CN114164270B - Application of CRIP2 in detecting resistance of prostate cancer to docetaxel and reversing resistance of prostate cancer to docetaxel - Google Patents
Application of CRIP2 in detecting resistance of prostate cancer to docetaxel and reversing resistance of prostate cancer to docetaxel Download PDFInfo
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Abstract
The invention belongs to the technical field of medical biology, and discloses a molecular marker for detecting the drug resistance of prostate cancer to docetaxel, wherein the molecular marker is CRIP2 gene or CRIP2 protein; the expression level of CRIP2 gene or CRIP2 protein in DTX drug-resistant prostate cancer cells is obviously higher than that of normal prostate cancer cells, and the difference has statistical significance, so that the expression level of CRIP2 gene or CRIP2 protein can be used as one of the basis for judging the drug resistance/sensitivity of a drug user to DTX drugs by measuring the expression level of CRIP2 gene or CRIP2 protein. In addition, the expression level of the CRIP2 gene in the DTX resistant prostate cancer cells is knocked down, so that the sensitivity of the DTX resistant prostate cancer cells to DTX can be improved, meanwhile, the proliferation of the DTX resistant prostate cancer cells can be obviously inhibited after the CRIP2 gene is interfered, therefore, the CRIP2 gene can be used as an action target point for reversing the DTX resistance of the prostate cancer, and the drug resistance of tumor cells to docetaxel can be reversed by inhibiting the expression and/or the function of the CRIP2 gene, so that the CRIP2 gene has important significance for treating the prostate cancer.
Description
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to application of a CRIP2 gene in detecting drug resistance of prostate cancer to docetaxel and reversing the drug resistance of the prostate cancer to docetaxel.
Background
Prostate Cancer (PC) is the most common malignancy in men and is also the 2 nd leading cause of cancer-related death in men worldwide. The early stage of the prostate cancer is often asymptomatic, and a simple, effective and specific screening mode is lacking, so that the early diagnosis of the prostate cancer is difficult, and most patients are in middle and late stages in diagnosis, so that the treatment is troublesome. Androgen ablation therapy is still the underlying treatment for metastatic prostate cancer, advanced carcinoma in situ, and hormone sensitive prostate cancer. Recent studies have found that the use of Docetaxel (DTX) chemotherapy for castration-resistant prostate cancer patients can significantly extend the overall survival of the patients. In the guidelines for diagnosis and treatment of urological disorders in China, 2019 edition, docetaxel-based treatment is suggested as a standard treatment regimen for castration-resistant prostate cancer.
Docetaxel (DTX) is a microtubule stabilizer that has an antitumor effect by disrupting the mitotic process of tumor cells by disrupting the dynamic equilibrium state of microtubules and tubulin dimers. Clones of Androgen Receptor (AR) insensitive prostate cancer cells may be involved in the progression of castration resistance. In hormone sensitive prostate cancer patients, androgen Deprivation Therapy (ADT) in combination with docetaxel can inhibit clonal growth of pre-existing androgen receptor insensitive cells, thereby allowing these cells to die before they replicate in large numbers and develop multiple escape mechanisms. Emerging preclinical data indicate that docetaxel can inhibit androgen receptor signaling. These cytotoxic drugs interfere with microtubule polymerization, preventing androgen receptor signaling and induced gene expression, and thus docetaxel may act synergistically with endocrine therapy and may destroy androgen receptor activity.
Docetaxel has better clinical effect as a first-line chemotherapeutic medicine for treating castration resistant prostate cancer. However, up to 95% of patients develop docetaxel acquired resistance after docetaxel treatment. Chemotherapy resistance is inevitable during tumor treatment, involving a variety of mechanisms including lowering the drug concentration of cells, increasing the cellular metabolism of drug detoxification proteins, activating survival signaling pathways, inhibiting apoptosis, etc. Searching for new docetaxel drug resistance molecular targets and possible mechanisms thereof has important significance for reversing chemotherapy drug resistance, improving chemotherapy efficiency and improving prognosis of patients.
According to the invention, after interference is carried out by taking the cysteine-rich intestinal protein 2 (CRIP 2) as a target, the interference CRIP2 reduces the drug resistance of the prostate cancer drug-resistant cells to docetaxel, reverses the drug resistance of the docetaxel drug-resistant prostate cancer cells, inhibits the proliferation of the prostate cancer drug-resistant cells, provides a new target for treating the prostate cancer, and provides a scientific basis for effectively resisting the drug resistance of the prostate cancer docetaxel.
Disclosure of Invention
In view of the problems and deficiencies in the prior art, one of the objects of the present invention is to study the application of CRIP2 gene in detecting and reversing the resistance of prostate cancer to docetaxel.
In order to achieve the aim of the invention, the technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides a molecular marker useful for detecting resistance of prostate cancer to docetaxel, the molecular marker being a CRIP2 gene or a CRIP2 protein (CRIP 2 protein is a protein encoded by the CRIP2 gene). The gene ID of CRIP2 gene in NCBI is 1397; the CRIP2 protein has the protein sequence number in NCBI: np_001303.1.
