CN112755190A - Target and diagnostic marker for inhibiting colorectal cancer growth and application thereof - Google Patents
Target and diagnostic marker for inhibiting colorectal cancer growth and application thereof Download PDFInfo
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Abstract
The invention relates to a target and a diagnostic marker for inhibiting the growth of colorectal cancer and application thereof, and particularly discloses application of a TEX11 expression promoter in preparing a medicament for inhibiting the growth of colorectal cancer, which is used for inhibiting the proliferation of colorectal cancer cells in vivo; the TEX11 expression promoter is an overexpression plasmid of TEX11, and is a pCDH-EF1-MCS-T2A-Puro-TEX11 plasmid. The invention shows that TEX11 can be used as a potential therapeutic target for colorectal cancer. The invention provides a brand-new diagnosis marker and a treatment target for colorectal cancer.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to discovery and application of a new tumor marker and a new target for inhibiting colorectal cancer growth, and more particularly relates to a new tumor marker and a new target for research on inhibiting colorectal cancer growth by using TEX11 and application thereof.
Background
Colorectal cancer (CRC) is a common malignant tumor of the digestive tract, and its incidence and mortality rate are rising year by year compared with other cancers in human, and the number of cases of death is 3 rd and 2 nd in men and women all over the world, and 4 rd and 3 rd in men and women all over the world. In 2015, about 38 million new cases and about 19 million deaths of colorectal cancer in China exist, the morbidity and the number of deaths account for 18.6 percent and 20.1 percent of the worldwide morbidity and the number of deaths respectively, and the colorectal cancer is the first in the world. Compared with the severe condition of colorectal cancer, the current treatment of colorectal cancer is mainly surgical resection, assisted by radiotherapy and chemotherapy and traditional Chinese medicine treatment, but the effect is not ideal, and the 5-year survival rate of patients is only 47.2%. In recent years, targeted therapy is more and more popular and becomes one of the main means of anti-tumor therapy, while targeted therapy also has certain defects, gene regulation networks are complex and diverse, the drug resistance phenomenon of targeted drugs is a ubiquitous problem, once the drug resistance situation occurs, new targeted drugs are required to be changed, although more action targets and targeted drugs thereof exist in various tumors including colorectal cancer at present, the current situation of clinical therapy cannot be met, so that the discovery of new molecular targets, the elucidation of the molecular mechanism of the action of the new molecular targets and the development of diversified targeted drugs are a great challenge in basic science and clinical practice at present.
The TEX11(Testis Expressed 11) gene, located on the X chromosome, encodes a 108KD protein. The TEX11 gene was originally studied and reported in male azoospermia as an X-linked gene. Since the previous research considers that the TEX11 gene is specifically expressed in male testis, the related research is only limited to the research on spermatogenesis such as male non-obstructive azoospermia and oligospermia, and besides, no related research report is provided for the TEX11 gene in other diseases or biological effects at home and abroad.
Disclosure of Invention
The invention aims to provide a target and a diagnostic marker for inhibiting colorectal cancer growth and application thereof.
In order to solve the technical problems, the invention provides an application of a TEX11 expression promoter in preparing a medicament for inhibiting the growth of colorectal cancer.
The improvement of the application of the TEX11 expression promoter in preparing the medicine for inhibiting the growth of colorectal cancer comprises the following steps:
inhibiting proliferation of colorectal cancer cells in vivo; colorectal cancer cells include SW480 cells, RKO cells.
The TEX11 expression promoter is an overexpression plasmid of TEX11, namely pCDH-EF1-MCS-T2A-Puro-TEX11 plasmid.
The invention also provides a composition for preventing or/and treating colorectal cancer, which comprises the following components:
(1) a TEX11 expression promoter;
(2) a pharmaceutically acceptable carrier.
As an improvement of the composition for preventing or/and treating colorectal cancer of the present invention: the TEX11 expression promoter is an overexpression plasmid of TEX 11.
The invention also provides a reagent for detecting the expression of TEX11, wherein the reagent for detecting the expression of TEX11 comprises a reagent based on a fluorescent quantitative PCR quantitative detection method, and the reagent for detecting the fluorescent quantitative PCR quantitative detection method comprises a pair of specific primers;
the specific primers are as follows:
f (upstream primer): 5'-CACTGATGCCCTACAATGGTACTAT-3'
R (downstream primer) 5'-GTTCCTAGGGTCATGTCGTTCAG-3'.
