Application of XPR1 inhibitor in preparation of product for inhibiting migration and/or proliferation of thyroid cancer cells
阅读说明：本技术 Xpr1抑制剂在制备抑制甲状腺癌细胞迁移和/或增殖的产品的应用 (Application of XPR1 inhibitor in preparation of product for inhibiting migration and/or proliferation of thyroid cancer cells ) 是由 陈恩东 尤捷 陈不染 陈盈盈 李�权 胡孝渠 项晓 蔡业丰 王青煊 杜远 于 2021-09-13 设计创作，主要内容包括：本发明属于肿瘤分子生物学领域,具体涉及XPR1抑制剂在制备抑制甲状腺癌细胞迁移和/或增殖的产品的应用。本发明发现甲状腺癌细胞中XPR1基因的表达显著高于正常甲状腺细胞,通过CCK-8实验、集落形成实验、Transwell迁移实验、Matrigel侵袭实验、流式细胞术、细胞凋亡实验和蛋白印迹实验验证了XPR1基因对甲状腺癌细胞迁移和增殖能力的影响。本发明发现敲减XPR1基因的表达能有效地降低甲状腺癌细胞的迁移和增殖能力。(The invention belongs to the field of tumor molecular biology, and particularly relates to an application of an XPR1 inhibitor in preparation of a product for inhibiting migration and/or proliferation of thyroid cancer cells. The invention finds that the expression of the XPR1 gene in the thyroid cancer cell is obviously higher than that of a normal thyroid cell, and the influence of the XPR1 gene on the migration and proliferation capacity of the thyroid cancer cell is verified through a CCK-8 experiment, a colony forming experiment, a Transwell migration experiment, a Matrigel invasion experiment, a flow cytometry, an apoptosis experiment and a western blot experiment. The invention discovers that the expression of the XPR1 gene is knocked down, so that the migration and proliferation capacity of thyroid cancer cells can be effectively reduced.)
1. Application of an XPR1 inhibitor in preparing products for inhibiting migration and/or proliferation of thyroid cancer cells.
2. Use according to claim 1, characterized in that: the product is a medicament.
3. Use according to claim 1, characterized in that: the thyroid cancer cell is one or more of TPC-1 cell, BCPAP cell and FTC cell.
4. Use according to claim 1, characterized in that: the XPR1 inhibitor refers to a product capable of reducing the content or expression level of XPR 1.
5. Use according to any one of claims 1 to 4, characterized in that: the XPR1 inhibitor is a specific small interfering Si-RNA targeting XPR 1.
6. A pharmaceutical composition for inhibiting migration and/or proliferation of thyroid cancer cells, comprising: which include XPR1 inhibitors.
7. The pharmaceutical composition of claim 6, wherein: the XPR1 inhibitor refers to a product capable of reducing the content or expression level of XPR 1.
8. The pharmaceutical composition according to claim 6 or 7, characterized in that: the XPR1 inhibitor is a specific small interfering Si-RNA targeting XPR 1.
Technical Field
The invention belongs to the field of tumor molecular biology, and particularly relates to an application of an XPR1 inhibitor in preparation of a product for inhibiting migration and/or proliferation of thyroid cancer cells.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Biomarkers (biomarkers) are biochemical markers that can mark changes or changes that may occur in the structure or function of systems, organs, tissues, cells, and subcellular structures and functions, and have a wide range of uses. Biomarkers can be used for disease diagnosis, tumor burden monitoring, risk assessment to determine tumor recurrence and metastasis, or to evaluate the safety and effectiveness of new drugs or therapies in a target population.
Thyroid Cancer (TC) is the most common endocrine tumor, and its incidence has increased rapidly and dramatically over the past decade worldwide. Thyroid cancer ranks fifth among female cancers. The prognosis after surgery is good for most patients with thyroid cancer, with a ten-year survival rate > 90%. However, some patients still experience relapse and metastasis after treatment. The overall survival rate for patients with local recurrence is about 70-85%, while the long-term survival rate for patients with distant metastasis is 30-60%. Therefore, it is very important to find and determine biomarkers that can distinguish patients with high-risk and low-risk thyroid cancer.
The xenogenic and polytypic retroviral receptor (XPR 1) is a 696 amino acid protein with multiple transmembrane domains. XPR1 was originally defined as a specific cell surface receptor for heterophilic and polyphhilic murine leukemia viruses. XPR1 has also been reported to mediate G protein recruitment and play a role in G protein-coupled signal transduction. XPR1 is also considered an inorganic phosphate export protein in human cells based on its homology to proteins involved in phosphate transport regulation, including PHO1, PHO90, and PHO 91. There have also been several studies finding that XPR1 has been shown to interact with the beta-type platelet-derived growth factor receptor (PDGFRB) and to play an important role in maintaining cellular phosphate balance in the brain, a causative gene involved in primary familial hereditary diseases cerebral calcification (PFBC). However, the role of XPR1 in human malignancies, particularly in thyroid cancer, is yet to be further elucidated.
