CN109856351B - Method for predicting that TAK1 and medicament synergistically influence apoptosis - Google Patents
Method for predicting that TAK1 and medicament synergistically influence apoptosis Download PDFInfo
- Publication number
- CN109856351B CN109856351B CN201910264363.9A CN201910264363A CN109856351B CN 109856351 B CN109856351 B CN 109856351B CN 201910264363 A CN201910264363 A CN 201910264363A CN 109856351 B CN109856351 B CN 109856351B
- Authority
- CN
- China
- Prior art keywords
- tak1
- tumor
- apoptosis
- plasmid
- tissue
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Abstract
The invention discloses a method for predicting that TAK1 and a medicament synergistically influence apoptosis. The method comprises the following steps: (1) treating tumor tissue with TAK1 antibody, and measuring the expression level of TAK1 in the tumor tissue to obtain R1(ii) a Meanwhile, normal tissues beside the tumor were treated with the TAK1 antibody, and the expression level of TAK1 in the normal tissues was determined to obtain R2(ii) a (2) Comparison of R1And R2If R is1＜R2If the difference is significant, TAK1 is predicted to be capable of promoting tumor cell apoptosis in cooperation with the medicine; on the contrary, if R1＞R2The difference is significant, so that the TAK1 is predicted not to be capable of promoting the tumor cell apoptosis by cooperating with the medicament, and the TAK1 inhibitor is predicted to promote the tumor cell apoptosis by cooperating with the medicament. The method can predict whether TAK1 can be used for promoting synergistic medicine based on the expression level of TAK1 in tumor and paraneoplastic tissueThe apoptosis lays a foundation for the subsequent scientific research.
Description
Technical Field
The invention relates to the field of intracellular signal transduction, in particular to a method for predicting that TAK1 and a medicament synergistically influence apoptosis.
Background
TAK1 (TGF-. beta. -activated kinase1), a serine threonine kinase of the MAPK family[1]. TAK1 is involved in the regulation of a range of cellular processes including development, differentiation, autophagy, apoptosis and survival. TAK1 is a key protein kinase in multiple intracellular signal transduction pathways such as JNK, p38, NF kappa B and the like, TAK1 and a binding protein TAB1(TGF-beta activated kinase1binding protein 1) play important roles in the apoptosis process through the JNK, p38 and NF kappa B signal transduction pathways[2-6]。
Paclitaxel can act on cells of multiple tissue sources such as ovarian cancer, breast cancer, lung cancer, gastric cancer, colon cancer, head and neck cancer, esophageal cancer, seminoma, lymphoma, etc., and induce typical apoptosis manifestations such as DNA fragmentation, cell nucleus contraction, apoptosis corpuscle formation, etc[7-8]. TAK1 acts in combination with paclitaxel to influence apoptosis through JNK, p38, NF kappa B signal transduction pathways.
At present, a plurality of documents report that TAK1 or TAK1 inhibitors influence the apoptosis of tumor cells of different tissue sources at home and abroad, including breast cancer, neuroblastoma, cervical cancer, acute lymphocytic leukemia, colon cancer, lung cancer and the like[9-16]. TAK1 is a key protein kinase in many cell signal transduction pathways, and it is predicted that TAK1 may exert different effects on cells of different tissue origins and even different individual origins through different intracellular signal pathways. TAK1, and possibly TAK1 inhibitors, promote apoptosis. In subsequent scientific studies, how to predict and evaluate whether TAK1 or an inhibitor thereof has a pro-apoptotic effect on cells derived from different tissues is a problem that we must face.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a method for predicting that TAK1 and a medicament synergistically influence apoptosis.
The invention also aims to provide application of the reagent for determining the expression level of TAK1 in tumor tissues and paraneoplastic tissues in preparing a kit for predicting whether TAK1 and medicaments can synergistically influence tumor cell apoptosis.
The purpose of the invention is realized by the following technical scheme: a method for predicting that TAK1 synergistically affects apoptosis with a drug, comprising the steps of:
(1) treating tumor tissue with TAK1 antibody, and measuring the expression level of TAK1 in the tumor tissue to obtain R1(ii) a Meanwhile, normal tissues beside the tumor were treated with the TAK1 antibody, and the expression level of TAK1 in the normal tissues was determined to obtain R2；
(2) Comparison of R1And R2If R is1＜R2If the difference is significant, TAK1 is predicted to be capable of promoting the apoptosis of the tumor cells with the same source as the tumor tissue in the step (1) in a synergistic manner with the medicine; on the contrary, if R1＞R2The difference is significant, and thus it is predicted that TAK1 cannot synergistically promote apoptosis of tumor cells of the same origin as the tumor tissue in step (1) with the drug, and that TAK1 inhibitor synergistically promotes apoptosis of tumor cells of the same origin as the tumor tissue in step (1) with the drug.
The tumor tissue in the step (1) is tumor tissue obtained by tumor tissue biopsy or complete or partial surgical excision; including kidney cancer, colon cancer, ovarian cancer, cervical cancer, breast cancer tissue, etc.
The volume ratio of the TAK1 antibody to the tumor tissue in the step (1) is 1:200 to 500.
The normal tissue beside the tumor in the step (1) is the normal tissue after the corresponding tumor is removed, and the normal tissue is confirmed by the surrounding of the tumor focus, the form and the pathological detection of the same individual without limitation.
The determination of the expression level of TAK1 described in step (1) can be carried out by conventional methods in the art; preferably, the determination is performed by at least one of immunohistochemistry, flow cytometry detection, quantitative PCR, and immunoblotting.
The immunohistochemistry is realized by the following steps: and calculating the percentage of positive cells, repeating the steps for more than 3 times, and calculating the mean value of the positive cells to obtain the expression level of the TAK 1.
The flow cytometry detection is preferably realized by the following steps: the determination calculates the percentage of positive cells, repeats for more than 3 times, and calculates the mean value to obtain the expression level of TAK 1.
