.
In the process, the invention discloses model cell lines of metastatic esophageal cancer cells, and the model cell lines can be applied to drug screening for metastasis of esophageal cancer cells, for example, a biocompatible targeting oligonucleotide or a carrier for over-expressing miR150 is used as a drug, or a drug for promoting miR150 expression is screened to provide model cells.
Drawings
FIG. 1 is tumor tissue (upper panel) and HE staining (lower panel) of tumor-bearing mice with esophageal cancer.
FIG. 2 is a microscopic image of screening EC-109-1 cell lines for metastatic esophageal cancer cells.
FIG. 3 is a microscopic view of primary fibroblasts after 24h of culture (left panel) and a microscopic view of third-generation fibroblasts (right panel).
FIG. 4 is a microscopic view of cells after incubation of FITC-labeled secondary antibodies of the fibrous ring cell slide in the same field, FIG. 4A is a bright field, and FIG. 4B is a dark field; FIG. 4C is a chart of staining of the fibrous ring cell immune tissue.
FIG. 5 is a comparative analysis scatter plot of miRNA chips; the T1 axis of the left graph of FIG. 5 represents the fluorescence signal intensity values of EC-109 cells, and the C1 axis represents the fluorescence signal intensity values of EC-109-2 cells, respectively; as shown in the right panel of FIG. 5, the T1 axis represents the fluorescence signal intensity values of EC-109 cells, and the C1 axis represents the fluorescence signal intensity values of EC-109-1 cells, respectively.
FIG. 6 is a result of RT-PCR expression detection of miR150 in EC-109 cells, EC-109-2 and EC-109-1 cells.
FIG. 7 shows the results of WB detection of the E-cadherin, vimentin and Claudin1 expression levels of cells (miR-CON) obtained by transfection of both miR 150-overexpressed EC-109-1 and miR 150-overexpressed EC-109-2 into the pSuper empty vector group.
FIG. 8 is an electrophoretogram of a target fragment (a band indicated by an arrow) of the recombinant vector pGW-miR150 subjected to single NdeI cleavage.
FIG. 9 is a fluorescent micrograph of stable transgenic cell growth of miR 150-knockdown lentivirus (miR 150-KDEV) infected with EC-109-1 cells (FIG. 9C) and EC-109 cells (FIG. 9D), respectively, and of lentivirus pSuper empty vector without miR150 knockdown sequence infected with EC-109-1 cells (FIG. 9A) and EC-109 cells (FIG. 9B), respectively.
FIG. 10 shows the results of WB detection of E-cadherin, vimentin and Claudin1 expression levels of cells (miR-CON) obtained by transfection of both miR 150-knockdown EC-109-1 and miR 150-knockdown EC109 into the pSuper empty vector group.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. The reagents not specifically and individually described in the present invention are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.
It should be noted that, the terms "first," "second," and the like in the description and the claims of the present invention and the above drawings are used for distinguishing similar objects, and are not necessarily used for describing a particular sequence or order, nor do they substantially limit the technical features that follow. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
1. Construction of tumor-bearing mice with esophagus cancer
Human esophageal cancer cell line human esophageal cancer EC-109 cell line, accession number HTX2246, was purchased from ATCC.
Construction of esophageal cancer tumor-bearing mice: taking human esophageal cancer EC-109 cells in logarithmic growth phase, digesting and counting, adjusting cell concentration to 5×10 in RPmL-1640 complete culture medium 6 And each mL. 5 BALB/C nude mice were inoculated subcutaneously with 0.2mLEC-109 cell suspension (about 1X 10) 6 And 7 d), carefully peeling off the tumor of the well-grown tumor-bearing mice in a sterile environment, shearing off the tumor tissue with good growth state, adding a proper amount of physiological saline, and preparing a single-cell suspension of the living tumor cells for later use. The experimental animal model modeling method for the transplanted solid tumor comprises the following steps: taking 0.2% trypan blue staining counting suspension aliveThe cell rate is not less than 95%, diluted and counted by the physiological saline for injection according to the proportion of 1:4, and the concentration is adjusted to be 5-20 multiplied by 10 6 Single cell suspensions of live tumor cells per mL in a sterile environment, 0.2mL of suspension was inoculated subcutaneously into the right armpit of each mouse to establish an animal model of tumor.
Observing the growth condition of the tumor-bearing mice, and visually observing the symptoms of slightly bad spirit, less static movement and the like in 4d of the nude mice inoculated with the tumor growth condition, and continuously representing phenomena of reduced foraging, lassitude, reduced quality, loss of luster of hair and the like. In addition, fresh tumor tissue (as shown on the left of FIG. 1) was fixed in 10% formalin solution, dehydrated with ethanol gradient, xylene-soaked until the tissue was transparent, paraffin-embedded, cut into sections with a thickness of 2 μm, HE stained (as shown on the right of FIG. 1), and observed under 200-fold microscope for pathological changes. After 35 days, the mice were sacrificed and dissected to find that 10 nude mice were tumor-bearing successfully and that lung metastases occurred, with 100% incidence of metastasis.