Disclosure of Invention
The invention mainly aims to overcome the defects and shortcomings of the prior art and provide an application of an XPR1 inhibitor in preparing a product for inhibiting migration and/or proliferation of thyroid cancer cells.
In a first aspect of the invention, the invention provides an application of an XPR1 inhibitor in preparing products for inhibiting migration and/or proliferation of thyroid cancer cells.
The product for inhibiting migration and/or proliferation of thyroid cancer cells is at least one of a-g;
wherein, a product for inhibiting migration and/or proliferation of thyroid cancer cells is prepared;
b, preparing a product for inhibiting the expression of Cyclin D1 (Cyclin D1) in thyroid cancer cells;
c, preparing a product for inhibiting expression of a MYC proto-oncogene (c-Myc) in a thyroid cancer cell;
d preparing a product for inhibiting the expression of matrix metalloproteinase 9 (MMP 9) in thyroid cancer cells;
e preparing a product for inhibiting the expression of matrix metalloproteinase 13 (MMP 13) in thyroid cancer cells;
f preparing a product for inhibiting the expression of the RelA protooncogene (p-p 65) phosphorylated in thyroid cancer cells;
g preparing the product for promoting the expression of apoptosis regulatory protein (Bax) of BCL-2 related X in thyroid cancer cells.
The product is a medicament.
The thyroid cancer cell is one or more of TPC-1 cell, BCPAP cell and FTC cell.
The XPR1 inhibitor is a specific small interfering Si-RNA targeting XPR 1.
The XPR1 inhibitor refers to a product capable of reducing the content or expression level of XPR 1.
In a second aspect of the invention, there is provided a pharmaceutical composition for inhibiting migration and/or proliferation of thyroid cancer cells comprising an inhibitor of XPR 1.
The XPR1 inhibitor is a specific small interfering Si-RNA targeting XPR 1.
The XPR1 inhibitor refers to a product capable of reducing the content or expression level of XPR 1.
In a third aspect of the invention, there is provided a product for inhibiting migration and/or proliferation of thyroid cancer cells, comprising a pharmaceutical composition as described above.
The invention has the following beneficial effects: the invention finds that the expression of the XPR1 gene in the thyroid cancer cell is obviously higher than that of a normal thyroid cell, and the influence of the XPR1 gene on the migration and proliferation capacity of the thyroid cancer cell is verified through a CCK-8 experiment, a colony forming experiment, a Transwell migration experiment, a Matrigel invasion experiment, a flow cytometry, an apoptosis experiment and a western blot experiment. The invention discovers that the expression of the XPR1 gene is knocked down, so that the migration and proliferation capacity of thyroid cancer cells can be effectively reduced.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is within the scope of the present invention for those skilled in the art to obtain other drawings based on the drawings without inventive exercise. The exemplary embodiments and descriptions of the present invention are provided to explain the present invention and not to limit the present invention.
Figure 1 XPR1 is overexpressed in thyroid cancer cells compared to normal thyroid cells;
FIG. 2 mRNA levels of XPR1 from the Si-XPR1 group were lower than those from the Si-NC group in all three thyroid cancer cell lines;
FIG. 3 at the protein level, the XPR1 expression for the Si-XPR1 group was lower than for the Si-NC group;
FIG. 4 CCK-8 assay for TPC-1, BCPAP, FTC three cell lines;
FIG. 5 clonogenic experiments on three cell lines TPC-1, BCPAP, FTC;
FIG. 6 migration experiments were performed for the Si-XPR1 and Si-NC groups of three cell lines;
FIG. 7 invasion experiments were performed on the Si-XPR1 and Si-NC groups of three cell lines;
figure 8 shows that XPR1 is associated with apoptotic pathways based on GSEA analysis from TCGA databases;
FIG. 9 demonstrates that knock-down of XPR1 promotes apoptosis in three thyroid cancer cell lines;
FIG. 10 silencing XPR1 increased Bax expression at the protein level while decreasing BCL-2 and p-p65 expression;
FIG. 11 isolation of cytoplasmic nuclear protein and detection of changes in expression of p65 in both the cytoplasm and the nucleus;
FIG. 12 analysis of expression changes of genes associated with cell proliferation and migration by real-time RT-qPCR.
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 accompanying drawings. It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. 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.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular is intended to include the plural unless the context clearly dictates otherwise. Further, it will be understood that the terms including and/or comprising, when used in this specification, specify the presence of stated features, steps, operations, and/or combinations thereof.
As described in the background, the mechanism of action of XPR1 in thyroid cancer in humans has yet to be fully elucidated, and in order to solve the above problems, in a first exemplary embodiment of the present invention, there is provided the use of an XPR1 inhibitor in at least one of the following a-g:
wherein, a product for inhibiting migration and/or proliferation of thyroid cancer cells is prepared;
b, preparing a product for inhibiting the expression of Cyclin D1 (Cyclin D1) in thyroid cancer cells;
c, preparing a product for inhibiting expression of a MYC proto-oncogene (c-Myc) in a thyroid cancer cell;
d preparing a product for inhibiting the expression of matrix metalloproteinase 9 (MMP 9) in thyroid cancer cells;
e preparing a product for inhibiting the expression of matrix metalloproteinase 13 (MMP 13) in thyroid cancer cells;
f preparing a product for inhibiting the expression of the RelA protooncogene (p-p 65) phosphorylated in thyroid cancer cells;
g preparing the product for promoting the expression of apoptosis regulatory protein (Bax) of BCL-2 related X in thyroid cancer cells.