The purpose of the present invention is to show that TEX11 can be applied as a diagnostic marker as well as a therapeutic target for colorectal cancer.
The technical scheme adopted by the invention is as follows: through a bioinformatics technical means, exon-Seq V2 sequencing data and clinical data of colon cancer (TCGA-COAD) in a TCGA database (https:// TCGA-data. nci. nih. gov /) are downloaded, and transcriptome data is deeply mined. Further validation of clinical tissue samples by RT-QPCR technique using 150 laboratory collections then revealed that in 150 clinical tissue samples, the expression of the TEX11 gene was significantly down-regulated in mRNA levels in cancerous tissues compared to paracancerous normal tissues, consistent with the TCGA database. Protein expression conditions of the TEX11 gene in clinical tissues and cell lines are respectively detected by an immunohistochemical experiment (IHC) experiment and a protein immunoblotting (Western Blotting), and the TEX11 gene is remarkably reduced in protein level expression in 150 pairs of clinical tissue samples, and meanwhile, the TEX11 is remarkably reduced in cancer cell lines such as HCT116 and the like compared with a normal colon epithelial cell CCD-18 Co.
Further, CRC patients in the TCGA database are grouped according to the high and low expression of the TEX11 gene by means of bioinformatics technology, and the prognosis of patients with high expression of TEX11 is obviously better than that of patients with low expression of TEX 11.
After construction of TEX11 overexpression vector and establishment of SW480 and RKO cell stable cell strain, through ATP activity determination experiment and soft agar experiment, in vitro research shows that the proliferation of SW480 and RKO cells is remarkably promoted by the down regulation of TEX 11.
A nude mouse ectopic transplantation tumor model is established by adopting a subcutaneous injection mode, and the growth conditions of SW480 cells and RKO cells under the nude mouse skin are observed. It was found that TEX11 significantly inhibited the proliferation capacity of colorectal cancer cells SW480 and RKO cells in vivo.
The invention has the following beneficial effects:
the TCGA database is analyzed by a bioinformatics means, and experimental techniques such as Q-PCR and the like and WB, IHC and the like find that TEX11 shows a remarkable down-regulation trend in the transcription level and the protein level in cancer tissues compared with paracancer normal tissues, and the expression level of TEX11 gene is remarkably related to the prognosis of a patient (CRC patient), which indicates that TEX11 can be used as a colorectal cancer diagnosis marker and becomes one of the prognosis indexes of the patient. Meanwhile, by further taking colorectal cancer SW480 and RKO cells as a model, the over-expression of TEX11 can obviously inhibit the in vivo and in vitro proliferation capacity of the colorectal cancer SW480 and RKO cells, and the result shows that TEX11 can be used as a potential therapeutic target of colorectal cancer. The invention provides a brand-new diagnosis marker and a treatment target for colorectal cancer.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows the down-regulation of TEX11 expression in clinical tissues and cell lines of colorectal cancer;
in the context of figure 1 of the drawings,
a is that the transcriptional expression level of TEX11 in colorectal cancer is obviously reduced compared with that in paracarcinoma tissues by bioinformatics means analysis of TCGA database;
b, detecting the transcriptional expression condition of TEX11 in 150 pairs of clinical samples by using specific primers, and further finding that the transcriptional expression level of TEX11 in colorectal cancer is remarkably reduced compared with that in a tissue beside cancer;
C. d is the expression level of TEX11 protein detected by an immunohistochemical experiment, and the expression level of TEX11 protein is found to be remarkably reduced in colorectal cancer compared with that in a tissue adjacent to the cancer;
and E, detecting the expression of TEX11 in the cell line by using western blot, and finding that the protein expression level of TEX11 in a colorectal normal cell line is remarkably reduced compared with that of a colorectal cancer cell line.
FIG. 2 shows the relationship between the expression level of TEX11 in cancer tissues and the prognosis of patients.
In the context of figure 2, it is shown,
a is the relation between TEX11 expression and the disease-free survival of colorectal cancer patients, and the higher the TEX11 expression is, the longer the disease-free survival of the patients is.