2. Screening of metastatic esophageal cancer cells isolation of EC-109-1
In-situ lung metastasis tissues of nude mice with the size of 0.5cm multiplied by 0.5cm are excised, placed into a 15mL centrifuge tube, added with 2-3 mL of 0.5% collagenase IV, and digested for 15min at 37 ℃ after shaking. After full digestion, repeatedly blowing with 1mL two glass straws until no obvious tissue blocks exist, and then placing into a centrifuge, and centrifuging at 700 Xg for 5min; removing supernatant, adding 10mL of RPMI1640 culture solution containing 10% foetal calf serum and 100 μl of double antibody (penicillin and streptomycin), blowing uniformly, and respectively placing in 2 pieces with size of 25cm 2 Placing in a carbon dioxide incubator (Thermo company of America) with a volume fraction of 0.05% at 37deg.C, culturing for 24 hr, and replacing liquid to continue culturing; after which the liquid is changed 1 time every 2-3 d. After cells were stably attached (48 h), the culture flask was placed under a phase contrast microscope with a green light source to observe the growth state of epithelial cells and mesenchymal cells. The epithelial cells in the 2 culture flasks were in the form of irregular polygons and shuttles, and the mesenchymal cells were in the form of stripes or ropes, both of which grew on the wall. After 5d, the adherent growth of epithelial and mesenchymal cells was seen under a phase contrast microscope to occupy 80% of the bottle bottom area. Diluting 0.25% pancreatin with PBS 10 times, adding into culture flask, and standing in greenObserving under a phase contrast microscope of a color light source, lightly blowing when the mesenchymal cells are changed into spheres from strip ropes, taking the blown cells as the mesenchymal cells, and sucking the mesenchymal cell suspension by using a liquid transfer device; transferring the rest cells into a cell with the size of 75cm by adopting a conventional cell digestion passage method 2 The cell morphology of the culture flask of (2) is shown in FIG. 2.
3. Transwell culture of high invasive esophageal cancer cell lines
(1) Isolation, culture and identification of the fibrous Ring cells (AF cells)
After the injection of the vein air at the edge of the rabbit ears is fatal, the rabbit ears are disinfected, skin, subcutaneous fascia and muscle tissues are cut layer by layer along the back median line of the rabbit, lumbar spinal column of the rabbit is exposed, the lumbar spinal column of the rabbit is taken out under the aseptic condition, and the spinal column is immediately transferred to an aseptic ultra-clean workbench. Soaking in iodophor for 15min, washing with D-Hanks solution for 3 times, separating intervertebral disc tissue with rongeur and tissue scissors, carefully removing fibrous ring and nucleus pulposus tissue with ophthalmic scissors and blades, placing the rest fibrous ring tissue in culture dish containing DMEM high sugar culture solution, penicillin and streptomycin, washing with D-Hanks solution, and cutting into 1mm pieces 3 Size, digestion is terminated by adding culture solution into a water bath box with 37 ℃ of 0.2% type II collagenase and 0.25% trypsin solution, evenly blowing, counting, inoculating into a 25mL culture flask, and adding CO with volume fraction of 5% at 37 DEG C 2 Primary culture was started in the incubator with 1 change of liquid every 3 d. The primary cultured cells are passaged when reaching 90% fusion, washed for 2 times by sterile PBS, added with 0.25% pancreatin for 2min at 37 ℃, and stopped from being digested by pancreatin when the cells begin to shrink, and the bottle wall cells are blown by a suction tube to form single cell suspension. After counting, the suspension was inoculated in 1:2 into two flasks for continuous cultivation. And taking the third generation AF cell line for identification or experiment.
Detecting type I collagen (1) climbing slices by adopting an indirect immunofluorescence method and a Hoechst nuclear staining method on third-generation AF cells: and taking well-grown second-generation fiber ring cells, digesting the well-grown second-generation fiber ring cells, and then transferring the well-grown second-generation fiber ring cells into a six-hole plate inner climbing piece. (2) fixing: after PBS film washing, fixing in a PBS solution of 4% paraformaldehyde for 15min; the PBS solution of 4% paraformaldehyde in each well was blotted and reused 3%H 2 O 2 Incubating for 15min at room temperature to eliminate the activity of endogenous peroxidase, and washing with PBS; permeabilizing the immobilized cells with a solution containing 0.2% Triton X-100 for 10min (room temperature), and washing with PBS after blotting; blocking with 10% normal goat serum (PBS dilution) and incubating at room temperature for 10min. (3) Antibody incubation, namely adding primary antibody (mouse anti-rabbit type I collagen antibody) at 4 ℃ overnight, and adding secondary antibody (goat anti-mouse FITC labeled IgG) at 37 ℃ for 30min. (4) Hoechst nuclear dyeing, namely adding Hoechst33258 nuclear dye with the concentration of 1 mug/mL for 2 hours, and then observing and shooting under a fluorescence microscope.