In one or some embodiments of the invention, the product is a medicament.
In one or some embodiments of the present invention, the XPR1 inhibitor refers to a product capable of reducing the content or expression level of XPR1, and includes antagonists, down-regulators, blockers, etc., and in some embodiments of the present invention, the XPR1 inhibitor specifically employs one or more of the following: a Si-RNA expression vector and a Cas9-sgRNA co-expression vector which are obtained by Si-RNA and a CRISPR-mediated gene knock-down strategy and used for inhibiting the expression level of XPR1, and a compound, a composition or a reagent for reducing the content or the expression level of XPR 1. Wherein, the skilled person constructs a Si-RNA expression vector and a Cas9-sgRNA co-expression vector for inhibiting the expression level of XPR1 by a knocking-down method according to the well-known conventional technical means in the technical field of biological engineering.
In one or some embodiments of the invention, inhibiting Cyclin D1 expression in a thyroid cancer cell refers to decreasing Cyclin D1 expression levels; inhibiting c-Myc expression in thyroid cancer cells refers to reducing the expression level of c-Myc; inhibiting MMP9 expression in thyroid cancer cells refers to decreasing MMP9 expression levels; inhibiting MMP13 expression in thyroid cancer cells refers to decreasing MMP13 expression levels; promoting Bax expression in thyroid cancer cells refers to increasing Bax expression levels.
In a second exemplary embodiment of the present invention, a pharmaceutical composition for inhibiting migration and/or proliferation of thyroid cancer cells is provided, wherein the active ingredient is an XPR1 inhibitor.
In one or more embodiments of the present invention, the pharmaceutical composition further comprises other drugs compatible with the inhibitor and pharmaceutically acceptable carriers and/or excipients.
In a third exemplary embodiment of the present invention, there is provided a product for inhibiting migration and/or proliferation of thyroid cancer cells, characterized in that: comprising the pharmaceutical composition.
In one or some embodiments of the invention, the product is a medicament.
In one or some embodiments of the invention, the product further comprises pharmaceutically acceptable auxiliary materials, wherein the auxiliary materials are conventional auxiliary materials in pharmaceutical preparations, such as lubricants, binders, disintegrating agents and the like, and are preferably one or more of starch, sodium carboxymethyl cellulose, glycerol, betaine and the like.
In one or some specific embodiments of the invention, the thyroid cancer cells are TPC-1 cells and/or BCPAP cells and/or FTC cells.
In the invention, the medicament can be any pharmaceutically acceptable dosage form, including tablets, capsules, oral liquid, syrup, granules, pills, powder, ointment, pellets, injection, suppositories, creams, sprays, dripping pills, sustained-release preparations, controlled-release preparations and the like.
In the present invention, the expression level refers to the expression level of a gene as measured by the amount of mRNA transcribed from the gene.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
Examples
Materials and methods
1.1 bioinformatics analysis
Thyroid cancer patient information with complete clinical case characteristics (age, sex, LNM, tumor size, clinical stage, etc.) was downloaded from the TCGA database for analysis. The GSEA software (GSEA v3.0, http:// www.broadinstitute.org/GSEA) was used to perform single gene enrichment analysis between the low XPR1 and high XPR1 expression sets of the TCGA database.
1.2 RNA extraction, reverse transcription and quantitative real-time fluorescence polymerase chain reaction (qRT-PCR)
1.2.1 RNA extraction
1.2.1.1 tissue specimens: the tube was removed from the vial, and placed in a 1.5 ml EP tube, and 1ml Trizol lysate was added. Then, the mixture was sufficiently disintegrated by a tissue homogenizer and then allowed to stand on ice. Cell specimen: discard the plate supernatant, add 1ml PBS to wash once, try to suck up the waste liquid, add 1ml Trizol lysate, after 5 min of standing blow to transfer to 1.5 ml EP tube.
1.2.1.2 precooling the centrifuge at 4 ℃, adding 200 mul of chloroform into the EP pipe, shaking violently for about 20 ℃, and then standing for five minutes. Followed by centrifugation at 12000 rpm for 20 minutes. Meanwhile, preparing EP tubes with the same number of tubes, adding 400 mu l of isopropanol in advance, and inserting the tubes on ice for cooling in advance.
1.2.1.3 taking supernatant liquid at the uppermost part of the layer in the EP tube, transferring the supernatant liquid to the EP tube prepared in advance, and paying attention not to draw liquid at other layers. The EP tube was then gently rotated and allowed to stand at-20 ℃ for at least 1 hour. Followed by centrifugation at 12000 rpm for 1 hour.