The quantitative PCR is preferably realized by the following steps: and calculating the copy number of the cDNA after reverse transcription, repeating for more than 3 times, and calculating the mean value to obtain the expression level of the TAK 1.
The immunoblotting is preferably carried out by: and measuring the expression quantity of the TAK1 in the whole cell extract, repeating the operation for more than 3 times, and calculating the mean value of the expression quantity to obtain the expression level of the TAK 1.
The significance of the difference in the step (2) is judged by the following method: firstly, carrying out the homogeneity test of the variances, if the variances are uniform, using the mean t test of two samples for comparison, if the variances are not uniform, using the t' test for comparison, and if P is less than 0.05, considering that the differences are significant.
The medicine in the step (2) is an anti-tumor medicine; preferably paclitaxel.
The TAK1 inhibitor in step (2) is preferably 5Z-7-oxozeaenol, and TAK1 gene editing plasmid can also be used to silence the TAK1 gene.
The gene editing plasmid of TAK1 is preferably TAK1/TAB1eCRISPR plasmid.
The method for predicting that TAK1 and the drug synergistically influence the apoptosis further comprises the step of further verifying after the step (2); the verification can be realized by any one or two methods as follows:
(a) immunoblotting
TAK1/TAB1 in combination with a drug: respectively transfecting the tumor cells with the same tumor tissue source as that in the step (1) with an unloaded plasmid, a plasmid containing TAK1 and a plasmid containing TAK1 and TAB1, culturing the transfected cells for 48 or 72 hours, adding a medicament for treating for 9-18 hours, cracking the cells to obtain a whole cell extract, and detecting PARP shearing by using an immunoblotting method; if the test results show that PARP cleavage of tumor cells transfected with the plasmid containing TAK1 and the plasmids containing TAK1 and TAB1 is enhanced compared to tumor cells transfected with the empty plasmid, it is verified that TAK1 synergistically promotes apoptosis with the drug; on the contrary, if the PARP cleavage of tumor cells transfected with the plasmid containing TAK1 and the plasmid containing TAK1 and TAB1 genes was reduced compared to tumor cells transfected with the empty plasmid, it was confirmed that TAK1 could not promote tumor cell apoptosis in cooperation with the drug;
(b) flow cytometry
TAK1/TAB1 in combination with a drug: respectively transfecting tumor cells with the same tumor tissue source as that in the step (1) with an unloaded plasmid, a plasmid containing TAK1, a plasmid containing TAK1 and TAB1 and/or an edited plasmid for transfecting a silent TAK1 gene, adding a TAK1 inhibitor into the tumor cells which are not transfected with the plasmids, culturing the cells for 48 or 72 hours, adding a medicament for treating for 9-18 hours, and detecting the apoptosis rate by using a flow cytometer; if the detection result shows that compared with the tumor cells transfected with the unloaded plasmids, the apoptosis rate of the tumor cells transfected with the plasmids containing TAK1 and the plasmids containing TAK1 and TAB1 is increased, the TAK1 and the medicament are verified to synergistically promote the apoptosis; if the detection result shows that the apoptosis rate of the tumor cells transfected by the plasmid containing TAK1 and the plasmid containing TAK1 and TAB1 is reduced compared with that of the tumor cells transfected by the unloaded plasmid, the verification is that the TAK1 cannot promote the apoptosis of the tumor cells in cooperation with the medicament; if the detection result shows that compared with the tumor cells transfected with the unloaded plasmids, the apoptosis rate of the cells added with the TAK1 inhibitor or the edited plasmids transfected with the silenced TAK1 gene is increased, the TAK1 inhibitor or the silenced TAK1 gene is verified to be cooperated with the medicine to promote the apoptosis.
The plasmid described in methods (a) and (b) is preferably pcDNA3.1 or pHBLV-MCS-3FLAG plasmid.
The plasmids containing the TAK1 gene in the methods (a) and (b) are eukaryotic cell expression plasmids; preferably pcDNA3.1-TAK1-myc plasmid or pHBLV-MCS-3FLAG-TAK1 plasmid.
The plasmid containing the TAB1 gene described in the methods (a) and (b) is a eukaryotic cell expression plasmid; preferably pcDNA3.1-TAB1-myc plasmid or pHBLV-MCS-3FLAG-TAB1 plasmid.
The editing Plasmid for silencing TAK1 gene described in methods (a) and (b) is preferably TAK1/TAB1eCRISPR Plasmid (available from Public Protein/Plasmid Library, China).
The TAK1 inhibitor described in method (b) is preferably 5Z-7-oxozeaenol.
The dosage of the added TAK1 inhibitor in the method (b) is calculated according to the final concentration of the inhibitor in a system of 5-10 mu M; preferably calculated as its final concentration in the system of 5. mu.M.
The silencing TAK1 gene in method (b) is the silencing TAK1 gene using TAK1 gene editing plasmid such as TAK1/TAB1eCRISPR plasmid.
The medicament in the methods (a) and (b) is an anti-tumor medicament; preferably paclitaxel.
The dosage of the added medicine in the methods (a) and (b) is preferably calculated according to the final concentration of the added medicine in the system of 3-10 mu M.
The application of the reagent for determining the expression level of TAK1 in tumor tissues and paraneoplastic tissues in preparing a kit for predicting whether TAK1 and medicaments can synergistically influence tumor cell apoptosis.
The tumor tissue is obtained by tumor tissue biopsy or complete or partial surgical excision.
The paraneoplastic tissue is a normal tissue after the corresponding tumor is removed, and the normal tissue is confirmed by the surrounding of the tumor focus, the form and the pathological detection of the same individual, and the range is not limited.
The reagent comprises TAK1 antibody, immunohistochemical reagent and the like.
The immunohistochemical reagent comprises a polymer molecule marked with horseradish peroxidase and rabbit and mouse resistant immunoglobulin, a DAB color developing agent, a color development buffer solution and the like.