In the early research, the inventor utilizes a human prostatic cancer cell line DU145 to construct a cell model of human prostatic cancer cell line DU145/DTX (docetaxel) drug resistance, and the inventor utilizes qRT-PCR to detect the amplification level of CRIP2 genes in DU145 parent and DU145/DTXR cells, so that the amplification level of CRIP2 genes in DU145/DTXR cells is obviously higher than that of DU145 parent, and the difference has statistical significance. Moreover, the inventor adopts Western Blot to detect the expression level of CRIP2 protein in DU145 parent and DU145/DTXR cell, and finds that the expression level of CRIP2 protein in DU145/DTXR cell is obviously higher than that of DU145 parent, and the difference has statistical significance. Therefore, the CRIP2 gene or CRIP2 protein can be used for detecting the drug resistance or the sensitivity of the prostate cancer to docetaxel, and the expression level of the CRIP2 gene or CRIP2 protein can be used as one of the bases for judging the drug resistance/sensitivity of a drug user to DTX drugs by measuring the expression level of the CRIP2 gene or CRIP2 protein.
Furthermore, the inventors of the present invention found that stable interference of CRIP2 gene expression in DTX-resistant prostate cancer cells can increase sensitivity of DTX-resistant prostate cancer cells to DTX, and therefore, CRIP2 gene can be an action target for reversing DTX resistance of prostate cancer.
In a second aspect the invention provides the use of a detection reagent for the CRIP2 gene or a protein encoded thereby in the manufacture of a product for the detection or co-detection of resistance/sensitivity of prostate cancer to docetaxel.
According to the above application, preferably, the product detects the expression level of the CRIP2 gene or protein encoded thereby in a sample by real-time quantitative PCR, in situ hybridization, northern blotting, chip, high throughput sequencing platform, western blot or enzyme linked immunosorbent assay.
According to the above application, preferably, the product contains specific primers for amplifying the CRIP2 gene, probes hybridizing to the nucleotide sequence of the CRIP2 gene or antibodies specifically binding to the CRIP2 protein.
According to the above application, it is preferable that specific primer sequences for amplifying CRIP2 gene are shown as SEQ ID NO.1 and SEQ ID NO. 2;
SEQ ID NO.1：AATGCCCCAAGTGCGACAA；
SEQ ID NO.2：GCTTCTCGTAGATGTAGGAGCC。
according to the above application, preferably, the product is a chip, a preparation or a kit.
According to the above application, preferably, the sample is tissue, cells or serum; more preferably, the sample is a cell, most preferably, the cell is a prostate cancer cell.
In a third aspect the invention provides the use of a substance inhibiting CRIP2 gene expression and/or function in the manufacture of a product for reversing the resistance of a tumour cell to docetaxel
According to the above application, preferably, the tumor cell is a prostate cancer cell.
According to the above application, preferably, the substance inhibiting CRIP2 gene expression and/or function comprises siRNA and/or shRNA specifically targeting CRIP2 gene.
According to the application, preferably, the shRNA specifically targeting the CRIP2 gene comprises shRNA1 and shRNA2, wherein the sequence of the shRNA1 is shown as SEQ ID NO.3, and the sequence of the shRNA2 is shown as SEQ ID NO. 4;
SEQ ID NO.3：GCAAGCCCAGGGCGAGTATTG；
SEQ ID NO.4：GGGCGTCCCATGATCCCTTCT。
in a fourth aspect, the invention provides the use of a substance which inhibits CRIP2 gene expression and/or function in the manufacture of a product for inhibiting proliferation of docetaxel resistant tumour cells or in inhibiting proliferation of docetaxel resistant tumour cells.
According to the above application, preferably, the tumor cell is a prostate cancer cell.
According to the above application, preferably, the substance inhibiting CRIP2 gene expression and/or function comprises siRNA and/or shRNA specifically targeting CRIP2 gene.
According to the above application, preferably, the shRNA specifically targeting the CRIP2 gene includes shRNA1 and shRNA2, the sequence of shRNA1 is shown in SEQ ID No.3, and the sequence of shRNA2 is shown in SEQ ID No. 4.
In a fifth aspect, the invention provides a medicament for reversing the resistance of prostate cancer to docetaxel, comprising a substance that inhibits CRIP2 gene expression and/or function.
According to the above medicament, preferably, the substance inhibiting CRIP2 gene expression and/or function comprises siRNA and/or shRNA specifically targeting CRIP2 gene.
According to the above-mentioned medicament, preferably, the shRNA specifically targeting CRIP2 gene comprises shRNA1 and shRNA2, the sequence of shRNA1 is shown as SEQ ID NO.3, and the sequence of shRNA2 is shown as SEQ ID NO. 4.
According to the above medicament, preferably, the medicament further comprises other medicaments compatible with the substance for inhibiting CRIP2 gene expression and/or function and pharmaceutically acceptable carriers and/or auxiliary materials.
Further, the carriers/excipients include (but are not limited to): diluents, excipients such as lactose, sodium chloride, dextrose, urea, starch, water and the like, fillers such as starch, sucrose and the like; binders such as simple syrups, dextrose solutions, starch solutions, cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; humectants such as glycerol; disintegrating agents such as dry starch, sodium alginate, laminarin powder, agar powder, calcium carbonate and sodium bicarbonate; absorption promoters quaternary ammonium compounds, sodium lauryl sulfate, and the like; surfactants such as polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, monoglyceride of stearic acid, cetyl alcohol, etc.; wetting agents such as glycerin, starch, and the like; adsorption carriers such as starch, lactose, bentonite, silica gel, kaolin, and bentonite; lubricants such as talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, and the like.