1.2.1.4 the supernatant was aspirated, 1ml of pre-cooled absolute ethanol was added and centrifuged at 12000 rpm for 5 minutes.
1.2.1.5, absorbing the supernatant as much as possible, showing white precipitate at the bottom, standing at room temperature for drying, and adding 50 mul of enzyme-removing water to lightly blow and mix uniformly when the gel turns into a gel shape. The samples were placed on ice.
1.2.1.6 the purity (optimal ratio of absorbance at 260 nm/280 nm between 1.8-2.0) and concentration of RNA were determined spectrophotometrically and recorded.
1.2.2 reverse transcription of RNA
1.2.2.1 reverse transcription of miRNA: the procedure follows the standard experimental procedure of miRNA reverse transcription kit from Ribo company. The preparation system of the reaction solution is 10 mul, 5 mul (1 mug of RNA template and enzyme removing water) +2 mul of reaction 1 Xbuffer +2 mul of enzyme and 1 mul of specific miRNA reverse transcription primer. And (3) lightly beating the mixed solution uniformly, and then carrying out reaction in a PCR machine: 60 min at 42 ℃, 5 min at 98 ℃ and maintaining at 4 ℃. The resulting cDNA can be stored in a refrigerator at-20 ℃.
1.2.2.2 qRT-PCR: the experiment was carried out according to the instructions of Takara PCR kit RR 820A. The system is 10 mul, 4 mul cDNA, 5 mul buffer, 0.5 mul positive and negative primers and 0.2 mul ROX dye. The following reaction was carried out in ABI 7500 fast: pre-denaturation at 95 ℃ for 30 s, followed by heat denaturation at 95 ℃ for 5 s and annealing extension at 60 ℃ for 34 s for 40 cycles. The miRNA primer and the control U6 thereof are provided by Ribo company, and the other genes and the reference gene GAPDH primer thereof are synthesized by Shanghai Biotech company. Finally use 2-△△CTDetecting the expression level of the gene relative to a reference. Δ CT is equal to CT of the target gene minus CT of the internal reference gene. The gene primers used are as follows:
XPR1：
Forward-TGTGTATCCACTTGCCCTTTAT (SEQ ID No:3 of the sequence Listing),
Reverse-CAGCCAAAACCGGGATTTATAG (SEQ ID No:4 of the sequence Listing);
Cyclin D1:
Forward-GCTGCGAAGTGGAAACCATC (SEQ ID No:5 of the sequence Listing),
Reverse-CATGGAGGGCGGATTGGAA (SEQ ID No:6 of the sequence Listing);
c-Myc：
Forward-CGACGAGACCTTCATCAAAAAC (SEQ ID No:7 of the sequence Listing),
Reverse-CTTCTCTGAGACGAGCTTGG (SEQ ID No:8 of the sequence Listing);
MMP9：
Forward-GACATCGTCATCCAGTTTGG (SEQ ID No:9 of the sequence Listing),
Reverse-TGGCCTTGGAAGATGAATGG (SEQ ID No:10 of the sequence Listing);
MMP13：
Forward-AGTTCCAAAGGCTACAACTT (SEQ ID No:11 of the sequence Listing),
Reverse-CGCCAGAAGAATCTGTCTTT (SEQ ID No:12 of the sequence Listing);
GAPDH：
Forward-GGTCGGAGTCAACGGATTTG (SEQ ID No:13 of the sequence Listing),
Reverse-ATGAGCCCCAGCCTTCTCCAT (SEQ ID No:14 of the sequence Listing).
1.3 cell culture
1.3.1 cell culture conditions: TPC-1 and KTC-1 were cultured in 1640 medium with 10% serum concentration, and FTC was cultured in 10% DMEM medium. The culture conditions were 37 ℃ and 5% CO295% air. The culture medium was changed at least 2 times per week.
1.3.2 cell recovery: the tube storing the cells was taken out of the liquid nitrogen and immediately placed in a 37 ℃ water bath, after no ice column was formed in the tube, it was drawn out and added to a 15 ml centrifuge tube containing 2 ml of fresh 10% serum culture medium and centrifuged at 2000 rpm for 5 minutes. And gently taking out the test tube, randomly removing the supernatant by using a pipette, adding 3 ml of culture solution, blowing, beating, uniformly mixing, transferring to a culture bottle, and shaking uniformly. One hour later or the next morning under the microscope to assess the overall condition of the cells.
1.3.3 cell passage and cryopreservation: the flask was aspirated, 1ml of PBS was added to wash out the remaining culture medium, and 1ml of PBS and 1ml of pancreatin were added. When the cells became round and partially fallen, 1ml of serum-containing culture medium was added to stop digestion. The residual cells were blown and collected and centrifuged at 1000 rpm for 5 minutes. After the supernatant was aspirated, 1ml of the culture medium was homogenized and resuspended, 1/4 cells were added to the cell culture flask to which the culture medium had been added. Shaking the cross evenly, and transferring to an incubator for culture. Or abandoning the supernatant, resuspending 2 ml of culture solution containing 10% dimethyl sulfoxide, adding 1ml of cells into a freezing tube, and transferring the tube to a programmed cooling box (containing isopropanol). The box was placed in a-80 ℃ freezer, the next day the cells were removed, registered, and transferred to liquid nitrogen.
