CN110938627B - Use of an ESCO2 inhibitor for the manufacture of a medicament for the treatment of hypopharyngeal cancer - Google Patents
Use of an ESCO2 inhibitor for the manufacture of a medicament for the treatment of hypopharyngeal cancer Download PDFInfo
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Abstract
The invention discloses an application of an inhibitor of ESCO2 in preparation of a medicine for treating hypopharyngeal cancer, and belongs to the field of biological medicines. The inhibitor is selected from antisense nucleic acid which takes the ESCO2 protein or the transcript thereof as a target sequence and can inhibit the protein expression or gene transcription thereof; or a construct capable of expressing or forming the antisense nucleic acid. The siRNA of the target gene can obviously inhibit the proliferation and invasion of the Fadu cell of hypopharynx cancer and inhibit the tumorigenicity of cells and the growth of tumor tissues in vivo through verification, and has good medicament potential for treating hypopharynx cancer.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to application of an ESCO2 inhibitor in preparation of a medicine for treating hypopharyngeal cancer.
Background
Hypopharyngeal Squamous Cell Carcinoma (HSCC) is a malignant tumor located in the hypopharynx region, with poor differentiation and hidden locations. The common disease parts are piriform crypts, the posterior annulus and the posterior hypopharynx wall, and the recurrence rate and the distant metastasis rate are higher.
At present, the clinical treatment mode of the upper and lower pharyngeal cancer is in a diversified trend, wherein the emphasis is placed on the conservative treatment mainly based on radiotherapy and chemotherapy, and the conservative treatment is one of the more researched treatment methods in recent years because the laryngopharynx function of the patient can be kept, which is beneficial to improving the life quality of the patient to be treated. Despite the considerable controversy over this treatment, it is now widely used. In recent years, the incidence rate of hypopharyngeal carcinoma is on the rising trend year by year, and the survival rate of patients suffering from the hypopharyngeal carcinoma is obviously reduced compared with that of patients suffering from other malignant tumors due to the early appearance of metastasis.
How to improve the cure rate of hypopharyngeal carcinoma so as to improve the survival rate of patients is one of the difficulties which need to be solved aiming at the acute hypopharyngeal carcinoma at present. The method which is beneficial to early discovery, early diagnosis and early treatment of hypopharynx cancer is searched, the biological characteristics of hypopharynx cancer cells are researched, the biological mechanism influencing the biological behavior of hypopharynx cancer cells is explored, and the targeted treatment method is urgently found.
Disclosure of Invention
The present inventors have conducted extensive theoretical and practical studies in order to solve at least one of the above-mentioned technical problems, and unexpectedly found that ESCO gene has a significant association with hypopharyngeal cancer, thereby completing the present invention.
The invention provides the application of an inhibitor of ESCO2 in preparing a medicament for treating hypopharyngeal cancer, wherein the inhibitor is selected from antisense nucleic acid which takes ESCO2 protein or transcripts thereof as target sequences and can inhibit protein expression or gene transcription thereof; or a vector capable of expressing or forming the antisense nucleic acid; or a recombinant cell containing said vector.
The cohesin and cohesin regulatory protein play important roles in the process of cohesiveness and separation of sister chromatids, and simultaneously participate in the repair and transcriptional regulation of DNA double-strand breaks. The cohesin protein 2(ESCO2) is mainly composed of a C2H2 zinc finger structure near the N-terminal and an acetyltransferase functional region, and is a conserved cohesin acetyltransferase. Once the ESCO2 gene is mutated, the corresponding human body is caused to suffer from the Roberts syndrome.
In some embodiments of the invention, the medicament is for:
a) inhibiting proliferation of hypopharyngeal cancer cells;
b) promoting the apoptosis of the hypopharyngeal cancer cells;
c) inhibiting hypopharyngeal cancer cell migration;
d) inhibiting hypopharyngeal cancer cell invasion; or
e) Inhibit the growth of tumor body in hypopharyngeal carcinoma patients.
In some embodiments of the invention, the inhibitor is an siRNA molecule having a nucleotide sequence as shown in SEQ ID No.5 or SEQ ID No. 6.
In other embodiments of the invention, the inhibitor is an shRNA molecule whose coding sequence is shown in SEQ ID No.7 and SEQ ID No.8, or SEQ ID No.9 and SEQ ID No. 10.