B is the relationship between TEX11 expression and the overall survival of colorectal cancer patients, and the higher the TEX11 expression is, the longer the overall survival of patients is.
FIG. 3 is a graph demonstrating the effect of TEX11 on the proliferation capacity of colorectal cancer cells in vitro, after over-expression of TEX11 in SW480 and RKO cells and WB identification (FIG. 3A), by ATP experiments (FIG. 3B, 3C) and soft agar (FIG. 3D-G) experiments.
FIG. 4 is a model of xenograft tumor of nude mice established by subcutaneous injection, and the growth of SW480 cells and RKO cells under the nude mice skin is observed;
in the context of figure 4, it is shown,
a is a tumor body solid picture obtained by carrying out subcutaneous injection on a nude mouse corresponding cell after over-expressing TEX11 in SW480 cell and carrying out experiment;
b, weighing the tumor formed by the SW480 cells of the experiment, analyzing and mapping, and comparing the tumor weight difference between the experiment group and the control group;
c, after TEX11 is over-expressed in RKO cells, corresponding cells are injected into nude mice subcutaneously, and a tumor body solid graph obtained by an experiment is photographed;
d, weighing the tumor formed by the RKO cells for the experiment, analyzing and mapping, and comparing the tumor weight difference between the experiment group and the control group;
e, carrying out volume measurement on subcutaneous tumors of the SW480 cell experimental group and the nude mice of the control group at different time points and drawing a growth curve;
f, carrying out volume measurement on subcutaneous tumors of the RKO cell experimental group and the nude mice of the control group at different time points and drawing a growth curve.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
examples 1,
The TCGA database was analyzed by bioinformatics for the relative upregulation of transcription level expression of TEX11 in colorectal cancer compared to paracancerous tissues (fig. 1A). Q-PCR and IHC detection of TEX11 transcription levels in 150 pairs of clinical samples (FIG. 1B), protein level expression (FIGS. 1C-D), and expression in cell lines (FIG. 1E)
1) Exon-Seq V2 sequencing data and clinical data were downloaded for colon cancer (TCGA-COAD) in the TCGA database (https:// TCGA-data. nci. nih. gov /).
2) First, genes expressed in low amounts in the data were knocked out (by filtering genes whose raw count was 80% or more and 0). Samples with cancer and paracancer paired tissues were selected as subjects, and differential analysis was performed. mRNA data were preprocessed using R-package edgeR (Version: 3.4, http:// www.bioconductor.org/packages/release/bioc/html/edgeR. html), respectively, raw count was normalized to log-CPM values, linear modeling was performed, and the mean variance relationship was adjusted using precision weights calculated from the voom function. Differential expression analysis was performed on mRNA data, Tumor VS Normal, using linear regression and empirical bayesian methods provided by limma package, respectively. All genes obtain corresponding P.value values, and the corrected p value, namely adj.P.value, is obtained by performing multiple inspection correction by using a Benjamini and Hochberg method. mRNA differential expression thresholds in this study were all adj.p. value <0.05 and | log2FC | > 2.
3) Tissue sample
Clinical tissue samples of colorectal cancer that have been diagnosed and surgically resected are provided by the first hospital affiliated with the university of medical science, and sample collection and utilization have been approved by the ethical committee of the first hospital affiliated with the university of medical science, and are collected and utilized strictly in accordance with relevant regulations and procedures. After a sample is collected, a part of tissues are stored in a liquid nitrogen tank in a liquid nitrogen quick-freezing mode, and a part of tissues are immediately fixed for 24-48 h by 4% PFA, wherein the specific treatment process comprises the following steps:
a. tissue dehydration: after the tissue was fixed with 4% PFA, the tissue was washed overnight with running water to remove residual PFA fixative. The tissue was then dehydrated in the order of 30% alcohol 1h → 50% alcohol 1h → 70% alcohol 4 ℃ overnight → 80% alcohol 1h → 90% alcohol 1h → 95% alcohol 1h → 100% alcohol I1 h → 100% alcohol II 1 h.