1.4 transfection of cells
1.4.1 plating: TPC-1, KTC-1 and FTC cells were digested according to the above passage procedure, and 10 ten thousand cells were plated on a six-well plate previously filled with 1ml of culture medium. The cross is put into a cell culture box after shaking up.
1.4.2 transfection: the next day, cells are removed and the density of cells is noted under the mirror, and if confluency reaches between 50% and 65%, the next transfection procedure can be performed. Preparing a transfection mixed solution by using an OPTI culture medium, carrying out transfection assisted by Lipofectamine RNAiMAX (3.5 microlitres per hole) by using 7 microlitres of Si-RNA per hole; approximately 1. mu.g of plasmid per well, each assisted with 2. mu.l Lipofectamine (TM) 3000 and p 3000. All liposomes need to be premixed and distributed evenly to each well. The culture medium will need to be replaced before the mixed solution is injected into the cells. Cells were harvested 48 hours after transfection for further experiments.
The Si-RNA sequence targeting XPR1 is:
sense 5'-GCAUGCUUCUUUGCUCCAATT-3' (sequence listing SEQ ID No: 1)
Antisense 5'-UUGGAGCAAAGAAGCAUGCTT-3' (sequence listing SEQ ID No: 2)
1.4.3 transfection efficiency assay: the transfection efficiency is detected by the methods of RNA extraction, reverse transcription and qRT-PCR.
1.5 colony formation assay and CCK-8 assay
The CCK-8 reagent contains WST-8, which can be reduced by succinate dehydrogenase in mitochondria to produce yellow substances. The absorbance of the color measured at a wavelength of 450 nm can indirectly determine the activity of the cell.
1.5.1 Collection of cells: and taking out the cells after the plating transfection, checking the cell condition under a mirror, and carrying out the next experiment if the state is good. The culture medium in the six-well plate is completely pumped out, washed once by 1ml PBS, then digested by adding 1ml pancreatin, the cell is stopped when the cell becomes round or part of the cell is separated from the wall, the cell is blown to a 15 ml centrifuge tube, and centrifuged for 5 minutes at 1000 rpm.
1.5.2 calculate cell mass: the supernatant was aspirated, resuspended in 1ml of culture medium, and 10 μ l was counted. The CCK-8 experiment measurement is performed before cells are plated in a 96-well plate, the colony formation experiment is performed before cells are plated in a 6-well plate, and the cell quantity requirements of the two experiments are as follows: 1000 cells per well, CCK-8 cells per group were measured for a total of 4 days, 5 secondary wells were set per day, 2 secondary wells per group for colony formation experiments, and 25 total wells for binding errors. 2.5 ten thousand cells were taken and mixed into 2.5 ml of the culture medium (1000 cells per 100. mu.l).
1.5.3 CCK-8 Experimental measurements: and (4) taking 100 mul of the prepared suspension, and paving the suspension into a 96-well plate along the side wall of the well. The medium is paved in the middle of a 96-well plate as far as possible, so that the influence of evaporation on cell growth is avoided. The measurements were carried out by adding CCK-8 reagent at 24, 48, 72 and 96 hours after plating, respectively: the culture solution in the holes needs to be measured on the day is firstly pumped out, then 10% of CCK-8 reagent is prepared by the culture solution, 100 mu l of the culture solution is added into each hole in a dark place and then placed in an incubator, and the culture solution is detected in an enzyme labeling instrument (with the wavelength of 450 nm) 3 h after the culture solution is added, so that a proliferation curve is drawn.
1.5.4 colony formation assay: adding 2 ml of culture solution into each hole of a six-hole plate, then adding 100 mu l of the suspension into the holes, standing for 5 min after the cross is uniformly shaken, and moving into an incubator. Taking out microscope for observation on 5-7 days, if there are more than 30 clone groups, each group has more than 50 cells, then the next operation can be performed. The culture was aspirated, washed once with PBS, and then fixed with 4% paraformaldehyde for 15 minutes. After the liquid is absorbed out, the liquid can be recycled and finally dyed by 0.1 percent crystal violet for 10 minutes. After sucking up the liquid, washing with pure water once, photographing and manually counting the number of cells.
1.6 Transwell migration experiment and Matrigel invasion experiment
1.6.1 Collection of cells: cells after plating transfection were collected using the method described above and counted.
1.6.2 calculate cell mass: the Transwell laboratory required 3.5 ten thousand cells, while the Matrigel Transwell chamber required 5 ten thousand cells.