In still further embodiments of the invention, the inhibitor is a vector comprising the sequences shown in SEQ ID No.7 and SEQ ID No.8, or SEQ ID No.9 and SEQ ID No. 10.
The second aspect of the invention provides an siRNA molecule for treating hypopharyngeal cancer, wherein the nucleic acid sequence of the siRNA molecule is shown as SEQ ID No.5 or SEQ ID No. 6.
In a third aspect, the invention provides an shRNA molecule for treating hypopharyngeal carcinoma, wherein the coding sequence of the shRNA molecule is shown as SEQ ID No.7 and SEQ ID No.8, or as shown as SEQ ID No.9 and SEQ ID No. 10.
In a fourth aspect, the invention provides a vector for the treatment of hypopharyngeal cancer, said vector comprising the sequences shown in SEQ ID No.7 and SEQ ID No.8, or comprising the sequences shown in SEQ ID No.9 and SEQ ID No. 10.
In a fifth aspect, the invention provides a recombinant lentivirus, said recombinant cell being obtained by cotransfection of a mammalian cell with a vector according to the fourth aspect of the invention together with a viral packaging helper plasmid pHelper 1.0 vector and a viral packaging helper plasmid pHelper2.0 vector.
In a sixth aspect, the present invention provides a host cell comprising at least one of the siRNA of the second aspect, the shRNA of the third aspect, the vector of the fourth aspect and the recombinant lentivirus of the fifth aspect. The present invention is not particularly limited with respect to the specific type of the host cell, and examples thereof include 293T cells.
In a seventh aspect, the present invention provides a pharmaceutical composition for treating hypopharyngeal cancer, comprising an siRNA molecule according to the second aspect of the present invention, an shRNA molecule according to the third aspect of the present invention, a vector according to the fourth aspect of the present invention, a recombinant lentivirus according to the fifth aspect of the present invention, or a host cell according to the sixth aspect of the present invention, and a pharmaceutically acceptable pharmaceutical carrier.
As used herein, the term "ESCO 2 inhibitor" includes antagonists, down-regulators, blockers, etc., as long as they are capable of down-regulating the expression level of ESCO 2. They may be chemical compounds, chemical small molecules, biological molecules. The biological molecules can be nucleic acid level (including DNA and RNA) or virus products for inhibiting the expression of the ESCO 2. The ESCO2 inhibitor is any substance which can reduce the activity of ESCO2, reduce the stability of ESCO2, reduce the expression of ESCO2 and reduce the effective action time of ESCO2, and the substances can be used in the invention and can be used as substances which are useful for reducing ESCO 2. For example, the inhibitor is: nucleic acid inhibitors, protein inhibitors, nucleases, nucleic acid binding molecules, provided that they are capable of down-regulating the expression of ESCO 2.
The invention has the advantages of
Compared with the prior art, the invention has the following beneficial effects:
1. the invention finds that the suppression of ESCO2 can inhibit the proliferation, migration and invasion of hypopharyngeal carcinoma cell strains and promote apoptosis, shows that the suppression of ESCO2 has the effect of suppressing the occurrence and development of hypopharyngeal carcinoma, can be used as an emerging therapeutic target, reduces the expression of ESCO2 aiming at patients with high expression of the hypopharyngeal carcinoma ESCO2, has a potential therapeutic effect, and the inhibitor of ESCO2 can be used for preparing a medicine for treating the hypopharyngeal carcinoma.
2. The siRNA provided by the invention can specifically reduce the expression of the ESCO2 gene, and has good potential in preparing medicines for treating hypopharyngeal cancer.
Drawings
FIG. 1 shows the expression of ESSCO2 in FaDu cells of hypopharyngeal carcinoma.
FIG. 2 shows the result of agarose gel electrophoresis of RNA interference vector positive clones. Lane 1: negative control (ddH)2O); lane 2: self-ligation control (empty vector self-ligation control group); lane 3: 250bp Marker: 5kb, 3kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp and 100bp are sequentially arranged from top to bottom; lanes 4-8: a: monoclonal psc45154-1, 2, 3, 4, 5, B: monoclonal psc45156-1, 2, 3, 4, 5.
Figure 3 shows the results of lentivirus titer detection.
FIG. 4 shows a fluorescent map of lentivirus-infected FaDu cells:
FIG. 5 shows the knockdown efficiency of the recombinant plasmid for the ESCO2 gene in FaDu.