The tumor is malignant tumor, including renal carcinoma, colon cancer, ovarian cancer, cervical cancer, breast cancer, etc.
The medicine is an anti-tumor medicine; preferably paclitaxel.
A kit for predicting whether TAK1 and a drug can synergistically affect tumor cell apoptosis, the kit comprising reagents for determining the expression level of TAK1 in tumor and paraneoplastic tissues.
The reagent comprises TAK1 antibody, immunohistochemical reagent and the like.
The immunohistochemical reagent comprises a polymer molecule marked with horseradish peroxidase and rabbit and mouse resistant immunoglobulin, a DAB color developing agent, a color development buffer solution and the like.
The tumor is malignant tumor, including renal carcinoma, colon cancer, ovarian cancer, cervical cancer, breast cancer, etc.
The medicine is an anti-tumor medicine; preferably paclitaxel.
Use of an agent for determining the expression level of TAK1 in tumor and paraneoplastic tissues in the manufacture of a kit for predicting whether TAK1 and a drug can be used together to treat a patient with a tumor.
The reagent comprises TAK1 antibody, immunohistochemical reagent and the like.
The immunohistochemical reagent comprises a polymer molecule marked with horseradish peroxidase and rabbit and mouse resistant immunoglobulin, a DAB color developing agent, a color development buffer solution and the like.
The tumor is malignant tumor, including renal carcinoma, colon cancer, ovarian cancer, cervical cancer, breast cancer, etc.
The medicine is an anti-tumor medicine; preferably paclitaxel.
The application of the reagent for measuring the expression level of TAK1 in tumor and paraneoplastic tissue in the preparation of a kit for screening the TAK1 inhibitor which has the effect of promoting tumor cell apoptosis in cooperation with medicaments.
The reagent comprises a TAK1 antibody, an immunohistochemical reagent and the like.
The immunohistochemical reagent comprises a polymer molecule marked with horseradish peroxidase and rabbit and mouse resistant immunoglobulin, a DAB color developing agent, a color development buffer solution and the like.
The tumor is malignant tumor, including renal carcinoma, colon cancer, ovarian cancer, cervical cancer, breast cancer, etc.
The medicine is an anti-tumor medicine; preferably paclitaxel.
The research field of the application is intracellular signal transduction, and the following findings are found: TAK1 and its binding protein TAB1 act in combination with paclitaxel to promote renal cell apoptosis; TAK1/TAB1 inhibited paclitaxel-induced apoptosis of colon-derived cells. Meanwhile, TAK1 is expressed less strongly in kidney cancer tissue than in paracancerous normal tissue; TAK1 was expressed more strongly in colon cancer tissues than in paracancerous normal tissues. The expression differences in the two directions are consistent with the effect of the combination of TAK1/TAB1 and paclitaxel on apoptosis, which suggests that the difference of the expression levels of TAK1 in different tumor/paraneoplastic tissues can predict whether TAK1 can effectively cooperate with drugs to promote apoptosis, and then the method is used for subsequent scientific research.
Compared with the prior art, the invention has the following advantages and effects:
1. the invention relates to a method for predicting and evaluating the apoptosis effect of TAK1 on different tissue sources in the prior art, which is used for predicting whether TAK1 affects apoptosis by detecting the expression level of TAK1 in tumor/paraneoplastic tissues and evaluating the expression difference.
2. The method of the invention can be subsequently used for research of scientific projects: for scientific research projects, the effect of the TAK1/TAK1 inhibitor synergistic medicine on tumor cell apoptosis can be predicted through the difference of the expression level of TAK1 in tissues in tumor/paracarcinoma tissues.
Drawings
FIG. 1 is a technical scheme of the present invention.
FIG. 2 is a graph of immunoblots of kidney-derived cells (293) after overexpression of TAK1/TAB1 in combination with paclitaxel.
FIG. 3 is a morphological diagram of the combined effect of TAK1/TAB1 over-expression and paclitaxel on the promotion of renal cell (293) apoptosis (in the figure: 5Z is TAK1 inhibitor 5Z-7-oxozeaenol, CTAB1CTAK1 is Cas9/CRISPR plasmid).
FIG. 4 is a graph showing that TAK1/TAB1 overexpression in combination with paclitaxel promoted apoptosis in kidney-derived cells (293); wherein, panel a is a flow cytogram; panel b is a statistical plot of early apoptosis, late apoptosis, and total apoptosis.
FIG. 5 is a graph of immunoblots of kidney-derived tumor cells (769P cells) after overexpression of TAK1/TAB1 in combination with paclitaxel.
FIG. 6 is a graph of the effect of TAK1/TAB1 in combination with paclitaxel (10 μ M; 9h and 18h) on apoptosis in renal derived tumor cells (769P cells); wherein, panel a is a flow cytogram; panels b and c are statistical plots of apoptosis rates after 9 hours and 18 hours of treatment of cells with TAK1/TAB1 in combination with paclitaxel.
FIG. 7 is a graph of immunoblots of TAK1/TAB1 over-expression inhibiting paclitaxel-induced apoptosis in colon-derived tumor cells (HCT 116).
FIG. 8 is a graph of TAK1/TAB1 overexpression inhibiting paclitaxel-induced apoptosis in colon-derived tumor cells (HCT 116); wherein, panel a is a flow cytogram; panel b is a statistical plot of early apoptosis, late apoptosis, and total apoptosis.
FIG. 9 is a graph showing the expression of TAK1 in renal cancer/paraneoplastic tissue, and in colon cancer/paraneoplastic tissue; wherein, the graph a shows the expression difference of TAK1 in colon cancer/paracarcinoma tissues; panel b shows the difference in expression of TAK1 in the cancer/paracarcinoma tissue of renal cancer.
FIG. 10 is a statistical graph showing the expression difference of TAK1 in renal cancer tissue/paraneoplastic tissue and colon cancer tissue/paraneoplastic tissue.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.