Further, immunohistochemistry was also used to detect type ii collagen in third generation AF cells: the climbing tablet and the fixing step are the same as above, the primary antibody (mouse anti-rabbit type II collagen antibody) is added into a six-hole plate with fixed cells, the temperature is 4 ℃ overnight, then secondary antibody (goat anti-mouse IgG) is added for incubation for 30min at 37 ℃, DAB color development liquid is dripped for color development, and the shooting tablet is observed under a microscope.
As shown in the left part of the graph in FIG. 3, a small amount of cells begin to adhere after 24 hours of morphological change of the cells of the annulus fibrosus, and the cells just adhered are in a quasi-circular shape and rapidly extend into a short fusiform shape; as shown in the right part of FIG. 3, the cell growth rate is obviously accelerated after the third generation, and part of cell attachment exists in 2 hours.
As shown in fig. 4A and 4B, the fibrous ring cell slide showed uniform green fluorescence in cells after incubation of FITC-labeled secondary antibody in the same field of view, suggesting that cells expressed type i collagen. As shown in FIG. 4C, the results of the immunohistochemistry of the fibrous ring cells showed that the cytoplasm was brown yellow, suggesting that the fibrous ring cells also expressed type II collagen. Thus, the isolated cells were described as AF cell lines.
(2) Co-cultivation
The above isolated third generation AF cells were taken at 1.6X10 per well 5 The plants are planted in a Transwell6 pore plate, and after 24 hours reach the logarithmic phase; regulating density of esophageal cancer cells EC-109-1 to 2.5X10 respectively 8 1.5mL of the culture medium per well was co-cultured in a Transwell chamber at 37℃in 5% CO 2 Is cultured in the incubator of (2) for 3 weeks. The liquid was changed 1 time every 3 d. Meanwhile, a control group 1 is arranged, the upper layer is EC-109-1 cells, and the lower layer is basal culture solutionIs a DMEM incompletely high sugar culture solution. Meanwhile, a control group 2 is arranged, the upper layer is EC-109-1 cells, the lower layer is a basic culture solution, and the basic culture solution is a DMEM incompletely high sugar culture solution containing 25 mu mol/L propofol. Cells after 3 weeks of culture in the experimental group were designated EC-109-2 5 And (3) performing a cell scratch experiment on the cell to detect the migration capability of the cell, and performing a Transwell invasion experiment to observe the cell invasion capability. The cells obtained by co-culturing the test group were designated as EC-109-2 cells.
The scratch test steps are as follows: cells were 1X 10 per well 5 Individual cells were added to 6-well plates with parallel lines of labeling, 3 wells per group. Adding 2mL of complete culture medium into each well, repeatedly blowing, and placing at 37deg.C with 5% CO 2 After culturing for 10-12 hours, the cells were scored with a 200. Mu.L gun head perpendicular to the drawn lines and the bottom surface of the 6-well plate. After the scratch is finished, the cells are washed for 3 times by PBS, dead cells after the scratch are removed, 2mL of serum-free culture medium is added, and the cells are placed in an incubator for continuous culture. After 24 hours, samples were taken and photographed, and cell mobility was calculated to observe cell migration ability. Experiments were repeated 3 times and the mean was taken. The mobility of each group of cells was calculated by measuring the scratch width of 3 groups of cells at 0, 24h, cell mobility= (0 h scratch width-24 h scratch width)/0 h scratch width×100%.
The Transwell invasion experimental steps are as follows: taking logarithmic phase cells, washing with PBS, adding trypsin for digestion, centrifuging at 1000rpm for 5min, re-suspending cell pellet with 1ml of LDMEM medium, counting under microscope with cell counting plate, and diluting cell suspension to 1×10 with DMEM medium 8 L -1 And (5) standby. Placing Matrigel glue into a refrigerator at 4 ℃ in advance for overnight, melting the Matrigel glue into a liquid state for later use, diluting the Matrigel glue with PBS according to a ratio of 1:8, taking 35 mu L of diluted Matrigel glue, uniformly adding the diluted Matrigel glue onto a film at the bottom of a Transwell cell, and standing the Matrigel glue for 1-2 h at normal temperature. Marks were made on the Transwell cells according to the foregoing packets. 200. Mu.L of each cell suspension 1X 10 was added to each well of the labeled cells 8 L -1 Adding DMEM medium containing 10% fetal bovine serum by volume fraction into lower chamber, standing at 37deg.C and 5% CO 2 Culturing for 24h, washing the membrane with PBS, and fixing 4% formaldehyde at room temperature for 2h4g/L crystal violet is used for dyeing for 30min, cells in the inner layer of the membrane are wiped off after washing, a picture is taken under a microscope, 6 different visual fields are randomly taken from each cell, and the number of cells penetrating the membrane is observed and recorded.