Fig. 6 shows the effect of knockdown ESCO2 on FaDu cell growth. Wherein shCtrl is a control group, shESCO2-1 and shESCO2-2 are experimental groups, A curve is the absolute number of cell growth, and B curve is the relative value of cell growth. (cell count/fold is the growth multiple of day1-day5 versus day1 for each experimental group).
Figure 7 shows the impact of knockdown ESCO2 on the FaDu cell growth cycle. Wherein shCtrl is a control group, and shESCO2-1 and shESCO2-2 are experimental groups.
Figure 8 shows the effect of knockdown ESCO2 on FaDu cell proliferation activity. Wherein shCtrl is a control group, shESCO2-1 and shESCO2-2 are experimental groups (OD490 reflects the number of cells with viability).
Figure 9 shows the effect of knockdown ESCO2 on FaDu apoptosis. Wherein shCtrl is a control group, and shESCO2-1 and shESCO2-2 are experimental groups.
Fig. 10 shows the effect of knockdown ESCO2 on the migratory capacity of FaDu cells. Wherein shCtrl is a control group, and shESCO2-1 and shESCO2-2 are experimental groups.
Fig. 11 shows the effect of knockdown ESCO2 on FaDu cell invasion (number of migrations). Wherein shCtrl is a control group, and shESCO2-1 and shESCO2-2 are experimental groups.
FIG. 12 shows the tumor formation and tumor size of the living animals. A: for comparison of animal pictures, from left to right, the animals are numbered as follows: NC: 4. 3, 10, 6, 9, 5, 7, 8, 1, 2, KD: 16. 20, 11, 12, 13, 14, 15, 17, 18, 19; b: comparing tumor body images, and numbering the tumor bodies from left to right as follows: NC: 4. 3, 10, 6, 9, 5, 7, 8, 1, 2, KD: 16. 20, 11, 12, 13, 14, 15, 17, 18, 19; c: comparing the images of the animals and the tumor body, the animals and the tumor body are numbered from left to right as follows: NC: 4. 3, 10, 6, 9, 5, 7, 8, 1, 2, KD: 16. 20, 11, 12, 13, 14, 15, 17, 18, 19. NC: a control group; KD: experimental group.
Figure 13 shows an animal in vivo imaging contrast. NC control group; KD experimental group.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.
Examples
The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
The experimental procedures in the following examples are conventional unless otherwise specified. The instruments used in the following examples are, unless otherwise specified, laboratory-standard instruments; the test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
In the examples described below, the reference human ESCO2 is numbered NM _ 001017420.
In the examples below, statistical analysis was performed using GraphPad Prism 6.0 software. All in vitro experiments were repeated three more times. Data are expressed as mean ± Standard Deviation (SD). P <0.05 was considered statistically significant.
Example 1 high expression of ESCO2 in the FaDu cell line of hypopharyngeal carcinoma
RNA extraction and reverse transcription quantitative PCR (RT-qPCR)
1. Total RNA extraction: taking out a 6-hole plate for culturing cells, pouring out the original culture medium, washing with PBS twice, blowing to fully crack the cells, transferring the cells into a 1.5mL EP tube without RNase, and standing for 5 min; adding 200 mu L/tube chloroform, reversing, mixing for 15 seconds, standing at room temperature for 10 minutes, and centrifuging at constant speed of 4 ℃ for 15 minutes at the rotating speed of 12800 rpm; separating supernatant liquid after centrifugation in an EP tube and uniformly mixing with isopropanol (precooling) with the same volume; standing at 4 ℃ for 10 minutes, and centrifuging for 12 minutes at the rotating speed of 12800 rpm; the supernatant after centrifugation was removed and rinsed with 75% 1mL ethanol (freshly prepared with DEPC water); centrifuging at constant temperature of 4 ℃ for 5 minutes at the rotating speed of 11800 rpm; removing the upper layer liquid after centrifugation, repeatedly centrifuging, and drying; RNase-free water was added to the cleared RNA precipitate to completely dissolve the precipitate, and the concentration was measured with a spectrophotometer (Nanodrop 2000/2000C).