The differences in expression levels of TAK1 in tumor/paracancerous tissues referred to in the examples can be first tested for homogeneity of variance, which is compared using a two-sample mean t test, and for variance which is compared using a t' test, i.e. when P < 0.05, the differences are considered significant.
Example 1
1. Expression level of TAK1 in 3 samples of renal tumor/tissue adjacent to carcinoma
Multi-tissue tumor chips (including tumor/paraneoplastic paraffin-embedded tissue sections such as kidney cancer, colon cancer, etc., OD-CT-Com03, available from Shanghai core Biotechnology Co., Ltd.; in this example, the paraneoplastic paraffin-embedded tissue sections are renal cancer) were deparaffinized with xylene I, II for 20min each; carrying out delixylation to hydration by using gradient alcohol, carrying out antigen repair (the purpose of the antigen repair is to break cross-linking among antigens caused by formaldehyde so that an antibody is easier to penetrate and the accessibility of the antigen is increased, and preparing an antigen repair solution with pH 6.0Citrate and pH 9.0Tris-EDTA), and washing; with 3% H2O2Treating for 10min, and washing with water; diluting TAK1 antibody (TAK1ab79354, Abcam) at a volume ratio of 1:200 for incubation, and rinsing with PBS for 5min × 2 times; staining with immunohistochemical instrument, taking down the slice, counterstaining with hematoxylin for 5min, differentiating with 1% hydrochloric acid alcohol for 40s, washing with running water, and sealing with neutral gum for 20 min; finally the sections of the neutral gum seals were observed under an optical microscope (fig. 9 and 10).
2. And (4) interpretation of results: in 3 samples (n ═ 3) of the same pathological type, the proportion of TAK 1-positive cells in tumor tissues and in paraneoplastic tissues was calculated by observation under an optical microscope. The results show that: the mean positive cell rate of TAK1 in the paraneoplastic tissue of kidney (R2) was 71%, which is higher than the positive cell rate of 33% in the neoplastic tissue (R1), and R1< R2, which are significant in difference (P < 0.05), and it is predicted that TAK1 can synergistically promote apoptosis of cells derived from kidney with antitumor drugs (e.g., paclitaxel) (fig. 9, 10 and table 1).
TABLE 1 differences in expression levels of TAK1 in renal tumor/paracarcinoma tissues
3. TAK1/TAB1 combined with paclitaxel was used for effect verification in 293 cells (human embryonic kidney cells)
(1) Immunoblotting
293 cells (purchased from Shanghai cell of Chinese academy of sciences) were individually transferredStaining pcDNA3.1 No-load plasmid (purchased from Addgene; abbreviated as pcDNA3.1)[17]pcDNA3.1-TAB1-myc plasmid (myc-TAB 1; TAB1 for short)[17]pcDNA3.1-TAK1-myc plasmid (TAK 1-myc; TAK1 for short)[17]pcDNA3.1-TAK1-myc plasmid + pcDNA3.1-TAB1-myc plasmid (TAB1+ TAK1 for short), TAK1 inhibitor 5Z-7-oxozeano (Calbiochem, 499610) was added to the cells without plasmid transfection, paclitaxel (final concentration of 10. mu.M) was added after 48 hours for treatment for 9 hours, whole Cell extracts were extracted, Western-blot was performed, and hybridization was performed with PARP antibody (Cell Signaling Technology, 9532S), to show full-length, post-cleavage PARP. The results show that PARP cleavage was significantly enhanced in cells transfected with TAK1 and TAB1+ TAK1 compared to cells transfected with pcdna3.1 empty plasmid, suggesting that TAK1 synergizes with paclitaxel to promote apoptosis in 293 cells (fig. 2).
(2)293 cells were transfected with pcDNA3.1-unloaded plasmid (pcDNA3.1), pcDNA3.1-TAB1-myc plasmid (TAB1), pcDNA3.1-TAK1-myc plasmid (TAK1), pcDNA3.1-TAK1-myc plasmid + pcDNA3.1-TAB1-myc plasmid (TAB1+ TAK1), TAK1/TAB1eCRISPR plasmid (CTAB1CTAK1), respectively, and after 48 hours they were treated with paclitaxel (10. mu.M) for 9 hours, and 5Z-7-oxozeaeno (Calbiochem, 499610) and paclitaxel (10. mu.M) were added to the untransfected plasmid wells, and the final concentration of 5Z-7-oxozeaeno (5Z) was 5. mu.M, showing that: after treatment of cells with paclitaxel, 293 cells transfected with TAK1 and TAB1+ TAK1 had decreased cell density, rounded cells, and increased nuclear compaction; among these, 293 cells transfected with TAB1+ TAK1 were evident (FIG. 3).
(3) Annexin V/PI double staining and flow cytometry are used for detecting the apoptosis cell rate.
293 cells were transfected with pcDNA3.1-unloaded Plasmid (Vector), pcDNA3.1-TAB1-myc Plasmid (TAB1), pcDNA3.1-TAK1-myc Plasmid (TAK1), pcDNA3.1-TAK1-myc Plasmid + pcDNA3.1-TAB1-myc Plasmid (TAB1+ TAK1), TAK1/TAB1eCRISPR Plasmid (CTAK1CTAB1, available from Public Protein/Plasmid Library, China), respectively, and after 48 hours, they were treated with paclitaxel (10. mu.M) for 9 hours, and additionally, untransfected Plasmid wells were provided with 5Z-7-oxozeano (Calbiochem, 499610) and paclitaxel (10. mu.M), 5Z-7-oxozeano (5Z) at a final concentration of 5uM, and after 9 hours, the apoptosis was detected by flow cytometry. Collecting cells, washing the cells once by PBS buffer at 1500rpm for 5 minutes, and removing supernatant; cells were filtered, 100. mu.l of 1 XBinding buffer was added to the reaction tube, 5. mu.l of Annexin V-FITC and 5. mu.l of PI were added, mixed well, incubated for 15 minutes at room temperature in the dark, 100. mu.l of 1 XBinding buffer was added, and the apoptosis rate was analyzed by flow cytometry (Annexin V FITC apoptosis detection kit, BD, 556570). The results show that: compared to 293 cells transfected with pcDNA3 empty plasmid, the apoptosis rate was significantly increased for TAK1 and TAB1+ TAK1 plasmids (fig. 4).