The results are shown in Table 1, and the cell mobility and invasion capacity of the EC-109-2 cell line obtained in the experimental group are significantly increased compared to the control group 1 and the control group 2.
TABLE 1
| Grouping | Cell mobility/% | Invasive ability (number of cells penetrating membrane)/number |
| Experimental group | 33.52±4.63 | 56.32±6.77 |
| Control group 1 | 11.63±1.35 | 27.09±5.43 |
| Control group 2 | 15.48±2.11 | 31.58±7.25 |
4. MiRNA analysis of EC-109-2 cell lines, EC-109-1 cell lines and EC-109 cell lines
(1) RNA extraction and quality inspection
Taking the EC-109-2 cell line prepared by the experimental group, carrying out cell precipitation on the EC-109-1 cell line and the EC-109 cell line, crushing by a freezing crusher, extracting total RNA by a Trizol method, completely detecting quality, adding 500 mu L of 1mmol/LTris-HCl, adding 1 mu L of adenine nucleoside triphosphate mixture (ATPmix), fully and uniformly mixing, centrifuging and placing on ice. Another centrifuge tube was added with 1. Mu.g total RNA and then 2. Mu.LRNASpikeControl oligonucleotides; transfer 5.0 μlpoly (a) TailingMix into the RNA & SpikeControl mixture described above and centrifuge; incubation at 37℃for 15min; finally adding 4 mu L of 5-time FlashTagLiationnMixBiotin into the reaction system; adding 2 mu LT4 DNAligenase, mixing and centrifuging; a warm bath at 25 ℃ for 30min; and finally, adding Stopsolution to terminate the reaction. Transient centrifugation was collected and placed on ice for miRNA biotin labeling.
(2) Chip hybridization
The chip was taken out and equilibrated to room temperature. Adding the hybridization solution into the labeled miRNA system, fully and uniformly mixing and performing instantaneous centrifugation. The hybridization solution was placed on a heating block and incubated at 99℃for 5min. The hybridization solution after incubation was transferred to another heating block and incubated at 45℃for 5min. The hybridization solution was removed from the heating block and centrifuged for 5min. 100. Mu.L of hybridization solution was injected into the chip, and the chip was placed in a hybridization oven in an equilibrated state, and hybridization was performed at 48℃and 60rpm for 16 hours.
(3) Chip dyeing and scanning
Before hybridization, establishing test information, including Barcode of an input chip, by using GCOS software; preparing fluidics station, and running Prime; split charging reagent from StainModule, box1, the volume is enough for one chip to use; after hybridization, the hybridization solution was aspirated, and 100. Mu.LArrayHoldingBuffer was injected; in the Fluidics dialog box, the ExperimentName is selected, the corresponding Protocol is selected according to different chip types, clicking operation is performed, and chip cleaning and dyeing are started.
(4) Image acquisition and data analysis of chip
Performing quality control analysis on the miRNA chip by using miRNAQCTool software; calculating the ratio of the fluorescence intensities of miRNA chips among samples of EC-109-2, EC-109-1 and EC-109, and analyzing the difference of miRNAs among 2 samples; and clustering analysis is carried out on the miRNA with differential expression by adopting Cluster3.0 software, so that the quantity of the miRNA with obvious difference between two samples is summarized.
(5) Verification of relative fluorescent quantitative PCR method
The total miRNAs of EC-109-2, EC-109-1 and EC-109 cells were extracted by E.Z.N.A.TMmiRNAkit (OMEGA), respectively, and were reverse transcribed into cDNA using a high efficiency reverse transcription kit (GeneCopoe-ia). PCR reaction system: the total volume was 20. Mu.L, containing 2 XSYBRmix 10. Mu.L, 2. Mu.L each of the miR150 upstream and downstream primer or internal reference gene U6 upstream and downstream primer (2. Mu. Mol/L), 3. Mu.L cDNA, and 3. Mu.L double distilled water, wherein the primers were purchased from GeneCopoeia. Real-time quantitative PCR (qRT-PCR) reaction conditions: 50℃2min,95℃1min, (94℃30s,60℃15s,72℃15 s) for a total of 40 cycles, 72℃5min. After the reaction is finished, the specificity of the product is confirmed according to melting curve analysis, U6 is used as an internal reference, and a sample 2 to be detected is used as an internal reference -△△CT The values represent the relative expression levels of the target genes (CT is the number of cycles that each reaction tube undergoes when the fluorescent signal in the tube reaches a certain threshold value; sample DeltaCT=target gene CT value-U6 CT value), and all experiments were repeated 3 times or more.