In a sixth aspect, the invention provides a medicament for the treatment of prostate cancer comprising docetaxel and a substance inhibiting the expression and/or function of CRIP2 gene.
According to the above-described medicament for treating prostate cancer, preferably, the substance inhibiting the expression and/or function of CRIP2 gene comprises siRNA and/or shRNA specifically targeting CRIP2 gene.
According to the above medicament for treating prostate cancer, preferably, the shRNA specifically targeting the CRIP2 gene includes shRNA1 and shRNA2, the sequence of shRNA1 is shown in SEQ ID No.3, and the sequence of shRNA2 is shown in SEQ ID No. 4.
According to the above medicament for treating prostate cancer, preferably, the medicament further comprises other medicaments compatible with the substance for inhibiting CRIP2 gene expression and/or function and pharmaceutically acceptable carriers and/or auxiliary materials. Further, the carriers/excipients include (but are not limited to): diluents, excipients such as lactose, sodium chloride, dextrose, urea, starch, water and the like, fillers such as starch, sucrose and the like; binders such as simple syrups, dextrose solutions, starch solutions, cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; humectants such as glycerol; disintegrating agents such as dry starch, sodium alginate, laminarin powder, agar powder, calcium carbonate and sodium bicarbonate; absorption promoters quaternary ammonium compounds, sodium lauryl sulfate, and the like; surfactants such as polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, monoglyceride of stearic acid, cetyl alcohol, etc.; wetting agents such as glycerin, starch, and the like; adsorption carriers such as starch, lactose, bentonite, silica gel, kaolin, and bentonite; lubricants such as talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, and the like.
Compared with the prior art, the invention has the positive beneficial effects that:
(1) The invention discovers for the first time that the expression level of CRIP2 gene/protein in DTX drug-resistant prostate cancer cells is obviously higher than that of parent prostate cancer cells, so that the determination of the expression level of CRIP2 gene or CRIP2 protein can be used for judging drug resistance/sensitivity of a drug user to DTX drugs.
(2) The invention aims at DTX drug-resistant prostate cancer tumor cells, takes CRIP2 genes as targets, interferes CRIP2 gene expression, reduces CRIP2 gene expression quantity, and can improve the sensitivity of DTX drug-resistant prostate cancer cells to DTX, thereby reversing the drug resistance of the prostate cancer; therefore, by inhibiting CRIP2 gene expression in prostate cancer cells, the resistance of prostate cancer to DTX can be reversed, and the efficiency of tumor treatment can be improved.
(3) The shRNA of the specific targeting CRIP2 gene can efficiently inhibit or knock down the expression of the CRIP2 gene in target cells, so that the proliferation of docetaxel resistant prostate cancer cells is inhibited, and the growth of the prostate cancer cells is further inhibited, therefore, the shRNA can be used for preparing medicines for reversing the drug resistance of tumor cells to docetaxel, and has important significance in the treatment of prostate cancer.
Drawings
FIG. 1 is a graph showing the results of cell resistance detection of DU145 parent and DU145/DTXR cells;
FIG. 2 is a graph showing the results of CRIP2 expression levels in DU145 parent and DU145/DTXR cells; wherein A is an amplification result graph of CRIP2 genes in DU145 parent and DU145/DTXR cells, and B is an expression level result graph of CRIP2 proteins in DU145 parent and DU145/DTXR cells;
FIG. 3 is a graph showing the results of gene amplification after CRIP2 expression in stable interference DU145/DTXR cells and a graph showing the expression level of CRIP2 protein; wherein A is a CRIP2 gene amplification result diagram; b is a CRIP2 protein expression level result graph;
FIG. 4 is a graph showing the results of changes in cell sensitivity to DTX after stable interference CRIP2 in DU145/DTXR cells;
FIG. 5 is a graph showing the results of the CCK-8 assay for DU145/DTXR cell proliferation capacity after stable interference CRIP 2;
FIG. 6 is a graph of the results of cloning experiments to detect the ability of DU145/DTXR cells to proliferate after stable interference with CRIP 2.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation 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.
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 present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, components, and/or combinations thereof.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
Embodiment one: investigation of expression of CRIP2 Gene/protein in prostate cancer cells and prostate cancer drug resistant cells
1. Cell culture:
prostate cancer cell line DU145 was purchased from marsupenario life technologies limited; DTX-resistant prostate cancer cells DU145 (designated DU145/DTXR cells) were established by induction in the laboratory (currently stored in the laboratory and available to the public, who could purchase itDU145/DTXR cells). Prostate cancer cell line DU145 and DTX resistant prostate cancer cell line DU145 were each prepared with 1640 medium containing 10% fetal bovine serum in 5% CO 2 Culturing in a constant temperature cell incubator at 37 ℃, wherein DTX with corresponding concentration is added into a DTX drug-resistant prostate cancer cell culture medium.