Example 2
1. Expression level of TAK1 in 3 kidney tumor/paracancerous tissue samples: the same as in example 1.
2. And (4) interpretation of results: the same as in example 1.
3. TAK1/TAK1 was combined with paclitaxel and tested for its effect in 769P cells (adenocarcinoma cells of renal clear cell), and the plasmids and packaged lentiviruses involved in this example were purchased from Shanghai Henan bioscience.
(1) Immunoblotting
769P cells (purchased from Shanghai Cell of Chinese academy of sciences) were transfected with pHBLV-MCS-3FLAG No-load plasmid (Vertor), pHBLV-MCS-3FLAG-TAB1 plasmid (TAB1), pHBLV-MCS-3FLAG-TAK1 plasmid (TAK1), pHBLV-MCS-3FLAG-TAK1 plasmid + pHBLV-MCS-3FLAG-TAB1 plasmid (TAB1+ TAK1), 5Z-7-oxozeano (10uM) was added to untransfected cells, paclitaxel (10uM) was added after 72 hours (expressed after 72 hours of lentiviral transfection plasmid transfection) for 9 hours, whole Cell extract was extracted, Western-blot was performed, and hybridization was performed with PARP antibody (Signaling Cell Technology, 9532S), indicating full length and post-cleavage PARP. The results showed a significant increase in splicing of the transfected TAB1 and TAB1+ TAK1 cells compared to the empty control group, suggesting that TAK1/TAB1 promotes 769P apoptosis in conjunction with paclitaxel (FIG. 5).
(2) Annexin V/PI double staining and flow cytometry are used for detecting the apoptosis cell rate.
769P cells (purchased from Shanghai cell of Chinese academy of sciences) are transfected with pHBLV-MCS-3FLAG no-load control (Vertor), pHBLV-MCS-3FLAG-TAB1(TAB1), pHBLV-MCS-3FLAG-TAK1(TAK1), pHBLV-MCS-3FLAG-TAK1 plasmid + pHBLV-MCS-3FLAG-TAB1(TAB1+ TAK1), 5Z-7-oxozeano (5Z; 10uM) is added to untransfected wells, and paclitaxel (10uM) is added after 72 hours, and the apoptosis rate is detected by flow cytometry after 9 hours and 18 hours of paclitaxel treatment. Collecting cells, washing the cells once by PBS buffer at 1500rpm for 5 minutes, and removing supernatant; cells were filtered, 100. mu.l of 1 XBinding buffer was added to the reaction tube, 5. mu.l of Annexin V-FITC and 5. mu.l of PI were added, mixed well, incubated for 15 minutes at room temperature in the dark, 100. mu.l of 1 XBinding buffer was added, and the apoptosis rate was analyzed by flow cytometry (Annexin V FITC apoptosis detection kit, BD, 556570). The results showed that the apoptosis rate of TAK1 transfected and TAB1+ TAK1 cells was increased after 9h, 18h treatment with paclitaxel compared to cells transfected with the unloaded plasmid (FIG. 6).
Example 3
1. Expression level of TAK1 in 3 colon tumor/paracancerous tissue samples
Multiple tissue tumor chips (including tumor/paraneoplastic paraffin-embedded tissue sections such as kidney cancer, colon cancer, and the like, OD-CT-Com03, all purchased from Shanghai core Biotechnology Co., Ltd., in this example, the paraneoplastic paraffin-embedded tissue sections are referred to as colon cancer and paraneoplastic paraffin-embedded tissue sections) were deparaffinized with xylene I, II for 20min each; carrying out delixylation to hydration by using gradient alcohol, carrying out antigen repair (the purpose of the antigen repair is to break cross-linking among antigens caused by formaldehyde so that an antibody is easier to penetrate and the accessibility of the antigen is increased, and preparing antigen repair liquid of pH 6.0Citrate and pH 9.0Tris-EDTA), and washing; with 3% H2O2Treating for 10min, and washing with water; diluting TAK1 antibody at a volume ratio of 1:500, incubating (ab79354, Abcam), rinsing with PBS for 5min × 2 times; staining with immunohistochemical instrument, taking down the slice, counterstaining with hematoxylin for 5min, differentiating with 1% hydrochloric acid alcohol for 40s, washing with running water, and sealing with neutral gum for 20 min; finally the sections of the neutral gum seal were observed under an optical microscope (fig. 9).
2. And (4) interpretation of results: the proportion of TAK1 positive cells in tumor tissues and tissues adjacent to the tumor was calculated by observing 3 samples of the same pathological type under an optical microscope. The results show that the mean positive cell rate of TAK1 in the tissue (R2) beside the colon cancer is 18%, which is lower than the positive cell rate 79% in the tumor tissue (R1), R1 is greater than R2(P is less than 0.05), the difference is significant, TAK1 is predicted to be incapable of promoting the apoptosis of the colon cancer source cells in cooperation with the antitumor drugs (such as paclitaxel and the like) (figure 9, 10 and table 2), the tumor cell apoptosis induced by the antitumor drugs can be inhibited, and the TAK1 inhibitor can promote the tumor cell apoptosis in cooperation with the drugs.