(6) Results
Comparing the analysis scatter diagrams of the miRNA chip can intuitively see the difference of miRNA expression between two samples. As shown in FIG. 5, each data point in the fluorescence signal scatter plot represents the hybridization signal of each gene point on the chip, the red-marked and green-marked data points respectively represent the Ratio values of T1/C1miRNA not less than 2 and not more than 0.5, are miRNAs with different expression, and the black-marked data points represent the T1/C1 values between 0.5 and 2, and have basically no difference in expression. The T1 axis of the left graph of FIG. 5 represents the fluorescence signal intensity values of EC-109 cells, and the C1 axis represents the fluorescence signal intensity values of EC-109-2 cells, respectively. As shown in the right graph of FIG. 5, the T1 axis represents the fluorescence signal intensity values of EC-109 cells, and the C1 axis represents the fluorescence signal intensity values of EC-109-1 cells, respectively.
Cluster3.0 software was used to map a cluster analysis graph based on the expression results of miRNAs between EC-109-2, EC-109-1 and EC-109 cells. The results show that there are 128, 119 and 132 miRNAs in the EC-109-2, EC-109-1 and EC-109 cell lines in this order. Results were up-regulated in EC-109 cells, while 1miRNA down-regulated in EC-109-2 and EC-109-1 was obtained as miR150.
Further, RT-PCR is adopted to detect the expression of miR150 (miR 150 primer and U6 primer refer to 'mechanism of matrine F for regulating proliferation and apoptosis of cervical cancer HeLa cells through miR1501-3 p) [ J Shanxi traditional Chinese medicine, 2022, 1 st month, 13 rd volume, 1 st phase') among EC-109-2, EC-109-1 and EC-109 cells. The results are shown in FIG. 6 with reduced expression of miR150 in both EC-109-2 and EC-109-1 cells, and lower expression of EC-109-2 relative to EC-109 cells. Thus, the miR150 expression level in the highly-invasive cells is obviously lower than that of the parent cells.
5. Construction of cell line for stably rotating and over-expressing miR150 in esophageal cancer
(1) Construction of an over-expression miR150 vector
RNA of EC-109 cell line was extracted as miR150 (ENSG 00000268000, precursor sequence) by the method described above
CUCCCCAUGGCCCUGUCUCCCAACCCUUGUACCAGUGCUGGGCUCAGAC CCUGGUACAGGCCUGGGGGACAGGGACCUGGGGAC, SEQ ID No.6, the precursor was used as a template, the upstream primer GTCTCCCAACCCTTGTACCAG, SEQ ID No.1 was designed, and the downstream primer TTGGGAGACAGGGCCATG, SEQ ID No.2, into which two cleavage sites BglII and XhoI (uppercase above) were introduced. The PCR amplification procedure was: pre-denaturation at 94℃for 5min, denaturation at 95℃for 30s, annealing at 580C for 30s, extension at 72℃for 30s,35 cycles, and extension at 72℃for 10min. BglII and XhoI (purchased from TaKaRa) were double digested and purified again to obtain the desired miR150 precursor fragment. After pSuper.gfp/neo vector is cut by BglII and XhoI double enzyme and purified, a miR150 precursor target fragment is inserted, and after the vector is constructed successfully, the pSuper/miR150 is sent to Boshang biological Co-Ltd for sequencing and identification.
(2) Construction of an over-expressed esophageal cancer cell line
EC-109-1 and EC-109-2 cells were cultured with RPMI-1640 (10% HYCLONE serum), respectively, 1d 6 well plates, 1X 10 cells per well, prior to transfection 5 Cells were transfected with Lipofectamine2000 liposomes at 90% confluence. After transfection of EC-109-1 and EC-109-2 cells with pSuper.gfp/neo empty vector and pSuper/miR150 vector for 24h, stably transfected cell lines were selected by adding a culture solution containing G418 (1000 mg/L), after 3-4wk clone formation, the fluorescence display condition of the clone was observed under a fluorescence microscope, and if the clone showed fluorescence in a concentrated manner, the clone was picked up and the colony was continued to be expanded, and G418 was changed to a maintenance concentration of 400 mg/L. Finally lead toExpression of miR150 was verified by quantitative PCR (detection method see examples above). The results show that the relative expression level of the EC-109-1 over-expressed by miR150 is 2.67+/-0.35, the relative expression level of the EC-109-2 over-expressed by miR150 is 4.36+/-0.68, and the expression level of miR150 is obviously increased compared with cells (miR-CON) obtained by transfecting pSuper empty vector groups.