2. Reverse transcription: 7 μ L RNase-Free H2O, 2.0 mu g of total RNA, 1 mu L of OligodT with the concentration of 0.5 mu g/mu L, evenly inverted up and down, centrifuged and bathed for 10 minutes at 70 ℃; after the warm bath was completed, annealing was performed in a rapid ice bath (ice bath was performed in an ice-water mixture). Then, 5 XTRT buffer 4. mu.L, 10mM dNTPs 2. mu. L, Rnasin (40U/. mu.L), 0.4. mu. L, M-MLV-RTase (200U/. mu.L), 1. mu. L, RNase-Free H were added2O2.6. mu.L. The reaction conditions are as follows: 42 ℃ for 1h, 70 ℃ for 10 min.
3. Quantitative PCR: each 12. mu.L reaction system contained 2 XPCR Mix (ABI) 6. mu.L, upstream and downstream primers 0.5. mu.L each, cDNA 1. mu.L, ddH2O4.0. mu.L. The reaction conditions are as follows: 15s at 95 ℃; 95 ℃ for 15s, 60 ℃ for 30s, 40 cycles. The sequences of the primers used in the experiments are shown in Table 1
TABLE 1 fluorescent quantitative RT-PCR primer sequences
As a result, it was found (as shown in FIG. 1) that ESSCO2 was expressed in high abundance in FaDu cells, a cancer of the hypopharynx.
Example 2 Effect of knocking down ESCO2 Gene expression on proliferation and/or growth, invasion and metastasis of FaDu cells of hypopharyngeal carcinoma
First, RNAi lentiviral vector preparation
1. Target design
Aiming at the sequence of the ESCO2 gene, 2 RNAi target sequences are designed according to the RNAi sequence design principle, as shown in Table 2.
TABLE 2 nucleotide sequences of 2 interfering target siRNA of ESCO2 gene
Table 3 shows the nucleotide sequences of the 2 shRNAs used in the examples, which are the nucleotide sequences of psc45154-1, psc45154-2, psc45156-1 and psc45156-2
2. Vector cleavage
A50. mu.L digestion system was prepared according to Table 5. Sequentially adding various reagents according to the sequence of a list, lightly blowing and uniformly mixing by using a pipette, carrying out instantaneous centrifugation, and reacting for 1h at 37 ℃. And (4) carrying out agarose gel electrophoresis on the vector enzyme digestion product, and recovering a target band.
TABLE 5 vector cleavage System
3. DNA annealing of shRNA to form double-stranded DNA
The synthesized single-stranded DNA oligo dry powder was dissolved in annealing buffer (final concentration 20. mu.M) and water-washed at 90 ℃ for 15 min. After naturally cooling to room temperature, a double strand with a cohesive end was formed.
4. Carrier attachment
The double-cut linearized vector and the annealed double-stranded DNA were ligated by T4 DNA ligase (T4 DNA ligase) according to the system of Table 6 for 1-3h at 16 ℃.
TABLE 6 Carrier attachment System
5. Transformation of
Adding 10 μ L of the ligation reaction product into 100 μ L of competent cells, flicking the tube wall, mixing, and standing on ice for 30 min; heat shock is carried out for 90s at 42 ℃, and incubation is carried out for 2min in ice bath; adding 500 μ L LB culture medium, and shake culturing at 37 deg.C for 1 h; taking a proper amount of bacterial liquid, uniformly coating the bacterial liquid on a flat plate containing corresponding antibiotics, and carrying out inverted culture in a constant-temperature incubator for 12-16 h.
6. Sequencing identification
Inoculating the identified positive clone transformant into a proper amount of LB liquid culture medium containing corresponding antibiotics, culturing at 37 ℃ for 12-16h, taking a proper amount of bacterial liquid for PCR amplification, detecting by using gel electrophoresis, and further identifying by sequencing.
As a result, it was found (as shown in FIG. 2) that the RNA interference vector positive clone can amplify a band of about 307bp, indicating that the shRNA fragment is successfully ligated to the vector.
The results of sequencing of RNA interference vector positive clones are analyzed as shown in Table 7:
TABLE 7 sequencing results for RNA interference vector positive clones
7. Plasmid transfection and lentivirus harvesting
Viral packaging involves a total of three plasmids: the vector comprises a tool vector plasmid GV115 vector (purchased from Shanghai Jikai gene science and technology Co., Ltd.), a virus packaging Helper plasmid Helper 1.0 vector (purchased from Shanghai Jikai gene science and technology Co., Ltd.) and a virus packaging Helper2.0 vector (purchased from Shanghai Jikai gene science and technology Co., Ltd.). 293T cells were co-transfected with the above three plasmids.