TABLE 2 differences in expression levels of TAK1 in colon/paracancerous tissues
3. TAK1/TAB1 was combined with paclitaxel and tested for efficacy in Hct116 cells (human colon cancer cells).
(1) Immunoblotting
Hct116 cells (purchased from Shanghai cell of Chinese academy of sciences) were transfected with pcDNA3.1 airborne plasmid (pcDNA3.1), pcDNA3.1-TAB1-myc plasmid (TAB1)[17]pcDNA3.1-TAK1-myc plasmid (TAK1)[17]pcDNA3.1-TAK1-myc plasmid + pcDNA3.1-TAB1-myc plasmid (TAB1+ TAK 1). After 48 hours paclitaxel (final concentration of 10 μ M) was added for 9h, whole Cell extracts were extracted and Western-blot was performed, followed by hybridization with PARP antibody (Cell Signaling Technology, 9532S) to reveal full-length, post-cleavage PARP. The results showed that the excision band of the cells transfected with TAB1 group and TAB1+ TAK1 cells was shallower compared to the cells transfected with pcDNA 3-empty plasmid, suggesting that TAK1 inhibits paclitaxel-induced apoptosis of HCT116 cells (FIG. 7).
(2) Annexin V/PI double staining and flow cytometry are used for detecting the apoptosis cell rate.
Hct116 cells (purchased from Shanghai cell of China academy of sciences) were transfected with pcDNA3.1 null plasmid (Vector), pcDNA3.1-TAB1-myc plasmid (TAB1)[17]pcDNA3.1-TAK1-myc plasmid (TAK1)[17]The plasmid pcDNA3.1-TAK1-myc + pcDNA3.1-TAB1-myc (TAB1+ TAK1) and TAK1/TAB1eCRISPR (CTAK1CTAB1) are respectively treated with taxol (the final concentration is 10 mu M) for 9h after 48 h, 5Z-7-oxozeano (Calbiochem, 499610) and taxol (10 mu M) are added into an untransfected plasmid hole, the final concentration of 5Z-7-oxozeano (5Z) is 5 mu M, and the apoptosis rate is detected by a flow cytometer after 9 h. Collecting cells, washing the cells once by PBS buffer at 1500rpm for 5 minutes, and removing supernatant; the cells were filtered and 100. mu.l of 1 XBi was added to the reaction tubeAnd binding buffer, adding 5 ul Annexin V-FITC and 5 ul PI, mixing, incubating for 15 minutes at room temperature in a dark place, adding 100 ul 1 XBinding buffer, and analyzing the apoptosis rate by a flow cytometer (Annexin V FITC apoptosis detection kit, BD, 556570). Compared with cells transfected with pcDNA 3-no-load plasmid, the apoptosis rate of cells transfected with TAK1 and TAB1+ TAK1 is remarkably reduced, and the apoptosis rate of cells transfected with TAK1/TAB1eCRISPR plasmid and TAK1 inhibitor 5Z-7-oxozeaano is increased (FIG. 8).
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Reference documents:
1Yamaguchi K,Shirakabe K,Shibuya H,et al.Identification of a member of the MAPKKK family as a potential mediator of TGF-βsignal transduction.Science.1995Dec 22；270(5244):2008-11.(PMID:8533096)
2.Hayden MS,Ghosh S(2008)Shared principles in NF-kappaB signaling.Cell.2008Feb 8；132(3):344-62..(PMID:18267068)
3.Sakurai H(2012)Targeting of TAK1in inflammatory disorders and cancer.Trends Pharmacol Sci.2012Oct；33(10):522-30.(PMID:22795313)
4.Mihaly SR,Ninomiya-Tsuji J,Morioka S(2014)TAK1control of cell death.Cell Death Differ.2014Nov；21(11):1667-76.(PMID:25146924)
5.Xia Y,Shen S,Verma IM(2014)NF-κB,an active player in human cancers.Cancer Immunol Res.2014Sep；2(9):823-30(PMID:25187272)
6.Kimura N,Matsuo R,Shibuya H et al(2000)BMP2-induced apoptosis is mediated by activation of the TAK1-p38kinase pathway that is negatively regulated by Smad6.J Biol Chem.2000Jun 9；275(23):17647-52.(PMID:10748100)
7Wang TH,Popp DM,Wang HS,et.al.Microtubule dysfunction induced by paclitaxel initiates apoptosis through both c-Jun N-terminal kinase(JNK)-dependent and-independent pathways in ovarian cancer cells.J Biol Chem.1999Mar 19；274(12):8208-8216.(PMID:10075725)
8Reshkin SJ,Bellizzi A,Cardone RA et al.Paclitaxel induces apoptosis via protein kinase A-and p38 mitogen-activated protein-dependent inhibition of the Na+/H+exchanger(NHE)NHE isoform 1 in human breast cancer cells.Clin Cancer Res.2003 Jun；9(6):2366-73.(PMID:12796407)
9.Fan Y1,Cheng J,Vasudevan SA et al(2013)TAK1 inhibitor 5Z-7-oxozeaenol sensitizes neuroblastoma to chemotherapy.Apoptosis.2013 Oct；18(10):1224-34(PMID:23700229)
10.Iriondo O,Liu Y,Lee G,et.al.TAK1 mediates microenvironment-triggered autocrine signals and promotes triple-negative breast cancer lung metastasis.Nat Commun.2018 May 18；9(1):1994.doi:10.1038/s41467-018-04460-w.(PMID:29777109)
11.Zhou J1,Zhong J1,Huang Z1,et.al.TAK1 mediates apoptosis via p38 involve in ischemia-induced renal fibrosis.Artif Cells NanomedBiotechnol.2018 Apr 16:1-10.doi:10.1080/21691401.2018.1442841.(PMID:29661023)
12.Weng T,Koh CG,POPX2 phosphatase regulates apoptosis through the TAK1-IKK-NF-κB pathway.Cell Death Dis.2017 Sep 14；8(9):e3051.doi:10.1038/cddis.2017.443.(PMID:28906490)
13.Guan S,Lu J,Zhao Y,et.al.TAK1 inhibitor 5Z-7-oxozeaenol sensitizes cervical cancer to doxorubicin-induced apoptosis.Oncotarget.2017 May 16；8(20):33666-33675.(PMID:28430599)
14.Li P,Zhao QL,Jawaid P,Enhancement of hyperthermia-induced apoptosis by 5Z-7-oxozeaenol,a TAK1 inhibitor,in Molt-4 cells.Int J Hyperthermia.2017 Jan 22:1-11.doi:10.1080/02656736.2017.1278629.(PMID:28111999)
15.McNew KL,Whipple WJ,Mehta AK,et.al.MEK and TAK1 Regulate Apoptosis in Colon Cancer Cells with KRAS-Dependent Activation of Proinflammatory Signaling.Mol Cancer Res.2016 Dec；14(12):1204-1216.(PMID:27655129)
16.Li P,Zhao QL,Jawaid P,et.al.Enhancement of hyperthermia-induced apoptosis by 5Z-7-oxozeaenol,a TAK1 inhibitor,in A549 cells.Cell Stress Chaperones.2016 Sep；21(5):873-81.(PMID:27448221)
17.Di Y,Li S,Wang L,Zhang Y,Dorf ME(2008)Homeostatic interactions between MEKK3 and TAK1 involved in NF-kappaB signaling.Cell Signal.Apr；20(4):705-713.(PMID:18206350)