(3) Invasion assay of miR150 over-expression esophageal cancer cell line
Referring to the above test examples, transwell invasion experiments were performed on miR150 overexpressed EC-109-1 and miR150 overexpressed EC-109-2, respectively, with pSuper empty vector transfected into EC-109-1 and EC-109-2 cells as control groups (miR-CON), respectively. The results indicate that both the cell invasiveness of the EC-109-1 overexpressed by miR150 and the cell invasiveness of the EC-109-2 overexpressed by miR150 are significantly reduced relative to the control group transfected with pSuper empty vector to the EC-109-1 and EC-109-2 cells, respectively.
(4) WB detection of E-cadherin, vimentin and Claudin1 expression levels in the respective cell lines
Taking cell sediment of EC-109-1 over-expressed by miR150 and EC-109-2 over-expressed by miR150 and a control group thereof, respectively adding tissue lysate to carry out full lysis on ice for 30min, centrifuging at the temperature of 4 ℃ for 15min at 12000rpm, taking supernatant, adding SDS-loadingbuffer with the volume of 5 times, boiling in a boiling water bath for 15min, centrifuging at 12000rpm for 2min, then carrying out transfer after SDS-PAGE electrophoresis is finished, and sealing a PVDF membrane in 5% skimmed milk at room temperature for 1h after the transfer is finished. E-cadherein (ab 40772, abcam), vimentin antibodies (ab 92547, abcam) and Claudin1 (ab 211737, abcam) antibodies and antibodies were diluted 1:1000 with blocking solution, respectively, and covered with PVDF membrane and incubated overnight at 4 ℃. The PVDF membrane is washed for 8min by PBST, changed in liquid, repeated for 3 times, then added with goat anti-mouse secondary antibody, incubated for 2h at room temperature, washed for 3 times again by PBST, coated with luminous liquid and developed. The expression changes of the fibrinectin protein in the two groups of tissues take action as an internal reference.
As shown in FIG. 7, vimentin was expressed down-regulated in cells of miR 150-overexpressed EC-109-1 and miR 150-overexpressed EC-109-2 relative to the control, and E-cadherin and Claudin1 were expressed up-regulated relative to the control. Thus, miR150 can down-regulate expression of Vimentin in esophageal cancer cells, and up-regulate expression of E-cadherein and Claudin 1.
6. Construction of cell line for stably transferring and knocking down miR150 of esophageal cancer
(1) MiR150 knock-down oligonucleotide sequence design
A pair of most complementary oligonucleotide fragments was designed based on miR150 mature body sequence (UCUCCCAACCCUUGUACCAGUG, SEQ ID NO. 3) using Invitrogen miR-RNAi on-line design tool.
Sense strand:
5'-tgctgCACTGGTACAAGGGTTGGGAGAgttttggccactgactgacGTGACCATGTTCC CAACCC-3', SEQ the sense strand has the first 4 bases 5' -TGCT as the hanging end, the italic part as the complementary strand of miR150 mature body, the middle part as the loop structure and the bold part as the mature body sequence lacking 2 bases in the middle.
Antisense strand:
5'-cctgGTGACCATGTTCCCAACCCTCTgtcagtcagtggccaaaacCACTGGTACAACC CAAGGGc-3', SEQ ID No.5, the antisense strand is the sense strand complement except that the 5' -end CCTG is the overhang end.
Each oligonucleotide was dissolved in water to 50. Mu. Mol/L. An equal amount of the pair of complementary oligonucleotide fragments was added to a 0.2mLEP tube, and after thoroughly mixing, annealing reaction was performed on a PCR instrument: 2min at 95 ℃ and 2min at 50 ℃ for 3 cycles, and finally cooling to 4 ℃ to obtain an annealing product which is a knockdown sequence.