Construction method of DTX drug-resistant prostate cancer cells DU 145/DTXR:
the invention uses docetaxel drug to induce human prostate cancer cells DU145, and changes the cells into a culture medium without drug for culture after 24 hours of drug administration until the cells can stably grow and passaged in the culture medium under the concentration; the docetaxel drug concentration was then steadily increased and the above-described induction culture steps were continued until a prostate cancer docetaxel drug-resistant cell line was obtained that was resistant to a docetaxel drug concentration of 50nM. The initial drug concentration was 1 nM, and the gradually steadily increasing drug concentrations were 4nM, 6nM, 8nM, 10nM, 15nM, 20nM, 25nM, 30nM, 35nM, 40nM, 45nM, 50nM, respectively. Finally, prostate cancer cells DU145 (i.e., DU145/DTXR cells) capable of stable growth and passage in culture medium containing a drug concentration of 50nM docetaxel were obtained.
2. Cell resistance analysis:
cell density was adjusted to 1X 10 by taking DU145 and DU145/DTXR cells in logarithmic growth phase 5 Inoculating the cells/mL into 96-well plate, adhering to the wall for 36 hr, adding docetaxel to obtain final concentrations of 0nM, 0.78125nM, 1.5625nM, 3.125nM, 6.25nM, 12.5nM, 25nM, 50nM, 100nM, 200nM, and setting 5 multiple wells per group at 37deg.C and 5% CO 2 After 48h, 10. Mu.L of CCK8 solution was added to each well, and after 2h in the incubator, the absorbance at 450nm was measured.
Drug Resistance Index (RI) =drug resistant cell (DU 145/DTXR) IC 50/parental cell (DU 145) IC50. The tolerance of human prostate cancer DU145 cells and drug resistant cell lines DU145/DTXR to docetaxel is shown in FIG. 1.
As can be seen from FIG. 1, DU145 cells are more sensitive to low dose docetaxel, the survival rate gradually decreases with increasing concentration, the IC50 of DU145 cells is 8.337 + -0.23 nM, the IC50 of DU145/DTXR cells is 61.39+ -0.29 nM, and the drug Resistance Index (RI) is 7.35, suggesting that the drug resistant cells DU145/DTXR have good tolerance to docetaxel.
qRT-PCR detection of CRIP2 Gene expression in DU145 cells and DU145/DTXR resistant cells:
(1) Experimental method
1) Total RNA extraction:
a) The cells in logarithmic growth phase DU145 and DU145/DTXR were removed, the medium was aspirated, and after washing the cells 2 times with PBS pre-chilled at 4℃the residual PBS was aspirated. And (3) absorbing a proper amount of Trizol according to the cell number, rapidly lysing the cells in an orifice plate or a culture dish, blowing the cells, then separating the cells, collecting the cells into a 1.5mL enzyme-free EP tube, and standing the cells at room temperature for 5min.
b) 200 mu L of chloroform is correspondingly added into 1mL of Trizol, and the mixture is stirred and mixed for 1min by intense shaking and stands for 10min at room temperature; after the completion of the standing, the mixture was transferred to a centrifuge at 4℃and centrifuged at 13000rpm for 15 minutes.
c) After centrifugation, three obvious layers are visible, the top supernatant is gently collected by the gun head, and the lower two layers can not be sucked by less suction, so that pollution can be avoided, and the supernatant is used for the following steps: isopropanol was added at a ratio of 1:1, and the mixture was gently inverted to mix the mixture, and after leaving on ice for 20min, the mixture was centrifuged at 13000rpm at 4℃for 8 min.
d) After centrifugation, a little white precipitate is found at the bottom of the centrifuge tube, the supernatant is removed by sucking with a gun head, the precipitate is prevented from being sucked away, after the supernatant is sucked, 75% ethanol prepared by 700 mu L DEPC water is gently added, the mixture is placed under the condition of being slightly inverted up and down, and after standing for 2min at room temperature, the mixture is centrifuged at 4 ℃ and 7500rpm for 5min.
e) The above procedure was repeated and washed once more with 75% ethanol. After centrifugation, the supernatant was removed by the same method, 700. Mu.L of 75% ethanol was gently added, and after inversion with gentle inversion, the mixture was allowed to stand at room temperature for 2min, and centrifuged at 4℃and 7500rpm for 5min.
f) Sucking and discarding ethanol by using a gun head, opening a clean bench to blow for airing, adding a proper amount of DEPC water for dissolving after airing, and immediately placing on ice after dissolving.
g) Measuring the concentration and purity of RNA by a measuring instrument, marking, avoiding repeated freezing and thawing of RNA as much as possible, packaging according to the situation, and transferring to a refrigerator at-80 ℃ for preservation after packaging.