Description of the drawings: i and II represent the serial numbers of glass bottles, and the alcohol reagent is not different; the same applies to the following.
b. And (3) tissue transparency: after gradient dehydration, the tissue is put into a mixed solution glass jar of 50 percent absolute ethyl alcohol and 50 percent dimethylbenzene for 5min, then the tissue is transferred into dimethylbenzene I for 5min, and then transferred into dimethylbenzene II for 5 min.
Description of the drawings: i and II represent the numbers of glass bottles, and the xylene reagent is not different.
c. Tissue waxing: after the tissue was clear, the tissue was immersed in soft wax for 1h, followed by hard wax for 1 h.
d. Tissue embedding: taking out the tissue from the plastic embedding box, putting the tissue into a metal embedding box, covering the plastic embedding box on the plastic embedding box, dropwise adding a proper amount of hard wax to enable the hard wax to fully wrap the plastic embedding box, continuously transferring the wax block into an ice box after the hard wax is slightly solidified to enable the wax block to be separated from the metal embedding box, taking out the wax block, and storing the wax block at normal temperature or 4 ℃ for a long time.
4) Tissue total RNA extraction
a. Colorectal cancer clinical samples were removed from the ultra-low temperature refrigerator, approximately 50mg of each sample was taken in an EP tube, mixed well with 700. mu.l of Qiazol, and the tissue was minced and thoroughly disrupted with a tissue disruptor.
b. 200 μ l of chloroform was added, shaken vigorously for 15s, and allowed to stand on ice for 5 min. The centrifuge was precooled to 4 ℃ in advance. Centrifuge at 4 ℃ at 12000g for 15 min.
c. The supernatant was aspirated with a 200. mu.l removal tip and transferred to a new EP tube, approximately 400. mu.l. Add equal volume of 400. mu.l isopropanol, reverse mix and let stand on ice for 10 min. Centrifugation was carried out at 4 ℃ at 12000g for 10min, and the supernatant was discarded.
d. Preparing 75% alcohol with DEPC water, adding 1ml of prepared 75% alcohol into the precipitate, blowing the precipitate, centrifuging at 4 deg.C, 12000g, 5min, discarding supernatant, and repeating the steps.
e. Discarding the supernatant, then performing air separation for 5min, sucking the residual supernatant by a small enzyme-removing gun head, and leaving white sediment at the bottom. And opening the cover and airing, and adding enzyme-removing water after the white precipitate at the bottom is transparent. Dissolving at 4 deg.C for 2 hr, and determining RNA concentration.
5)RT-QPCR
After extraction and determination of the RNA concentration according to step 4), SuperScript purchased from Invitrogen was usedTMIV, carrying out reverse transcription by using the reverse transcription kit, wherein the reverse transcription reaction system and the steps are as follows according to a reagent use instruction:
adding the components into a PCR tube according to an instruction, oscillating and uniformly mixing, then placing the mixture into a PCR instrument, and setting a first-step reaction program of the PCR instrument: 65 ℃ for 5 min. After the reaction is finished, standing on ice for more than 1min, uniformly mixing the components according to the system shown in the table, adding the mixture into the product obtained in the first step, and carrying out the second step of PCR reaction.
Adding the components into a PCR tube according to an instruction, oscillating and uniformly mixing, then placing the mixture into a PCR instrument, and setting a second-step reaction program of the PCR instrument: 50-55 deg.C for 10min, and 80 deg.C for 10 min. After the cDNA is obtained, the cDNA is stored at the temperature of minus 80 ℃ in a sealing film sealing way or is stored after the next experiment is finished. After obtaining cDNA from the desired cells, PCR was carried out using a kit purchased from Qiagen, and the PCR reaction was as follows (4 ℃ procedure):
and (3) fully and uniformly mixing the components according to the reaction system, adding the mixture into a 384-well plate, setting 3 multiple wells for each sample, centrifuging for 1000g for 1min to uniformly mix the components, depositing the components at the bottom of the well, and placing the well in a Q6 fluorescent quantitative PCR instrument for detection. The detection primers are specific primers as follows:
f (upstream primer): 5'-CACTGATGCCCTACAATGGTACTAT-3'
R (downstream primer) 5'-GTTCCTAGGGTCATGTCGTTCAG-3'.
The PCR reaction conditions were pre-denaturation: 95 ℃, 30s, denaturation: 95 ℃, 5 seconds, annealing: 58 ℃, 30 seconds, extension: at 72 deg.C for 30 seconds, 40cycles in total were set.