(2) Cloning
The knockdown sequence is connected to pcDNA6.2-GW/EmGFP-miR carrier, and the reaction system is that: knock-down sequence 2. Mu.L, pcDNA6.2-GW/EmGFP-miR vector 2. Mu.L, 10×T4ligaseBuffer 1. Mu.L, T4ligase (200U/. Mu.L) 1. Mu.L, ddH 2 O4. Mu.L, 16℃overnight. Transforming DH5 alpha competent cells by heat shock method, adding 2 μl of ligation product into freshly thawed 50 μLDH5 alpha competent cells, flicking and mixing, ice-bathing for 10min, heat shock for 40s at 42deg.C, immediately placing on ice for 2min, adding 300 μLLB culture medium, incubating at 200rpm and 37deg.C for 1 hr, centrifuging the pre-cultured bacterial liquid at 4000rpm for 5min, discarding supernatant, collecting 100 μl culture medium suspension, uniformly coating on LB/spectinomycin solid culture plate, and concentrating at 37deg.CCulturing overnight. Single colony is picked up on a solid culture plate and shaken overnight, bacterial liquid is sent to sequence, and the clone with correct sequencing result extracts plasmid (named pGW-miR 150). The pCDH-CMV-MCS-EF 1-copGGFP is subjected to ClaI/SalI double-enzyme digestion electrophoresis to recover a large fragment, pGW-miR150 is subjected to ClaI/XhoI double-enzyme digestion electrophoresis to recover a small fragment, xhoI and SalI are isotail enzymes, and ClaI sites on a pcDNA6.2-GW/EmGFP-miR carrier are obtained through mutagenesis and are positioned in front of a CMVprotter sequence. The small fragment recovered from pGW-miR150 and the large fragment recovered from pCDH-CMV-MCS-EF1-copGFP vector are connected by T4ligase at 16 ℃ overnight, stabl3 competent cells are transformed, uniformly coated on an LB/ampicillin solid culture plate, cultured overnight at 37 ℃, single colony is picked on the solid culture plate, and the single colony is shaken overnight at 37 ℃ to extract plasmids (named miR 150-KDEV). NdeI single enzyme digestion identification is carried out at 37 ℃ for 15min. And (3) carrying out enzyme digestion, identifying and then carrying out sequencing, and freezing at-80 ℃ for later use on cloning with a correct sequencing result.
The result is shown in figure 8, the sequence of the pGW-miR150 recombinant vector is correct, the miR150-KDLV recombinant vector is subjected to NdeI single digestion, and the digestion identification and sequencing results show that the vector construction is successful. The expression cassette of the recombinant lentivirus comprises: CMVPromoter, emGFP reporter gene, 5 'miRNAflavangregion, inserted knockdown sequence, 3' miRNAflavangregion and transcription termination sequence.
(3) Construction of cell line for stably knocking down miR150 in esophageal cancer
24 hours before lentivirus packaging, the EC-109-1 cells and the EC-109 cells are respectively inoculated into a 10cm culture dish, and the cells are suitable for transfection when the confluence degree of the cells reaches 70% -80%. Fresh DMEM medium (containing 10% FBS, no diabody) was replaced 1h before transfection, 12mL/10cm dishes. Transfection system: DMEM1mL, miR150 KDEV/pCDH (also named control LV) 10 μg, GMeasy LentiviralMix10 μl, HGTransgenereagent60 μl. Mixing, standing at room temperature for 20min, dripping into cell culture medium, and standing at 37deg.C in 5% CO 2 Culturing in an incubator. After 16-20 h of transfection, carefully sucking the cell culture solution, discarding the cell culture solution in a waste liquid cup containing the disinfectant, and then adding 10mL of fresh culture medium for continuous culture. After 48h of liquid exchange, the cell supernatant is sucked into a 50mL centrifuge tube, centrifuged for 10min at 1000rpm at 4 ℃, and the supernatant is added into amiconIn the concentration column, 4000 Xg centrifugal 25min, get about 250 u L virus concentrate, determination of lentivirus titer. The method comprises the following steps: 293T well grown was 1X 10 before transfection 5 the/mL density was seeded into 96-well plates at 100. Mu.L/well for a total of 10 wells. At 37℃5% CO 2 Culturing is continued in the incubator. The virus concentrate was diluted at a 10-fold gradient in a 1.5mL sterile EP tube for 10 dilutions in succession. The original culture medium in the 96-well plate is discarded, and diluted virus liquid is added. After 24h of infection, 100 mu LDMEM complete medium is replaced per well, which is beneficial to cell growth. On the 4 th day of infection, observing the number of cells containing fluorescence in each hole under a fluorescence microscope, collecting the concentrated miR150 knockdown lentivirus and negative control thereof to infect EC-109-1 cells and EC-109 cells, concentrating supernatant, and then measuring the titer, and observing and calculating the infection efficiency under an inverted fluorescence microscope, wherein the infection efficiency refers to the percentage of the fluorescent cells in the total cells.
As shown in FIG. 9, the results show that the cells expressing green GFP fluorescent protein account for more than 80% of the total number of cells when the EC-109-1 cells and the EC-109 cells knocked down by miR150 and negative control thereof are infected under a fluorescence microscope, which indicates that the cells grow well. After verification of miR150 expression by quantitative RT-PCR (the detection method is referred to the above examples), the results show that both miR150 knockdown EC-109-1 and miR150 knockdown EC109 are cells (miR-CON) obtained by transfection of pSuper empty vector groups, and the miR150 expression level is obviously reduced.