2) Reverse transcription synthesis of cDNA:
a) The extracted RNA was thawed on ice and the volume of no more than 500ng was calculated as the concentration.
b) The forceps were burned on an alcohol burner for about 10 seconds, and after cooling, a 0.2mL PCR tube of RNase free was taken out of the storage box.
c) 5X gDNA Eraser Buffer, 5X PrimeScript RT Master Mix of the PrimeScript RT Master Mix (Perfect Real Time) kit were removed, centrifuged briefly and placed on ice.
d) The reaction solution for removing genomic DNA was prepared according to the composition shown in Table 1 and was performed on ice:
TABLE 1 reaction liquid System for removing genomic DNA
| Reagent(s) | Usage amount |
| 5×gDNA Eraser Buffer | 4μL |
| gDNA Eraser | 1μL |
| Total RNA | * |
| RNase Free dH 2 O | up to 10μL |
e) And (3) the reaction conditions of the machine: 42 ℃ 2min and 4 ℃ infinity.
f) RT reaction solutions were prepared according to the compositions in Table 2 and were run on ice:
TABLE 2 reverse transcription reaction system
g) After the prepared mixed solution is gently and evenly mixed, the PCR instrument is opened, the samples are put into the instrument one by one according to the following circulation set program, and the reverse transcription reaction is started, wherein the reverse transcription reaction program is as follows: after the reverse transcription reaction, cDNA is synthesized at 37 ℃ for 15min,85 ℃ for 5s and 4 ℃ for infinity, and the cDNA is marked and stored at-20 ℃. The synthesized cDNA was diluted 1:3 for qPCR reaction (RT reaction solution was added to the next Quantitative Real-time PCR (qRT-PCR) reaction system in an amount not exceeding 1/10 (V/V) of the volume of qRT-PCR reaction).
3) Fluorescent quantitative detection:
the amplification of CRIP2 gene in DU145 parent and DU145/DTXR was detected by qRT-PCR reaction using GAPDH as an internal reference, and the difference between the two was determined.
The nucleotide sequence of the CRIP2 specific amplification primer is as follows:
an upstream primer: 5-AATGCCCCAAGTGCGACAA-3' (SEQ ID NO. 1);
a downstream primer: 5-GCTTCTCGTAGATGTAGGAGCC-3' (SEQ ID NO. 2).
The nucleotide sequence of the specific amplification primers for GAPDH is as follows:
an upstream primer: 5'-CTGGGACGACATGGAGAAAA-3';
a downstream primer: 5'-AAGGAAGGCTGGAAGAGTGC-3'.
The qRT-PCR reaction system (20. Mu.L system) was prepared in the proportions shown in Table 3.
TABLE 3 qRT-PCR reaction System
| Name of the name | Volume of |
| FastStart Universal SYBR Green Master(Rox) | 10μL |
| Upstream primer (10. Mu.M) | 0.5μL |
| Downstream primer (10. Mu.M) | 0.5μL |
| cDNA | 1μL |
| ddH 2 O | To 20 mu L |
And (3) the reaction conditions of the machine: 1. pre-denaturation: 95 ℃ for 30s; PCR reaction: 95 ℃ for 5s,60 ℃ for 30s (fluorescence acquisition), 40 cycles; dissociation: 15s at 95 ℃, 30s at 60 ℃ and 15s at 95 ℃. Copying data after finishing the machine, according to 2 -△△CT And analyzing the relative expression quantity of the corresponding CRIP2 in each sample.
(2) Data processing and analysis
Experiments were performed in 3 replicates, and the data obtained were expressed as mean ± standard deviation, and were statistically analyzed using SPSS 18.0 statistical software, the differences between the two using t-test, and were considered statistically significant when P < 0.05.
(3) Experimental results
qRT-PCR detected gene amplification of CRIP2 in DU145 parental cells and DU145/DTXR cells, the detection results are shown in FIG. 2A. As can be seen from FIG. 2A, the CRIP2 gene amplification level in DU145/DTXR cells was significantly higher than that of DU145 parent cells, and the differences were statistically significant (P < 0.05).
Western blot detection of CRIP2 protein expression in DU145 parent and DU145/DTXR cells:
(1) The experimental method comprises the following steps:
1) SDS-PAGE gel electrophoresis: according to methylene bisacrylamide: a 30% gel stock solution was prepared at a ratio of acrylamide=1:29, and SDS-PAGE gel was prepared from the stock solution, and separation gels at different concentrations were prepared according to the size of the target protein. Mixing the protein samples evenly, and loading the protein samples after instantaneous centrifugation. Preparing electrophoresis liquid (10 times of electrophoresis liquid 100mL+10%SDS 10mL +deionized water to 1000 mL), firstly performing electrophoresis at constant voltage of 80V, separating by 20min-30min to Marker strips, and adjusting to constant voltage electrophoresis of 100V-120V; and (5) observing the separation condition of the strips, stopping electrophoresis until the target strips are separated, and cutting the corresponding gel.
2) Transferring: and (3) soaking a PVDF film with a proper size of 0.45 mu m in methanol for 15s for activation, sequentially placing the PVDF film from a negative electrode to a positive electrode according to sponge-filter paper-gel-film-filter paper-sponge, and removing bubbles by using a glass rod before placing the PVDF film into a film transferring device. 250mA is carried out under the ice bath condition, and the film is transferred for 60min-90min.
3) Closing: blocking was performed with 5% nonfat dry milk formulated with TBST for 2h at room temperature.
4) Incubation resistance: diluting the primary antibody with primary anti-dilution solution according to the recommended proportion of the antibody specification, soaking the sealed PVDF membrane with the primary antibody solution, and then incubating overnight in a refrigerator at 4 ℃.
5) Washing the film: PVDF membrane was washed 3 times with TBST for 15min each.