6) Immunohistochemistry (IHC)
a. Baking sheets and dewaxing: baking the slices in an oven at 65 ℃ for 3h, and fully melting wax, wherein xylene I (10min) → xylene II (10min) → 50% xylene and 50% absolute ethyl alcohol for 2 min.
b. Hydration: absolute ethanol 100% i → absolute ethanol 100% ii → 95% → 90% → 80% → 70% → 50% each for 90 seconds. ddH2O rinse for 10 min. The water was changed 3 times and the washing was sufficient.
c. Antigen retrieval (microwave retrieval method): adding a certain amount of antigen repairing solution with pH of 6.0 and 0.01M into a white slide repairing box, inserting the slices into a plastic rack, placing the plastic rack into a microwave oven, preheating to boil, adjusting the microwave firepower to 30P, and multiplying by 7min by 4.
d. Cooling to room temperature, throwing away the antigen repairing solution, washing with 1 × TBS for three times, each time for 5min, throwing away water stain of the slide after washing, and circling out the tissue by a special pen for grouping. 3% H was added dropwise to the slide2O2Blocking endogenous peroxidase, placing in a wet box, and incubating at room temperature for 30 min.
e. Get rid of H2O2Washed three times with 1 × TBS, 5 min/time. TBS was spun off, 5% BSA was added dropwise to block non-specific binding sites, and incubation at room temperature for 30 min.
f. BSA was spun off, without washing, the gun added primary antibody, 20. mu.l or so, TBS was added to the blank control, IgG of the same nature was added to the negative control, and incubation was carried out overnight at 4 ℃ in a wet box. Taking out the incubation wet box in a refrigerator at 4 ℃, re-warming for 30min at room temperature, and recovering the primary antibody.
g.1 × TBS three times, 5 min/time, TBS was spun off, biotinylated secondary antibody corresponding to the primary antibody was added and incubated at 37 ℃ in a wet box for 1 h. The secondary antibody was spun off and washed three times with 1 × TBS, 5 min/time.
h. SABC was added dropwise, incubated at 37 ℃ for 1h in a humidified chamber, spun off the SABC, and washed three times with 1 × TBS.
i. Color development: and (4) dropwise adding a proper amount of DAB color developing agent which is prepared in situ, controlling color development under a microscope and recording time. ddH2O was washed for 5min and water was changed three times.
j. Hematoxylin staining: hematoxylin was added dropwise to the slide tissue, staining for 2min, and the staining was stopped with tap water. Washing with tap water for 5 min.
k. And (3) dehydrating and decoloring: the slide glass was sequentially placed in 50% → 70% → 80% → 95% → 100% i → 100% ii ethanol for 90 seconds each.
l. transparent: 50% xylene + 50% absolute ethanol 2 min-xylene I (5min) -xylene II (5 min). Air drying in a fume hood for 30min, and sealing with neutral gum. Avoiding generating air bubbles, and placing the sealed piece in a fume hood for airing after the sealing is finished.
7) Western Blotting (Western Blotting technique)
a. Stably transfected cell strains (30 ten thousand per well) are inoculated into a 6-well plate, after the cells are attached to the wall, the cells are cultured for 12h by using a 0.1% FBS culture medium, and then the cells are continuously cultured for 24h by using a corresponding 10% FBS complete culture medium. When the cell density reached about 85%, the culture was terminated. Then the operation is carried out according to the following steps:
b. cell lysis: discarding the culture medium, washing with PBS once, discarding PBS, placing the six-hole plate on an ice box, adding about 100ul of BB cell lysate into each hole, and scraping the cells by cell scraping rapidly to ensure that the cells are rapidly and fully lysed. The lysate was pipetted into a previously labeled 1.5ml EP tube.
c. Protein sample treatment: the 1.5ml EP tube containing the protein sample was placed on a metal bath at 100 ℃ and the sample was boiled for 5 min. After the protein sample is cooled, the cells are disrupted by ultrasonication (1 s/time, 1s interval, 30 times in succession) until the sample changes from viscous state to non-viscous liquid state. And (3) measuring the protein concentration of the sample by using a Biodrop instrument, measuring each sample twice, averaging, and requiring that the difference of the concentration measured twice is less than 0.1, then using BB and 6 XSB to determine the protein of each sample as a uniform concentration, after the volume fixing is finished, placing the protein sample on a metal bath at 100 ℃ again, and boiling the sample for 5 min.