24h before transfection, 293T cells in the logarithmic growth phase were trypsinized and cell density was adjusted to about 5X 10 in medium containing 10% serum615mL, reseeding in 10cm cell culture dish, 37 deg.C, 5% CO2Culturing in an incubator; the cell can be used for transfection after 24 hours when the cell density reaches 70-80%; replacing the medium with a serum-free medium 2h before transfection; adding each DNA solution (20 μ g of GV115 plasmid, 15 μ g of pHelper 1.0 vector plasmid, 10 μ g of pHelper2.0 vector plasmid) into a sterilized centrifuge tube, uniformly mixing with a corresponding volume of Gecky transfection reagent, adjusting the total volume to 1mL, and incubating for 15min at room temperature; the mixed solution is slowly dripped into 293T cell culture solution, mixed evenly and treated at 37 ℃ with 5% CO2Culturing in a cell culture box; culturing for 6h, discarding the culture medium containing the transfection mixture, adding 10mL of PBS for washing once, gently shaking the culture dish to wash the residual transfection mixture, and then pouring and discarding; slowly adding 20mL of cell culture medium containing 10% serum, and heating at 37 deg.C with 5% CO2Culturing in the incubator for 48-72 h.
8. Lentivirus concentration and purification and quality control
Collecting 293T cell supernatant 48h after transfection (which can be counted as 0h after transfection) according to cell states; centrifuging at 4000g for 10min at 4 deg.C to remove cell debris; the supernatant was filtered through a 0.45 μm filter into a 40mL ultracentrifuge tube; respectively balancing samples, putting the ultracentrifuge tubes with virus supernatant into a Beckman ultracentrifuge one by one, centrifuging for 2h at 4 ℃ and 25000 rpm; after the centrifugation is finished, removing the supernatant, removing the liquid remained on the tube wall as much as possible, adding a virus preservation solution, and lightly and repeatedly blowing and resuspending; after full dissolution, centrifuging at high speed 10000rpm for 5min, taking the supernatant and subpackaging according to the requirement.
The virus titer detection was performed by fluorescence microscopy, and the result showed (as shown in FIG. 3) that the virus titer was 2X 108TU/mL。
Second, Lentiviral transfection
To ensure gene interference efficiency, this example designs 2 RNA interference targets (siRNA) for ESCO2 gene, and performs lentiviral packaging on 2 plasmids carrying different targets, respectively, to detect the knockdown efficiency of the target gene after virus infection of cells.
Cell resuspension 24 hours before experiment after trypsinization of logarithmically growing FaDu cells: the cell concentration was 2X 105one/mL cell suspension, using 10mL MEM + 10% FB medium. The resuspended cell fluid was inoculated into 6-well culture plates with a plating amount of about 15-30%, and the culture medium and virus fluid were added in the proportions shown in Table 8 for the experiments.
TABLE 8 lentivirus infection virus dose
And (3) observing the green fluorescence expression condition of GFP by a fluorescence microscope after the GFP is cultured for 72 hours, if the positive rate is more than 80%, subpackaging the cells for subsequent experiments, otherwise, adjusting infection conditions, repeating the experiments for infection for 3 days, taking the cells in the logarithmic growth phase, and checking the biological functions of the cells.
Fluorescence microscopy showed (FIG. 4) that more than 80% of the cells in each experimental group showed green fluorescence, i.e., 80% of the recombinant lentiviruses were transfected.
The knockdown efficiency of the target gene of the cell after virus infection is detected, and the result shows (as shown in figure 5) that the expression level of the ESCO2 gene in the FaDu cell of shESCO2-1 and shESCO2-2 experimental groups is inhibited at the mRNA level 3 days after shRNA lentivirus infection.
Thirdly, checking the biological function of cells
(1) Cell counting experiments
Pancreatin digests each experimental group of cells, counts the cells after cell resuspension, sets 96-well plate with 3 wells in each group, lays the culture system with 100 mul/well equivalent cells on a standard of 2000 cells/well, cultures with room temperature, and detects and reads the plate 1 time/day from the next day for 5 consecutive days. Adjusting detection parameters by counting the number of cells in the scanning well; and (6) performing data statistical mapping.