6) Secondary antibody incubation: TBST with 1% skim milk powder was prepared at 1: HRP-labeled goat anti-rabbit antibody was diluted at a ratio of 5000, and the membrane was immersed in a secondary antibody solution and incubated for 2h at room temperature.
7) Washing the film: PVDF membrane was washed 3 times with TBST for 15min each.
8) Exposure: starting up the C400 exposure instrument, mixing the ECL color development liquid A and the ECL color development liquid B according to the equal volume of 1:1, horizontally placing the film on an exposure plate, uniformly dripping the mixed ECL color development liquid on the film, putting the film into the exposure instrument, exposing and preserving the picture.
(2) Data processing and analysis
Experimental data are expressed as mean ± standard deviation, using SPSS 18.0 statistical software for statistical analysis, and the difference between the two is determined by t-test, which is considered statistically significant when P < 0.05.
(3) Experimental results
Western Blot examined CRIP2 protein expression in DU145 parental cells and DU145/DTXR cells, the results of which are shown in FIG. 2B. As can be seen from fig. 2B, the protein expression level of CRIP2 protein in DU145/DTXR cells was significantly increased compared to DU145 parental cells, suggesting that CRIP2 protein expression may be associated with prostate cancer docetaxel resistance.
The detection results of qRT-PCR and Western Blot show that the change of the expression level of CRIP2 gene or CRIP2 protein can be used as one of the bases for judging the drug resistance/sensitivity of a drug user to DTX drugs.
Embodiment two: construction of cell lines stably interfering with CRIP2 in DU145/DTX resistant cells
1. Cell culture:
the specific operation of cell culture is the same as that of example one, and will not be described here again.
shRNA design
The shRNA lentiviral vector name of CRIP2 gene is:
negative control shRNA (noted sh-NC) sequence: GGGCAAGACGAGCGGGAAG;
sequence of shRNA 1: GCAAGCCCAGGGCGAGTATTG (SEQ ID NO. 3);
sequence of shRNA 2: GGGCGTCCCATGATCCCTTCT (SEQ ID NO. 4).
3. Cell stable transfection:
cells were transfected according to the transfection Reagent Lipofectamine 3000Reagent (Invitrogen) instructions. The method comprises the following specific steps:
(1) 293T cells were inoculated into T25 flasks at 37℃,5％CO 2 The cells were cultured in the incubator for 24h to reach a cell confluence of 80% and fresh DMEM medium was replaced with 3.8mL before transfection.
(2) Transfection: lipofectamine 3000Reagent (inventory) was removed from the refrigerator at 4℃in advance, shRNA1, shRNA2, sh-NC were removed from the refrigerator at-20℃and dissolved on ice, and 150. Mu.L of Opti-MEM medium was added to each labeled EP tube.
a. 5 μg of shRNA was blended with packaging plasmids PSPAX2 and PMD2G in a ratio of shRNA: PSPAX2: PMD2 G=5:4:1 (10 μg total) and added to EP tube (tube A) containing 150 μl of Opti-MEM medium;
b. 25. Mu.L of Lipofectamine 3000Reagent (Invitrogen) was added to EP tube (tube B) containing 100. Mu.L of Opti-MEM medium;
c. sucking out all the liquid in the pipe A by using a pipetting gun, adding the liquid into the pipe B, and incubating for 15min at room temperature;
d. then the liquid in the B tube of the liquid transferring gun is completely sucked out, evenly dripped into a 6-hole plate, evenly mixed by shaking, the 6-hole plate is put back into a 37 ℃ incubator for continuous culture, and after 8 hours, the fresh DMEM culture medium containing 10% FBS is replaced. After 72h of culture, the mixture was filtered with a 0.45 μm filter membrane and sub-packaged into 1 mL/tube for storage in a refrigerator at-80℃for further use.
(3) Virus infection and stable strain screening:
a. DU145 cells were packed in 2.0X10-fold 5 Plating into six-well cell culture plate at 37deg.C with 5% CO 2 Culturing cells in an incubator for 24 hours, and carrying out virus infection when the cell confluence reaches 50%;
b. the DU145 medium was replaced with 2. Mu.L/well polybrane containing virus solution at 37℃with 5% CO 2 Culturing cells in an incubator for 48 hours;
c. each well was subjected to stable selection by adding 5. Mu.g/mL puromycin, and the cells were cultured by continuous liquid exchange to select stably transfected cell lines.
Among them, DU145/DTXR cells stably transfected with shRNA-NC (designated as sh-NC), DU145/DTXR cells stably transfected with shRNA1 (designated as sh-CRIP2# 1), and DU145/DTXR cells stably transfected with shRNA2 (designated as sh-CRIP2# 2).
qrt-PCR to detect the level of gene amplification of CRIP2 in transfected prostate cancer cells:
(1) Experimental method
1) Cell RNA extraction:
the specific procedure for cellular RNA extraction is the same as in example one and will not be described in detail here.
2) Reverse transcription synthesis of cDNA:
the specific procedure for the synthesis of cDNA by reverse transcription is the same as that of example one and will not be described here.
3) Fluorescent quantitative detection:
the specific operation steps of the fluorescent quantitative detection are the same as those of the first embodiment, and are not described herein.