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis: performing gel electrophoresis on a protein sample of about 100 μ g, adjusting voltage to 150v for about 15min when running concentrated gel, adjusting voltage to 125v when the sample enters separation gel, and continuing to run gel for about 1h45 min; and stopping electrophoresis when the bromophenol blue indicator reaches a position 1cm away from the bottom of the gel plate.
e. Film transfer: PVDF membrane with length of 8cm and width of 6cm is activated by methanol for about 15s, and then soaked in membrane transfer buffer. The assembly sequence from the cathode to the anode is as follows: the method comprises the following steps of sponge pad-filter paper-glue-PVDF membrane-filter paper-sponge pad, wherein bubbles are prevented from being generated in the process, and after the electrophoresis device is assembled, the voltage is set to be 25v for 4.5 h. The proteins on the gel were transferred to PVDF membrane by wet transfer method.
f. And (3) sealing: and (3) after the membrane conversion is finished, removing the membrane conversion device, marking corresponding experimental information on the PVDF membrane, washing the membrane for 5min by TBS, and then adding 5% skimmed milk, sealing for 1h by a shaking table at room temperature.
g. Primary antibody incubation: after the blocking, the membrane was washed with TBS for 5min, three times, thoroughly washing away the milk residues, adding the diluted primary antibody corresponding to the detection protein, and incubating overnight at 4 ℃. The primary antibody was recovered the next day and washed 3 times with 0.1% TBST for 5min each time.
h. And (3) secondary antibody incubation: adding Alkaline Phosphatase (AP) -labeled secondary antibody of corresponding species at appropriate concentration, and incubating at 4 deg.C for 2-3h according to antibody strength. The secondary antibody was recovered and washed 3 times with 0.1% TBST for 15min each time and 3 times with 0.1% TBS for 5min each time.
i. And (3) developing: preparing ECF developing solution of an AP substrate according to the ratio of 1:10, putting a PVDF film into the developing solution to react for 5s to 1min according to the strength difference of different antibodies, putting the PVDF film into a plastic film (avoiding bubbles), scanning the PVDF film by a Typhoon7000 film scanner (GE company) at different exposure degrees to obtain images, and storing the analysis and record results.
The results obtained were: TEX11 down-regulated in colorectal cancer clinical tissues and cell lines; therefore, it can be seen that: TEX11 can be used as a diagnostic marker for colorectal cancer.
Example 2 patients with high expression of TEX11 had a significantly better prognosis than patients with low expression of TEX11, as shown in FIG. 2.
The clinical information related to prognosis of colorectal cancer patients in the TCGA database is collated, and the clinical information comprises total survival time (OS), survival state (OS status), disease-free survival time (DFS) and disease-free survival state (DFS status). The mRNA of TEX11 was divided into two groups according to the expression levels of the tumor groups, respectively: high and low expression, and log-rank statistical test, setting p <0.05 as the statistical significance threshold. mRNA was analyzed for patient prognosis and K-M survival curves were plotted.
The results obtained were: the higher the expression of TEX11, the longer the disease-free survival and overall survival of the patient; therefore, it can be seen that: TEX11 can be used as a prognostic marker for colorectal cancer.
Example 3, TEX11 significantly inhibited the proliferative capacity of colorectal cancer cells in vitro, as shown in figure 3.
1) SW480 cells and RKO cells, Fenghui biological company (https:// www.fenghbio.cn /) are selected to construct pCDH-EF1-MCS-T2A-Puro-TEX11 overexpression plasmids, then the pCDH-EF1-MCS-T2A-Puro-TEX11 plasmids are utilized to overexpress TEX11 in SW480 and RKO cells, and stably transfected cells SW480-TEX11, RKO-TEX11 and comparison SW480-Vector, RKO-Vector and western-blot experiments thereof are established to verify the overexpression efficiency (figure 3A).