1.6.3 Transwell Chamber Placement and harvesting: the Transwell cells were placed in a 24-well plate containing 600 μ l 10% serum culture medium at an oblique angle to avoid the formation of air bubbles. Adding liquid corresponding to the amount of cells into the chamber, and supplementing with serum-free culture mediumThe liquid in the upper chamber of each well is 300 mu l. And (5) lightly beating the mixture evenly by using a 100 mu l pipette. Then placing the 24-hole plate at 37 ℃ and 5% CO2An incubator. After 20 hours the chamber was removed, the upper medium was discarded and washed once with PBS. The lower surface was fixed dyed. The operation is 1.5.4. Then wipe off the residual liquid and cells on the upper chamber surface with a cotton swab, air dry, place under an inverted microscope for observation (objective 20 ×), and randomly select five fields for counting.
1.6.4 Matrigel cell placement and retrieval: the Matrigel chamber was taken out of the-20 ℃ refrigerator, and the chamber was placed in advance in a 24-well plate containing 600 μ l of 10% serum culture solution, at an oblique angle, to avoid the generation of air bubbles. The 24-well plate was then transferred to an incubator for 30 minutes to liquefy the frozen matrigel. And then taking out, adding the cell suspension with the cell amount into the upper chamber, and supplementing the liquid of each hole in the upper chamber to 300 mu l by using a serum-free culture solution. And then, uniformly beating the mixture by using a 100 mu l pipette. The 24-well plate was then moved into the incubator. After 24 hours, the cells were removed, and the culture medium was discarded and washed with PBS. The lower surface was fixed and dyed as above. Then wipe off the residual liquid and cells on the upper chamber surface with a cotton swab, air dry, place under the mirror for observation (objective 20 ×), randomly select five fields for counting.
1.7 detection of apoptosis
1.7.1 Collection of cells: cells 48 hours after transfection were removed, and 15 ml centrifuge tubes corresponding to the number of tubes were prepared and labeled. The liquid was drained and transferred to a centrifuge tube. Adding 1ml PBS for washing once, collecting the washing liquid in a centrifuge tube, adding 1ml PBS and 1ml pancreatin for digestion, adding serum culture solution when the cells are slightly rounded or slightly flutter, blowing and collecting in the centrifuge tube, and centrifuging for 5 minutes at 1000 rpm.
1.7.2 washing cells: the supernatant was discarded, washed once with 1ml PBS and centrifuged at 1000 rpm for 5 minutes. The washing was repeated three times. Then using ddH2O prepared 1 Xbuffer.
1.7.3 Annexin V staining: and (3) sucking up the supernatant, sucking the supernatant to the greatest extent, adding 300 mul of 1 Xbuffer solution, slightly blowing and beating the mixed cells, transferring the suspension to a flow tube, adding 3 mul of Fluorescein Isothiocyanate (FITC), and incubating for 15 minutes in a dark place.
1.7.4 PI (propidium iodide) staining: 3 mul PI was added and incubated for 5 minutes in the dark.
1.7.5 computer and analysis: cells were collected at high speed using a flow cytometer and recorded for storage. The data results were analyzed by Flowjo and the rate of Apoptosis (apoltosis rate)% = percent early Apoptosis (third quadrant) + percent late Apoptosis (second quadrant) was calculated.
2.1 Western blotting
2.1.1 extraction of cellular proteins: cells were harvested 48 hours after transfection, the procedure was as in 1.5.1. 1000 μ l RIPA lysate (strong) was added, left to stand for 5 minutes, cells were collected with a spatula into a 1.5 ml EP tube, centrifuged at 12000 rpm for 15 min, and the supernatant was removed to a new 1.5 ml EP tube.
2.1.2 protein concentration determination: and drawing a standard curve according to the specification. A system for determining the concentration of a protein sample is configured, (5 mul of sample +95 mul double-distilled water), 5 mul of sample to be detected is added into a 96-well plate, and 95 mul double-distilled water is added and fully blown. Adding a BCA reagent B and a BCA reagent A according to the proportion of 1: 50 to prepare a BCA color developing solution, adding 200 mu l of newly prepared BCA color developing solution into the holes of the sample respectively, and incubating for 30 min in a constant temperature coating box at 37 ℃, wherein the BCA color developing solution needs to be prepared for use. And (3) measuring the absorbances of all the standard products and the samples at the A562 position by using a multifunctional microplate reader, combining with a protein standard curve, and calculating the concentration of the protein in the corresponding samples according to the absorbances of the protein samples to be detected.
2.1.3 preparation of the protein sample
To the total protein extracted from the cells, an appropriate volume of 5 XBuffer (1/4 volumes of 5 XBuffer for total protein was added to make the final concentration of Buffer in total protein 1X), after shaking and mixing, the protein sample was boiled for 10 min on a metal sample cooker at 97 ℃, taken out and placed on ice for cooling. The loading of each sample was adjusted to 20 μ g according to the measured protein concentration, and then the volume of all protein samples was filled to 20 μ l with 1 × Buffer.