(2) Experimental results
The qRT-PCR detection results are shown in FIG. 3A. As can be seen from fig. 3 a, the CRIP2 gene amplification levels of the sh-crip2# 1 and sh-crip2# 2 experimental groups stably interfering with CRIP2 were significantly down-regulated compared to the negative control group (sh-NC group), indicating that the knockdown cell line stably interfering with CRIP2 was successfully obtained by lentiviral infection of DU145/DTXR cells.
Western blot detection of CRIP2 protein expression level of transfected prostate cancer cells:
(1) Experimental method
1) Protein extraction and concentration determination:
the specific operations of protein extraction and concentration measurement are the same as those of the first embodiment, and will not be described here.
2)Western blot：
The specific operation of Western blot is the same as that of the first embodiment, and will not be described in detail here.
(2) Experimental results
The Western blot detection results are shown as B in FIG. 3. As can be seen from fig. 3B, protein expression levels of CRIP2 were significantly down-regulated in the sh-CRIP2# 1, sh-CRIP2# 2 experimental group, which stably interfered with CRIP2, compared to the negative control group (sh-NC group); it was shown that a stable interfering CRIP2 knockdown cell line was successfully obtained by lentiviral infection of DU145/DTXR cells. .
Embodiment III: changes in DTX sensitivity of DTX-resistant prostate cancer cells following stable interference with CRIP2
IC50 values (median lethal concentration) of DTX against interfering CRIP2 pre-and post-prostate cancer cells were compared using CCK8 method.
1. Cell culture:
the cells used in this experiment were DU145/DTXR cells stably transfected with sh-NC (designated as sh-NC), DU145/DTXR cells stably transfected with shRNA1 (designated as sh-CRIP2# 1) and DU145/DTXR cells stably transfected with shRNA2 (designated as sh-CRIP2# 2).
The specific operation of cell culture is the same as that of example one, and will not be described here again.
2. Experimental method
(1) The experiments were divided into 3 groups, namely a negative control group (sh-NC group), sh-CRIP2# 1 group and sh-CRIP2# 2 group, each group was provided with 10 DTX concentration gradients of 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.5625nM, 0.78125nM and 0nM, and each concentration gradient was provided with 5 multiplex wells.
(2) DU145 DTX cells after stable interference with CRIP2 were cultured for 72h, counted with 0.25% pancreatin digestion, cells were seeded in 96-well plates at a DU145 cell concentration of 1.0X10% 5 Per mL, 100 μl of each well was added, and after inoculation was completed, 37℃and 5% CO were added 2 Culturing in an incubator for 36h.
(3) Taking out 96-well plate after 36 hr, sucking out culture medium, adding culture medium containing DTX with corresponding concentration according to the concentration gradient designed in advance, adding 37 deg.C and 5% CO 2 Culturing in an incubator is continued for 48 hours.
(4) After 48h of culture, the culture medium is used as follows: CCK8 reagent = 10:1, the reaction solution was prepared in the same ratio, and the cells in the 96-well plate were subjected to liquid exchange and then cultured for 2 hours, and the OD value at a wavelength of 450nm was measured.
3. Experimental results
CCK8 detected and calculated IC50 values of DTX for cells before and after interference with CRIP2, each set of experiments was repeated three times and the experimental results are shown in fig. 4. As can be seen from fig. 4, the IC50 values of the experimental group cells of sh-crip2# 1 and sh-crip2# 2 for DTX were significantly reduced (P < 0.05) compared to the negative control group (sh-NC group). This demonstrates that DU145 DTX cells have increased sensitivity to DTX after stably interfering with CRIP 2. Thus, CRIP2 can be a potential target for reversing DTX resistance of prostate cancer cells.
Embodiment four: effects on cell proliferation of DTX-resistant prostate cancer cells after stable interference with CRIP2
1. CCK8 assay for cell proliferation
(1) Cell culture:
the cells used in this experiment were DU145/DTXR cells stably transfected with sh-NC (designated as sh-NC), DU145/DTXR cells stably transfected with shRNA1 (designated as sh-CRIP2# 1) and DU145/DTXR cells stably transfected with shRNA2 (designated as sh-CRIP2# 2).
The specific operation of cell culture is the same as that of example one, and will not be described here again.
(2) Experimental method
1) The experiments were divided into 3 groups, namely a negative control group (sh-NC group), a sh-CRIP2# 1 group and a sh-CRIP2# 2 group, and each group is provided with 4 compound holes.
2) DU145 DTX cells after stable interference with CRIP2 were cultured for 72h, counted with 0.25% pancreatin digestion, cells were seeded in 96-well plates at a DU145 cell concentration of 1.0X10% 5 Per mL, 100. Mu.L per well;
3) Under the light-shielding condition, the culture medium is prepared by the following steps: CCK8 reagent = 10:1, the reaction solution was prepared in the same ratio, and the cells in the 96-well plate were subjected to liquid exchange and then cultured for 2 hours, and the OD value at a wavelength of 450nm was measured.
(3) Experimental results
The experimental results of CCK8 detection of cell proliferation are shown in FIG. 5. As can be seen from fig. 5, the experimental groups of sh-crip2# 1 and sh-crip2# 2 showed significant inhibition of cell proliferation (P < 0.05) compared to the negative control group (sh-NC group). Therefore, the stable interference CRIP2 has obvious inhibition effect on DU145/DTXR cell proliferation capacity.