2) The ATP experiment is adopted to detect the growth activity change of the SW480-TEX11 and RKO-TEX11 of the tumor cells compared with the growth activity change of the SW480-Vector and RKO-Vector of a control (figure 3B-C), and the specific steps are as follows: digesting the cells in the logarithmic growth phase by using 0.25% pancreatin, blowing and beating the cells into single cell suspension by using a culture medium, counting, taking the cells with the corresponding suspension volume, and adding the cells into a 96-well plate according to the corresponding cell number. And (4) taking out corresponding cells from the incubator after the cells are attached to the wall, and observing the state under a microscope. The ATP detection reagent was removed from-20 ℃ and dissolved at room temperature. The old medium in the 96-well plate was spun off, 25. mu.l of PBS was added to each well, and 25. mu.l of ATP detection reagent was added to each well, and the plate was protected from light. Away from light, shake for 3min on the oscillator, and stand at room temperature for 10 min. West cell lysates from 96-well plates were transferred to photophobic plates with 40. mu.l per well. And (6) performing detection on the machine.
3) Tumor cells SW480-TEX11, RKO-TEX11 were evaluated for their anchorage-independent malignant proliferative capacity compared to control SW480-Vector, RKO-Vector cells using a soft agar clonogenic (soft agar) assay (FIG. 3D-G). The method comprises the following specific steps: 1.2ml of 1.25% agarose solution and 1.8ml of prepared culture medium (namely medium) are taken from each hole and put into a 15ml centrifuge tube, and the mixture is lightly blown and uniformly mixed and then added into the holes of a 6-hole plate, so that air bubbles are prevented from being blown and blown, and the plate is laid flat and uniformly, and the air bubbles are prevented from being generated. After standing for at least 2h, paving the upper layer glue according to the following system:
first, 1.25% agarose gel and 2X cell culture medium are mixed evenly and put into a 42 ℃ water bath for preheating, then 0.25% pancreatin is used for digesting the cells in logarithmic phase, the cells are blown and beaten into single cell suspension by using the culture medium, after counting, the cells with the corresponding suspension volume are added into the agarose gel and 2X cell culture medium with the corresponding cell number of 1.25%, and then the plates are paved. Standing for 1-2 hours, sealing the 6-hole plate with a sealing film, then placing the 6-hole plate into a 5% carbon dioxide cell incubator at 37 ℃ for continuous culture, beginning to observe the growth state of the clone after about 7 days, taking a picture by using a microscope 5-fold mirror when the clone grows to a proper size, counting, and calculating the formation rate of the cell colony number.
The results obtained were: the in vitro proliferation capacity of colorectal cancer cells can be obviously inhibited by over-expressing TEX 11; therefore, it can be seen that: TEX11 can be used as a new therapeutic target for inhibiting colorectal cancer proliferation.
Example 4, overexpression of TEX11 significantly inhibited the proliferative capacity of colorectal cancer cells in vivo, as shown in figure 4.
1) Animal feeding
BALB/C-nu female nude mice, week age 3-4 weeks, weight 15 + -0.5 g, experimental animals purchased from Jiangsu Jiejiaokang Biotech limited, and bred in SPF level experimental area of Experimental animals center of Wenzhou university of medical science. The animal experiments performed have been approved by the ethical committee of experimental animals at the university of medical science in wenzhou and the experimental procedures comply with the ethical requirements of the ethical committee on animals.
2) Subcutaneous injection
0.25% pancreatin SW480-Vector, SW480-TEX11, RKO-Vector, RKO-TEX11 cells in logarithmic growth phase; terminating digestion by using a culture medium, collecting cells of all culture dishes into a 50ml centrifuge tube, centrifuging at 1500rpm for 5min, then discarding a supernatant culture medium, washing the cells once by using PBS (phosphate buffer solution) for heavy suspension, centrifuging at 1500rpm for 5min again, then discarding the PBS, adding 1ml of PBS for heavy suspension, diluting the cells according to a certain proportion, filling a pool for counting, and calculating the required cell amount. Each nude mouse was injected subcutaneously with 100. mu.l of cell suspension containing a cell mass of 300 ten thousand. The subcutaneous injection part of the nude mice is wiped and disinfected by 75 percent alcohol, the cells are fully and uniformly mixed before inoculation, 100ul of cell suspension is absorbed by a 1ml sterile insulin syringe and is uniformly injected at the subcutaneous position of the right back of the mice, and 5 nude mice are injected in each group.