(4) Invasion assay and WB assay of miR150 knockdown esophageal cancer cell line
The attack test and WB test refer to the above test examples. As shown by the invasion test result, the cell invasion capacities of the EC-109-1 knockdown miR150 and the EC109 cell knockdown miR150 are obviously improved relative to the control group for transfecting the pCDH-CMV-MCS-EF 1-copGGFP empty vector into the EC-109-1 and EC109 cells respectively.
As shown in FIG. 10, the expression level of Vimentin in miR 150-knockdown EC-109-1 and miR 150-knockdown EC109 cells was up-regulated relative to that in the control group, while the expression levels of E-cadherin and Claudin1 were down-regulated relative to that in the control group. Thus, miR150 can down-regulate expression of Vimentin in esophageal cancer cells, and up-regulate expression of E-cadherein and Claudin 1.
The relationship between the expression quantity of miR150 and the expression quantity of signal factors of invasion and metastasis capacity of related cell transfer boxes is found, and miR150 has direct influence on the metastasis and invasion of esophageal cancer cells, can obviously inhibit the metastasis of esophageal cancer cells, and can be used as a marker for esophageal cancer treatment or prognosis.
Claims (10)
1. An esophageal cancer model cell line with strong metastatic invasion capability, which is characterized in that the esophageal cancer model cell line is one of miR150 knockdown EC-109-1 cells, miR150 knockdown EC-109-2 cells or miR150 knockdown EC-109 cells.
2. A method for constructing a strong invasive esophageal cancer model cell line, the method comprising:
in situ lung metastasis tissue cells EC-109-1 isolated from the nodulation of EC-109 cell esophageal carcinoma nude mice were placed in a Transwell six-well plate;
the cells of the annulus fibrosus were placed in a Transwell chamber at 37℃with 5% CO 2 Co-culture for 3 weeks, 1 change of liquid every 3 d.
3. A kit for knocking down a miR150 in a esophageal cancer model cell line, comprising a lentiviral vector carrying a miR150 knockdown sequence and a reagent for transfecting a cell, wherein the miR150 knockdown sequence comprises a pair of mostly complementary oligonucleotide fragments having a sense strand as shown in SEQ ID No.4 and an antisense strand as shown in SEQ ID No. 5.
4. The kit of claim 5, wherein the expression cassette of the lentiviral vector carrying the miR150 knockdown sequence comprises a CMVPromoter, emGFP reporter gene, a 5 'mirnas flankingregion, an inserted knockdown sequence, a 3' mirnas flankingregion, and a transcription termination sequence.
5. A preparation method of a kit for knocking down a miR150 in an esophageal cancer model cell line, which is characterized by comprising the steps of preparing a lentiviral vector carrying a miR150 knocking-down sequence and preparing a cell transfection reagent; the preparation method of the lentiviral vector carrying the miR150 knockdown sequence comprises the following steps:
obtaining a miR150 knockdown sequence, wherein the miR150 knockdown sequence comprises a sense strand shown as SEQ ID NO.4 and an antisense strand shown as SEQ ID NO. 5;
connecting the miR150 knockdown sequence to a pcDNA6.2-GW/EmGFP-miR carrier, transforming DH5 alpha competent cells, selecting positive clones, sequencing and identifying to obtain plasmid pGW-miR150;
the small fragment recovered by ClaI/SalI double digestion of pGW-miR150 is reacted with
The large fragment recovered by double digestion of pCDH-CMV-MCS-EF 1-copGGFP is connected with T4ligase at 16 ℃ overnight to transform Stabl3 competent cells, select positive clone, sequence and identify to obtain the lentiviral vector carrying miR150 knockdown sequence.
6. A weak invasive esophageal cancer model cell line, characterized in that the esophageal cancer model cell line is a miR150 over-expressed EC-109-1 cell and/or a miR150 over-expressed EC-109-2 cell.
7. The kit for detecting the metastasis capability of the esophageal epithelial cells and/or esophageal tumor cells is characterized by comprising a miRNA extraction reagent, a PCR reaction reagent and a PCR product electrophoresis reagent, wherein the PCR reaction reagent comprises a primer pair for amplifying and amplifying miR150 reverse transcription cDNA, and the primer pair is shown as SEQ ID NO. 1-2.
8. The kit for the miR150 overexpression of the esophageal cancer model cell line is characterized by comprising a miR150 overexpression vector and a reagent for transfecting cells, wherein the miR150 overexpression vector comprises a miR150 precursor sequence shown in SEQ ID NO. 6.
9. A reagent or a drug for inhibiting invasion of an esophageal cancer cell metastasis box, which is characterized by comprising a miR150 over-expression vector, wherein the miR150 over-expression vector comprises a precursor sequence of miR150, and the precursor sequence is shown as SEQ ID NO. 6.
10. The marker is miR150, and the nucleotide sequence of miR150 is shown in SEQ ID NO. 3.
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