2. Cloning formation experiments
(1) Cell culture:
the cells used in this experiment were DU145/DTXR cells stably transfected with shRNA-NC (designated sh-NC), DU145/DTXR cells stably transfected with shRNA1 (designated sh-CRIP2# 1) and DU145/DTXR cells stably transfected with shRNA2 (designated sh-CRIP2# 2).
The specific operation of cell culture is the same as that of example one, and will not be described here again.
(2) Experimental method
1) The experiments were divided into 3 groups, namely a negative control group (sh-NC group), a sh-CRIP2# 1 group and a sh-CRIP2# 2 group, and each group is provided with 3 compound holes.
2) Culturing DU145/DTXR cells for 72 hr after CRIP2 interference is stabilized, performing cell count by 0.25% pancreatin digestion, inoculating cells into 6-well plate, spreading 1500 cells per well, arranging 3 repeated wells per group, adding 2mL of complete culture medium per well in advance, calculating the required cell volume per group according to the count concentration, adding into corresponding wells, mixing uniformly, and placing into 37 deg.C and 5% CO 2 Culturing in an incubator, changing the liquid once about 5 days, and forming macroscopic cell colonies about 10 days.
3) Removing the culture medium, cleaning once with PBS, adding 1mL of pre-cooled 4% paraformaldehyde PBS into each hole, fixing for 15min, removing the fixing solution after fixing, adding 0.5% crystal violet, dyeing for more than 20min, removing crystal violet, slightly flushing residual crystal violet with clear water after dyeing, airing, and photographing with a camera under bright light.
(3) Experimental results
The experimental results are shown in FIG. 6. Wherein, A in FIG. 6 is a clone forming phenotype, and as can be seen from A in FIG. 6, the experimental groups of sh-CRIP2# 1 and sh-CRIP2# 2 have significantly fewer cell clones than the negative control group (sh-NC group); in fig. 6, B is a statistical comparison of the numbers of clones of different knockdown groups, and as can be seen from fig. 6, cell proliferation was significantly inhibited (P < 0.05) in experimental groups of sh-crip2# 1 and sh-crip2# 2 compared to the negative control group (sh-NC group). Therefore, the stable interference CRIP2 has obvious inhibition effect on DU145/DTXR cell proliferation capacity.
In conclusion, the expression level of CRIP2 protein in DU145/DTXR resistant cells is obviously higher than that of DU145 parent, and the difference has statistical significance, so that CRIP2 gene or CRIP2 protein can be used for detecting drug resistance or sensitivity of prostate cancer to docetaxel, and the expression level of CRIP2 gene or CRIP2 protein can be used as one of the basis for judging drug resistance/sensitivity of a drug user to DTX drugs by measuring the expression level of CRIP2 gene or CRIP2 protein. In addition, the invention discovers that stable interference of CRIP2 gene expression can improve the sensitivity of DTX drug-resistant prostate cancer cells to DTX, and meanwhile, the proliferation capacity of DTX drug-resistant prostate cancer cells can be obviously inhibited after the CRIP2 gene is interfered, so that the CRIP2 gene can be used as a potential target point for reversing the DTX drug resistance of the prostate cancer, and the drug resistance of tumor cells to docetaxel can be reversed by inhibiting the expression and/or the function of the CRIP2 gene, thereby having important significance for treating the prostate cancer.
The above description is merely illustrative of the preferred embodiments of the present invention and is not intended to limit the invention to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.
Sequence listing
<110> Hospital for people in Henan province
<120> use of CRIP2 for detecting and reversing resistance of prostate cancer to docetaxel
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Claims (8)
- Use of a crip2 gene or a protein encoded thereby for the preparation of a product for detecting or aiding in the detection of prostate cancer resistance/sensitivity to docetaxel.
- 2. The use according to claim 1, wherein the product is used for detecting the expression level of CRIP2 gene or protein encoded thereby in a sample by real-time quantitative PCR, in situ hybridization, northern blotting, chip, high throughput sequencing platform, western blot or enzyme linked immunosorbent assay.
- 3. The use according to claim 1, wherein the product comprises specific primers for amplifying the CRIP2 gene, probes hybridising to the nucleotide sequence of the CRIP2 gene or antibodies specifically binding to the CRIP2 protein.
- 4. The use according to claim 3, wherein the specific primer sequences for amplifying the CRIP2 gene are shown in SEQ ID NO.1 and SEQ ID NO. 2.
- 5. Use of a substance that inhibits CRIP2 gene expression and/or function for the preparation of a product that reverses the resistance of a tumor cell to docetaxel, said tumor cell being a prostate cancer cell.
- 6. Use of a substance that inhibits CRIP2 gene expression and/or function for the manufacture of a product for inhibiting proliferation of docetaxel resistant tumor cells, said tumor cells being prostate cancer cells.
- 7. The use according to claim 5 or 6, wherein the substance that inhibits CRIP2 gene expression and/or function comprises siRNA and/or shRNA that specifically targets CRIP2 gene.
- 8. The use according to claim 7, wherein the shRNA specifically targeting the CRIP2 gene comprises shRNA1 and shRNA2, the sequence of shRNA1 is shown in SEQ ID No.3, and the sequence of shRNA2 is shown in SEQ ID No. 4.
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