The results are shown in FIG. 6, where cells were plated in 96-well plates at 2000 cell plating numbers 3 days after shRNA lentivirus infection. Celigo continuously tests for 5 days, and found that the proliferation rate of the FaDu cells in shESCO2-1 and shESCO2-2 experimental groups is remarkably inhibited. Showing that the ESCO2 gene is significantly related to the proliferation capacity of FaDu cells.
(2) Cell cycle experiments
Cells were passaged on day 3 post infection, starved on day 4, and examined on day 5. When the cells of each experimental group grow to the coverage rate of about 80%, the cells are digested by pancreatin, and the cells are collected in a 5mL centrifuge tube after heavy suspension, wherein each group is provided with three multiple holes. Centrifuging the centrifuge tube in a centrifuge for 5min, discarding the washing precipitate for 1 time, rotating at 1300 deg.C, and pre-cooling the washing liquid at 4 deg.C to obtain D-Hanks (pH 7.2-7.4).
Centrifuging the D-Hanks mixed solution for 5min at the same rotating speed again, precooling 75% ethanol at 4 ℃, and fixing the centrifuged cell sediment for 1 h. And removing the fixing liquid at the same rotating speed again, and repeating the steps. Preparing a cell stain: 40 XPI stock (2 mg/mL): 100 XRNase stock solution (10mg/mL) 1 XD-Hanks 25:10: 1000. The cells are subjected to staining treatment by staining solution after being resuspended, and the cell passing rate when the cells are loaded on the machine is 300-800 cells/s. And (4) performing computer detection and data analysis.
The results are shown in fig. 7, after 5 days of shRNA lentivirus infection, the cells in S phase of shESCO2-1 group were decreased (P <0.05), the cells in G1 phase were increased (P <0.05), and the cells in G2/M phase were not significantly changed; the shESCO2-2 group had no significant change in cells in S phase, increased cells in G1 phase (P <0.05), and decreased cells in G2/M phase (P <0.05), indicating that the ESCO2 gene is associated with the FaDu cell cycle distribution.
(3) Cell proliferation assay
Experimental cells grown logarithmically were trypsinized 24 hours prior to the experiment and were counted after complete medium resuspension. Passage was carried out on day 3 by shRNA lentivirus infection and detection was started on day 5. Cells were plated in 96-well plates with a cell plating number of 2000. 20mL MTT was added to the cell culture wells at a concentration of 5 mg/mL. After adding MTT 4h, the excess culture medium was removed, and 100 μ L of formazan particles were dissolved in DMSO. Transferring the mixed solution into a shaking instrument for 5min, and detecting the OD value at 490nm by using an enzyme-labeling instrument.
The results are shown in FIG. 8, where cells were plated in 96-well plates at 2000 plates 3 days after shRNA lentivirus infection. After 5 days of continuous detection, the proliferation rate of FaDu cells in shESCO2-1 and shESCO2-2 experimental groups is remarkably inhibited. Showing that the ESCO2 gene is significantly related to the proliferation capacity of FaDu cells.
(4) Apoptosis assay
Cells were passaged 3 days after infection and detection started 5 days. Plating on a 96-well plate, wherein the plating number is 2000, the cell suspension is resuspended after the cell growth is reached to 80 percent fusion degree and the cell suspension is induced to undergo apoptosis and pancreatin digestion, the cell suspension and the supernatant cells are loaded into a centrifuge tube (5mL), and the culture system is as follows: 6 well plates, 2 mL/well, 3 negative wells per group. Centrifuging the centrifuge tube in a centrifuge for 5min, discarding the washing precipitate for 1 time, rotating the centrifuge at 1300rpm, and washing with 4 deg.C pre-cooled D-Hanks (pH 7.2-7.4). The centrifuged cell pellet was collected, washed with 1 × binding buffer and centrifuged at the same speed for 3 min. Collecting cell precipitate, resuspending 10 μ L Annexin V-APC with 200 μ L1 XBinding buffer, mixing with the cell suspension, staining at room temperature in dark for 10-15min, and analyzing the experimental result.
The result is shown in fig. 9, when shRNA lentivirus is detected after being infected by 3 days, the shESCO2-1 and shESCO2-2 experimental groups have obviously increased cells undergoing apoptosis, which indicates that the ESCO2 gene is obviously related to the apoptosis of FaDu cells.