Claims (8)
1. a method for predicting that TAK1 synergistically affects apoptosis with drugs for non-disease diagnostic and therapeutic purposes, comprising the steps of:
(1) treating tumor tissue with TAK1 antibody, and measuring the expression level of TAK1 in the tumor tissue to obtain R1(ii) a Meanwhile, normal tissues beside the tumor were treated with the TAK1 antibody, and the expression level of TAK1 in the normal tissues was determined to obtain R2；
(2) Comparison of R1And R2If R is1＜R2If the difference is significant, TAK1 is predicted to be capable of promoting the apoptosis of the tumor cells with the same source as the tumor tissue in the step (1) in a synergistic manner with the medicine; on the contrary, if R1＞R2If the difference is significant, the TAK1 is predicted not to be capable of promoting the apoptosis of the tumor cells with the same tumor tissue source as that in the step (1) in a synergistic way with the medicament, and the TAK1 inhibitor is predicted to promote the apoptosis of the tumor cells with the same tumor tissue source as that in the step (1) in a synergistic way with the medicament;
the tumor in the step (1) is kidney cancer, and the used cell is 769P human renal clear cell adenocarcinoma cell;
the tumor tissue and the normal tissue beside the tumor in the step (1) are kidney cancer and paraneoplastic paraffin-embedded tissue sections OD-CT-Com 03;
the drug in the step (2) is paclitaxel.
2. The method of claim 1, wherein:
the normal tissue beside the tumor in the step (1) is the normal tissue after the corresponding tumor is removed;
the TAK1 inhibitor in the step (2) is 5Z-7-oxozeaenol, or TAK1 gene is silenced by using TAK1 gene editing plasmid.
3. The method of claim 1, wherein:
the expression level of TAK1 in the step (1) is determined by an immunohistochemical method;
the significance of the difference in the step (2) is judged by the following method: firstly, carrying out the homogeneity test of the variances, if the variances are uniform, using the mean t of the two samples to test and compare, if the variances are not uniform, using the t' to test and compare, and if the variances are not uniform, using the mean t of the two samples to test and compareP＜0.05The difference was considered significant.
4. The method of claim 1, wherein: a step of further verification is included after step (2); the verification can be realized by any one or two methods as follows:
(a) immunoblotting
TAK1/TAB1 in combination with a drug: respectively transfecting the tumor cells with the same tumor tissue source as that in the step (1) with an unloaded plasmid, a plasmid containing a TAK1 gene and a plasmid containing TAK1 and TAB1 genes, culturing the transfected cells for 48 or 72 hours, adding a medicament for treating for 9-18 hours, cracking the cells to obtain a whole cell extract, and detecting PARP shearing by using an immunoblotting method; if the detection result shows that the PARP shearing of the tumor cells transfected by the plasmid containing the TAK1 gene and the plasmid containing the TAK1 and TAB1 genes is enhanced compared with the tumor cells transfected by the unloaded plasmid, the TAK1 and the medicament are verified to synergistically promote the apoptosis; on the contrary, if the PARP cleavage of the tumor cells transfected with the plasmid containing TAK1 gene and the plasmids containing TAK1 and TAB1 gene was reduced compared to the tumor cells transfected with the empty plasmid, it was confirmed that TAK1 could not promote tumor cell apoptosis in cooperation with the drug;
(b) flow cytometry
TAK1/TAB1 in combination with a drug: respectively transfecting tumor cells with the same tumor tissue source as that in the step (1) with an unloaded plasmid, a plasmid containing a TAK1 gene, a plasmid containing TAK1 and TAB1 genes and/or an edited plasmid for transfecting and silencing the TAK1 gene, adding a TAK1 inhibitor into the tumor cells of the untransfected plasmids, culturing the cells for 48 or 72 hours, adding a medicament for treating for 9-18 hours, and detecting the apoptosis rate by using a flow cytometer; if the detection result shows that compared with the tumor cells transfected with the unloaded plasmids, the apoptosis rate of the tumor cells transfected with the plasmids containing the TAK1 genes and the plasmids containing the TAK1 and TAB1 genes is increased, the TAK1 and the medicament are verified to synergistically promote the apoptosis; if the detection result shows that the apoptosis rate of the tumor cells transfected by the plasmid containing the TAK1 gene and the plasmid containing the TAK1 and TAB1 genes is reduced compared with the tumor cells transfected by the unloaded plasmid, the verification is that the TAK1 cannot promote the apoptosis of the tumor cells in cooperation with the medicament; if the detection result shows that compared with the tumor cells transfected with the unloaded plasmids, the apoptosis rate of the cells added with the TAK1 inhibitor or the edited plasmids transfected with the silenced TAK1 gene is increased, the TAK1 inhibitor or the silenced TAK1 gene is verified to be cooperated with the medicine to promote the apoptosis;
the medicament in the methods (a) and (b) is an anti-tumor medicament;
the dosage of the added medicine in the methods (a) and (b) is calculated according to the final concentration of the medicine in the system of 3-10 mu M;
the inhibitor of TAK1 in method (b) is 5Z-7-oxozeaenol;
the dosage of the added TAK1 inhibitor in the method (b) is calculated according to the final concentration of the inhibitor in the system being 5-10 mu M.