2.1.4 protein electrophoresis
Preparing 5% concentrated glue and 10% separation glue, placing the glue on a rack in an electrophoresis tank, adding 1 × electrophoresis liquid into the electrophoresis tank, immersing the sample loading hole on the inner side of the prefabricated glue, and adding the sample into the sample loading hole. Pouring 1 Xelectrophoresis solution into the outer tank of the electrophoresis tank, running gel by 50V constant-pressure electrophoresis, turning off the power supply when bromophenol blue in protein appears at the bottom of the gel, stopping electrophoresis, taking down the glass plate, taking out the gel containing the protein sample, and putting the gel into the membrane transferring solution to prepare for membrane transferring.
2.1.5 transfer film
The PVDF membrane is cut into a proper size before membrane conversion, soaked in methanol and placed on a shaking table for activation for 5 min. Soaking thick filter paper and thin filter paper in the pre-cooled membrane transferring liquid, and putting the thick filter paper and the thin filter paper on a membrane transferring clamp according to the following sequence: the method comprises the steps of coating a white board, a spongy cushion, 1 layer of thick filter paper, 2 layers of thin filter paper, PVDF (polyvinylidene fluoride) membrane, gel containing a protein sample, 2 layers of thin filter paper, 1 layer of thick filter paper, 1 layer of spongy cushion and a black board with a film rotating clamp, wherein when the layers are laminated, the bubbles among the layers are carefully discharged, then the film rotating clamp is locked, the white board surface of the film rotating clamp faces the red surface of a red-black box with the film rotating clamp, then the red surface is placed at the positive pole of the film rotating groove, then precooled 1 x electric transfer liquid is added, so that the position of the gel in the film rotating groove is immersed in the electric transfer liquid, the ice is placed in the film rotating groove, and the film is rotated overnight at constant voltage of 25V.
2.1.6 sealing
After the membrane transfer is finished, taking out the PVDF membrane by using a pair of tweezers, soaking the PVDF membrane in 1 xTBST, comparing the size of a protein Marker, cutting the PVDF membrane according to the size of the needed protein, then putting the cut membrane into 5% skimmed milk (about 10 ml for each membrane) prepared in advance, slightly shaking the membrane on a shaking table, sealing the membrane for 90 min at normal temperature, recovering the milk, and washing the membrane for 4 times by using 1 xTBST, wherein the 5 min is carried out each time.
2.1.7 incubation Primary antibody
The primary antibody was used in the proportions recommended in the antibody specification, and a 1 × TBST solution containing 5% BSA was used as an antibody diluent. Soaking the washed membrane in diluted primary antibody, incubating on shaking table of refrigerator at 4 deg.C for 8-10 h, recovering primary antibody, storing in refrigerator at 4 deg.C, washing membrane with 1 × TBST for 4 times, each for 5 min.
2.1.8 incubation Secondary antibody
Respectively selecting secondary antibodies of the corresponding anti-species, and diluting the secondary antibodies (the concentration is 1: 10000) by using 5% skimmed milk recovered after sealing according to the antibody specification of the secondary antibodies. And after membrane washing, discarding TBST, putting PVDF into the diluted secondary antibody, placing on a low-speed shaking table at room temperature for incubation for 60 min, after incubation, discarding the secondary antibody, and washing the membrane with 1 × TBST for 4 times, 5 min each time, and washing the membrane with 1 × TBST for two times, 4 min each time.
2.1.9 Exposure
After addition of the developing reagent, exposure was performed with an Odyssey scanner.
2.2 statistical analysis
All experimental part contents were repeated three times separately. For metrology data, the data were tested for normal distribution using Shapiro-Wilk. Two sets of data normally distributed were compared using the t-test, while two sets of data not normally distributed used the Mann-Whitney rank sum test. Two-way anova was used for data analysis for CCK-8 detection. From all statistical analyses, significant differences were considered for the results with a p value <0.05 (. p <0.05,. p <0.01,. p <0.001,. p < 0.0001).
Second, experimental results
Through the above experimental procedure, the following results can be found:
1. XPR1 is highly expressed in thyroid cancer cell lines and promotes the proliferation thereof, and an XPR1 inhibitor can effectively inhibit the proliferation capacity of thyroid cancer cell lines
First, the mRNA level of XPR1 was examined in thyroid cancer cells and normal thyroid cells. XPR1 was found to be significantly highly expressed in three cell lines, TPC-1, BCPAP and FTC, compared to normal thyroid cell HTORI-3 (FIG. 1). Small interfering RNA (Si-RNA) was then used to knock down XPR1 in three cell lines, TPC-1, BCPAP and FTC. In three cell lines, Si-RNA was able to reduce the expression of XPR1 at both mRNA level and protein level (FIGS. 2-3). Then, the effect of XPR1 on the proliferative capacity of thyroid cancer cells was evaluated. Using a cell counting kit (CCK-8) assay, it was found that knock-down of XPR1 decreased the proliferative capacity of thyroid cancer cells (fig. 4). In addition, the same conclusion was shown in the clonogenic experiments (FIG. 5). Therefore, decreasing the expression of XPR1 in thyroid cancer cell lines inhibits its proliferative capacity. Namely, the high-expression XPR1 can promote the proliferation of thyroid cancer cells, and the XPR1 inhibitor can effectively inhibit the proliferation capacity of thyroid cancer cell lines.