(5) Cell migration assay
The Oris TM obstructive matter is soaked in alcohol for sterilization, aired and placed in a 96-well plate at a rate of 100 mu L/well (cell inoculation number of 50000 cells/well); each hole is added with 3 multiplied by 104(ii) infected cells; the next day, Oris TM occlusion was carefully removed, gently rinsed 2-3 times with PBS, and cultured with 1% FBS medium; celigo floor sweeping, 5% CO at 37 ℃2Culturing in incubator, and taking pictures at 0h, 24h, and 48 h. The area of the green-fluorescent cells was calculated by integrating the input parameters of the analysis settings. The difference in tumor cell migration capacity can be measured by comparison based on the cell area values and time points.
As a result, as shown in FIG. 10, the migration ability of the FaDu cells in experimental groups shESCO2-1 and shESCO2-2 was reduced by the migration analysis of shRNA lentivirus 3 days after scratching for 24 hours. Indicating that the ESCO2 gene is significantly related to the migratory capacity of FaDu cells.
(6) Cell invasion assay
The Transwell kit was removed, and the laboratory chamber with 100. mu.L/well in the inner chamber and 600. mu.L/well in the outer chamber was placed in a 24-well plate and incubated in an incubator for 1 hour. Collecting cells with good growth state, digesting, collecting, and preparing into single cell suspension with serum-free culture medium, wherein the cell concentration is adjusted to 106And L. 600. mu.L of 30% FBS medium was added to the lower chamber, and 100. mu.L of the above cell suspension was added to the upper chamber (the medium remaining in the upper chamber was removed before adding the cells). Culturing at constant temperature of 37 ℃ for 48 h. Excess medium and non-transferred cells in the chamber were removed and fixed in 4% paraformaldehyde solution for 30 min. Taking out the small chamber, sucking dry the fixing solution in the small chamber, dyeing and transferring cells for 1min by using the dyeing solution, washing the small chamber, and drying in the air. Taking a picture by a microscope: each transwell cell was photographed with a randomly selected field of view at 100 x, 200 x. Data analysis the number of transferred cells in each group was counted according to 200 × image count, and statistical analysis was performed.
The results are shown in FIG. 11, where shESCO2-1 and shESCO2-2 experimental group FaDu cells were significantly inhibited in their transfer ability 3 days after shRNA lentivirus infection. Indicating that the ESCO2 gene is significantly related to the transfer capacity of FaDu cells. .
Fourth, animal in vivo experiment
To further observe the effect of ESCO2 on the tumorigenicity of cancer cells, this study constructed an in vivo tumorigenic model, as follows: preparing tumorous cells according to experimental groups → injecting animals subcutaneously with a certain cell amount → feeding animals to visualize tumor bodies → measuring animal weight, tumor body size (long diameter and short diameter) → live imaging → sacrifice, digital photography → tumor extraction, weighing → tumor body tissue preservation → data result processing.
A sufficient number of cells were prepared and the complete medium was resuspended into a cell suspension after trypsinization of the tumor cells of each experimental composition in logarithmic growth phase. Cells were infected with pre-packaged lentiviruses. Generally, after 24-48h of infection, the cell is screened by using a culture medium containing antibiotics (such as puromycin), and the survival rate of the cells is the positive infection rate.
Cells were counted on a hemocytometer and finally resuspended in a volume of D-Hanks or PBS to give a cell suspension concentration of 2E +7 cells/mL. After tumor cells were prepared (2E +7/mL), the cells were aspirated using a disposable sterile syringe and injected subcutaneously into animals at 200. mu.L each. According to the cell tumor forming capacity, after 5 to 20 days, the tumor forming condition is observed and the tumor size and the animal body weight are measured. The tumor size and animal body weight frequency are determined according to the tumor proliferation condition, and at least 5 times of measurement data are ensured. If the administration is needed, when the tumor volume reaches the administration condition, the regular administration is carried out by the administration modes such as gavage, intraperitoneal injection and the like according to the administration scheme.