3) Determination of photographs
PBS was well absorbed by the nude mice for about 1 week, the tumor cells had primarily formed tumors, at which time the tumor volume was calculated by measuring the tumor size with a vernier caliper (tumor volume V: 0.52X (a. times. B2); tumor volume was measured at various time points and growth curves were plotted (FIGS. 4E-F). when the nude mice were inoculated with tumor cells and grown for about 3 weeks, the nude mice were anesthetized with 0.5% sodium pentobarbital and then sacrificed, the tumors were dissected out, photographed (FIGS. 4A, C) and weighed (FIGS. 4B, D).
The results obtained were: the over-expression of TEX11 can obviously inhibit the proliferation capacity of colorectal cancer cells in vivo; it was therefore further demonstrated that TEX11 could be a novel therapeutic target for inhibiting colorectal cancer proliferation.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Sequence listing
<110> first Hospital affiliated with Wenzhou medical university
Wenzhou Medical University
<120> target and diagnostic marker for inhibiting colorectal cancer growth and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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cactgatgcc ctacaatggt actat 25
<210> 2
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gttcctaggg tcatgtcgtt cag 23
Claims (6)
- Use of a TEX11 expression promoter in the manufacture of a medicament for inhibiting the growth of colorectal cancer.
- 2. Use of a promoter of TEX11 expression in the manufacture of a medicament for inhibiting the growth of colorectal cancer according to claim 1, wherein: inhibiting proliferation of colorectal cancer cells in vivo.
- 3. Use of a promoter of expression of TEX11 in the manufacture of a medicament for inhibiting the growth of colorectal cancer according to claim 1 or 2, wherein: the TEX11 expression promoter is an overexpression plasmid of TEX11, and is a pCDH-EF1-MCS-T2A-Puro-TEX11 plasmid.
- 4. A composition for use in the prevention or/and treatment of colorectal cancer, wherein the composition comprises:(1) a TEX11 expression promoter;(2) a pharmaceutically acceptable carrier.
- 5. Composition for the prevention or/and treatment of colorectal cancer according to claim 4, characterized in that: the TEX11 expression promoter is an overexpression plasmid of TEX 11.
- 6. An agent for detecting the expression of TEX11, which is characterized in that: the reagent for TEX11 expression comprises a reagent based on a fluorescent quantitative PCR quantitative detection method, and the reagent for the fluorescent quantitative PCR quantitative detection method comprises a pair of specific primers;the specific primers are as follows:f (upstream primer): 5'-CACTGATGCCCTACAATGGTACTAT-3'R (downstream primer) 5'-GTTCCTAGGGTCATGTCGTTCAG-3'.
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|---|---|---|---|---|
| CN116735875A (en) * | 2022-07-14 | 2023-09-12 | 宁波大学 | Application of protein INF2 in preparation of liver cancer diagnosis marker |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110097423A1 (en) * | 2009-10-22 | 2011-04-28 | Vanderbilt University | Gene Prognosis Predictor Signature for Colorectal Carcinoma |
| CN105506110A (en) * | 2015-12-31 | 2016-04-20 | 杭州艾迪康医学检验中心有限公司 | Method and primer for detecting 8th and 25th whole exons of TEX11 gene |
-
2020
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110097423A1 (en) * | 2009-10-22 | 2011-04-28 | Vanderbilt University | Gene Prognosis Predictor Signature for Colorectal Carcinoma |
| CN105506110A (en) * | 2015-12-31 | 2016-04-20 | 杭州艾迪康医学检验中心有限公司 | Method and primer for detecting 8th and 25th whole exons of TEX11 gene |
Non-Patent Citations (2)
| Title |
|---|
| TIANCHENG LUO ET AL.: "Network cluster analysis of protein–protein interaction network identified biomarker for early onset colorectal cancer", 《MOL BIOL REP》 * |
| 李洪岩: "《温州医科大学申请硕士学位论文答辩会》", 21 May 2020 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116735875A (en) * | 2022-07-14 | 2023-09-12 | 宁波大学 | Application of protein INF2 in preparation of liver cancer diagnosis marker |
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Application publication date: 20210507 |