The over-expression of XPR1 promotes the migration and invasion capacity of thyroid cancer cells, and an XPR1 inhibitor can effectively inhibit the migration and invasion capacity of thyroid cancer cell lines
To evaluate the effect of XPR1 on the migration and invasion capacity of thyroid cancer cells, we performed a Transwell migration experiment and a Matrigel invasion experiment. The migration and invasion capacity of TPC-1, BCPAP and FTC after transfection with Si-XPR1 was significantly reduced compared to cells transfected with Si-NC (FIGS. 6-7). Therefore, it is concluded that the highly expressed XPR1 can promote the invasion and metastasis of thyroid cancer cells, and the XPR1 inhibitor can effectively inhibit the migration and invasion capacity of thyroid cancer cell lines.
3. XPR1 can inhibit apoptosis in thyroid cancer cell lines and activate expression of genes related to cell proliferation and migration, and XPR1 inhibitor can effectively promote apoptosis of thyroid cancer cell lines
To explore the mechanism of carcinogenesis of XPR1 in thyroid cancer, single gene enrichment analysis (GSEA) was used to find genes in thyroid cancer tissues that are likely to be regulated by XPR 1. Thyroid cancer patients in the TCGA database were classified into high-expression and low-expression groups according to the XPR1 expression level. Genes involved in apoptosis were found to be associated with expression of XPR1 (fig. 8). Therefore, the degree of apoptosis of thyroid cancer cells was first examined by flow cytometry after knock-down of XPR 1. The apoptosis rate was found to be higher in the knockdown group than in the control group for each cell line, and was dominated by an increase in late apoptosis (fig. 9). Further exploring the mechanism of knocking down XPR1 to accelerate thyroid cancer apoptosis, it was found that knocking down XPR1 decreased p65 phosphorylation (fig. 10), and inhibited nuclear translocation of p65 (fig. 11). The RT-qPCR results shown in fig. 12 show that XPR1 regulates expression of genes associated with cell proliferation and migration. Namely, the high-expression XPR1 can inhibit the apoptosis of thyroid cancer cells and activate the expression of genes related to cell proliferation and migration, and the XPR1 inhibitor can effectively promote the apoptosis of thyroid cancer cells.
Third, discussion and summary
The incidence of Thyroid Cancer (TC) has increased more than three times in the last 30 years, being the most common endocrine tumor. Although most thyroid cancers can be effectively controlled by surgery and radioiodine, there are some well-differentiated thyroid cancers and a high mortality rate in most poorly differentiated and undifferentiated thyroid cancers. The overall survival rate for patients with local recurrence is about 70-85%, while the long-term survival rate for patients with distant metastasis is 30-60%. Therefore, it is important to find and determine biomarkers that can distinguish patients with high-risk thyroid cancer from those with low-risk thyroid cancer.
The xenogenic and polytypic retroviral receptor (XPR 1) is a 696 amino acid protein with multiple transmembrane domains. XPR1 was originally defined as a specific cell surface receptor for heterophilic and polyphhilic murine leukemia viruses. XPR1 has also been reported to mediate G protein recruitment and play a role in G protein-coupled signal transduction. XPR1 is also considered an inorganic phosphate export protein in human cells based on its homology to proteins involved in phosphate transport regulation, including PHO1, PHO90, and PHO 91. There have also been several studies finding that XPR1 has been shown to interact with the beta-type platelet-derived growth factor receptor (PDGFRB) and to play an important role in maintaining cellular phosphate balance in the brain, a causative gene involved in primary familial hereditary diseases cerebral calcification (PFBC). However, the role of XPR1 in human malignancies, particularly in thyroid cancer, has yet to be further investigated. The invention discovers that the high-expression XPR1 can promote the proliferation, invasion and metastasis of thyroid cancer cells and inhibit the apoptosis of the thyroid cancer cells. It was found that the XPR1 gene exerts its oncogenic effect by activating the expression of genes involved in cell proliferation and migration. The knock-down of the XPR1 gene can effectively reduce the proliferation and migration capacity of thyroid cancer. Similarly, the flowing cell experiment also proves that the knock-down of the XPR1 gene can obviously promote the apoptosis of thyroid cancer cells.
In conclusion, the XPR1 gene can promote the proliferation and migration capacity of thyroid cancer cells, and the XPR1 inhibitor can effectively inhibit the proliferation and migration capacity of thyroid cancer cells.
The above disclosure is only a preferred embodiment of the present invention, and certainly should not be taken as limiting the scope of the present invention, and other changes, modifications, substitutions, combinations and simplifications which do not depart from the spirit and principle of the present invention are intended to be equivalents thereof, which may be made by those skilled in the art and are still within the scope of the present invention.
Sequence listing
<110> first Hospital affiliated with Wenzhou medical university
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