In vivo imaging: live imaging of animals during the experiment or prior to sacrifice: the animals are anesthetized by intraperitoneal injection of 0.7% sodium pentobarbital in an amount of 10ul/g, and after a few minutes, the animals are anesthetized (or isoflurane gas anesthesia is carried out by using a living body imaging instrument with a gas anesthesia system), the animals are placed under the living body imaging instrument for imaging, fluorescence is observed, and data are stored. After 28 days of subcutaneous injection or according to the actual tumor formation condition and animal welfare ethical regulation (the length and the short diameter of a tumor body are less than 20mm), experimental animals are euthanized by injecting 2% of sodium pentobarbital in excess, and cervical dislocation is carried out to confirm death. Animals are arranged on the white board and a scale is placed on the left and above with 2 to view the specific scale, and a picture of the animals is taken with a digital camera.
The results are shown in FIG. 12: in the control group, the nude mice can be found to have good tumor formation, the ESCO2 inhibition experiment composition has poor tumor formation, and the tumor is obviously reduced compared with the control group.
In vivo imaging is shown in figure 13: compared with a control group, the experimental group has poorer in vivo tumor formation and obviously reduced tumor volume.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
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Claims (5)
- Use of an inhibitor of ESCO2 selected from the group consisting of antisense nucleic acids targeting ESCO2 protein or its transcript and capable of inhibiting protein expression or gene transcription thereof for the manufacture of a medicament for the treatment of hypopharyngeal cancer; or a vector capable of expressing or forming the antisense nucleic acid; or a recombinant cell containing said vector.
- 2. The use according to claim 1, wherein the medicament is for:a) inhibiting proliferation of hypopharyngeal cancer cells;b) promoting the apoptosis of the hypopharyngeal cancer cells;c) inhibiting hypopharyngeal cancer cell migration;d) inhibiting hypopharyngeal cancer cell invasion; ore) Inhibit the growth of tumor body in hypopharyngeal carcinoma patients.
- 3. The use according to claim 1, wherein the inhibitor is an siRNA molecule having the nucleotide sequence shown in SEQ ID No.5 or SEQ ID No. 6.
- 4. The use according to claim 1, wherein the inhibitor is an shRNA molecule whose coding sequence is shown in SEQ ID No.7 and SEQ ID No.8, or SEQ ID No.9 and SEQ ID No. 10.
- 5. The use according to claim 1, wherein the inhibitor is a vector comprising the sequences shown in SEQ ID nos. 7 and 8, or SEQ ID nos. 9 and 10.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911166039.XA CN110938627B (en) | 2019-11-25 | 2019-11-25 | Use of an ESCO2 inhibitor for the manufacture of a medicament for the treatment of hypopharyngeal cancer |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105586389A (en) * | 2014-10-21 | 2016-05-18 | 天津华大基因科技有限公司 | Kit and application thereof in detection on hereditary bone disease genes |
| US20180357378A1 (en) * | 2017-06-13 | 2018-12-13 | Alexander Bagaev | Systems and methods for generating, visualizing and classifying molecular functional profiles |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105586389A (en) * | 2014-10-21 | 2016-05-18 | 天津华大基因科技有限公司 | Kit and application thereof in detection on hereditary bone disease genes |
| US20180357378A1 (en) * | 2017-06-13 | 2018-12-13 | Alexander Bagaev | Systems and methods for generating, visualizing and classifying molecular functional profiles |
Non-Patent Citations (5)
| Title |
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| Chen HM等.ESCO2 knockdown inhibits cell proliferation and induces apoptosis in human gastric cancer cells.《Biochemical and Biophysical Research Communications》.2018,第496卷(第2期), * |
| Defects in the Fanconi Anemia Pathway and Chromatid Cohesion in Head and Neck Cancer;Stoepker, C等;《CANCER RESEARCH》;20150930;第75卷(第17期);第3543-3553页 * |
| ESCO2 knockdown inhibits cell proliferation and induces apoptosis in human gastric cancer cells;Chen HM等;《Biochemical and Biophysical Research Communications》;20180205;第496卷(第2期);第476页左栏第4-6段 * |
| Purα基因过表达和RNAi慢病毒载体的构建及应用;贾中发等;《宁夏医科大学学报》;20140630(第6期);摘要和第623页左栏第3段 * |
| TRIM58/cg26157385 methylation is associated with eight prognostic genes in lung squamous cell carcinoma;Zhang, WM等;《ONCOLOGY REPORTS》;20180731;第40卷(第1期);摘要 * |
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