5. Use of a reagent for determining the expression level of TAK1 in tumor tissue and paraneoplastic tissue in the manufacture of a kit for predicting whether TAK1 and a drug can synergistically affect tumor cell apoptosis, characterized in that:
the tumor cell is 769P human kidney clear cell adenocarcinoma cell;
the tumor tissue and the paraneoplastic tissue are kidney cancer and paraneoplastic paraffin embedded tissue sections OD-CT-Com 03;
the medicine is paclitaxel.
6. Use according to claim 5, characterized in that:
the reagent is TAK1 antibody and immunohistochemical reagent;
the immunohistochemical reagent is polymer molecule marked with horseradish peroxidase and rabbit and mouse resisting immunoglobulin, DAB color developing agent or color developing buffer solution.
7. Use of an agent for determining the expression level of TAK1 in tumor and paraneoplastic tissues in the manufacture of a kit for predicting whether TAK1 and a drug can be used together to treat a patient with a tumor, characterized in that:
the reagent is TAK1 antibody and immunohistochemical reagent;
the immunohistochemical reagent is polymer molecules marked with horseradish peroxidase and rabbit and mouse resisting immunoglobulin, and DAB color developing agent or color developing buffer solution;
the tumor is kidney cancer, and the used cell is 769P human renal clear cell adenocarcinoma cell;
the tumor and the paraneoplastic tissues are kidney cancer and paraneoplastic paraffin-embedded tissue sections OD-CT-Com 03;
the medicine is paclitaxel.
8. The application of the reagent for measuring the expression level of TAK1 in tumor and paraneoplastic tissues in the preparation of a kit for screening the TAK1 inhibitor which has the effect of promoting tumor cell apoptosis in cooperation with medicaments is characterized in that:
the reagent is TAK1 antibody and immunohistochemical reagent;
the immunohistochemical reagent is polymer molecules marked with horseradish peroxidase and rabbit and mouse resisting immunoglobulin, and DAB color developing agent or color developing buffer solution;
the tumor cell is 769P human renal clear cell adenocarcinoma cell;
the tumor and the paraneoplastic tissues are kidney cancer and paraneoplastic paraffin-embedded tissue sections OD-CT-Com 03;
the medicine is paclitaxel.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910264363.9A CN109856351B (en) | 2019-04-03 | 2019-04-03 | Method for predicting that TAK1 and medicament synergistically influence apoptosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910264363.9A CN109856351B (en) | 2019-04-03 | 2019-04-03 | Method for predicting that TAK1 and medicament synergistically influence apoptosis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109856351A CN109856351A (en) | 2019-06-07 |
| CN109856351B true CN109856351B (en) | 2022-05-06 |
Family
ID=66903156
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910264363.9A Active CN109856351B (en) | 2019-04-03 | 2019-04-03 | Method for predicting that TAK1 and medicament synergistically influence apoptosis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109856351B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0803571A3 (en) * | 1996-04-24 | 1999-07-28 | Chugai Seiyaku Kabushiki Kaisha | Tab1 protein & dna coding therefor |
| JP2007089496A (en) * | 2005-09-29 | 2007-04-12 | Institute Of Physical & Chemical Research | Method for screening medicine |
| CN103255213A (en) * | 2013-04-01 | 2013-08-21 | 哈尔滨体育学院 | Molecular biological method for prediction of excellent ice and snow players' flexibility by detecting expression level of TGF-beta in osteoblasts |
| CN103462896A (en) * | 2008-04-11 | 2013-12-25 | 细胞研究有限公司 | Methods and use of inducing apoptosis in cancer cells |
| CN109420170A (en) * | 2017-08-25 | 2019-03-05 | 中国科学院上海生命科学研究院 | Novel tumor microenvironment related target TAK1 and its application in inhibition tumour |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8163709B2 (en) * | 2008-01-28 | 2012-04-24 | Board Of Regents Of The University Of Texas System | TAK1-D mediated induction of cell death in human cancer cells by specific sequence short double-stranded RNAS |
-
2019
- 2019-04-03 CN CN201910264363.9A patent/CN109856351B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0803571A3 (en) * | 1996-04-24 | 1999-07-28 | Chugai Seiyaku Kabushiki Kaisha | Tab1 protein & dna coding therefor |
| JP2007089496A (en) * | 2005-09-29 | 2007-04-12 | Institute Of Physical & Chemical Research | Method for screening medicine |
| CN103462896A (en) * | 2008-04-11 | 2013-12-25 | 细胞研究有限公司 | Methods and use of inducing apoptosis in cancer cells |
| CN103255213A (en) * | 2013-04-01 | 2013-08-21 | 哈尔滨体育学院 | Molecular biological method for prediction of excellent ice and snow players' flexibility by detecting expression level of TGF-beta in osteoblasts |
| CN109420170A (en) * | 2017-08-25 | 2019-03-05 | 中国科学院上海生命科学研究院 | Novel tumor microenvironment related target TAK1 and its application in inhibition tumour |
Non-Patent Citations (8)
| Title |
|---|
| Expression of TAK1/TAB1 expression in non-small cell lung carcinoma and adjacent normal tissues and their clinical significance;Jiang Zhu等;《Int J Clin Exp Pathol》;20151215;第8卷(第12期);全文 * |
| Identification of TGF-b-activated kinase 1 as a possible novel target for renal cell carcinoma intervention;Meng Fandong等;《Biochemical and Biophysical Research Communications》;20140926;全文 * |
| Inactivation of transforming growth factor-b-activated kinase 1 promotes taxol efficacy in ovarian cancer cells;Li Bo等;《Biomedicine & Pharmacotherapy》;20161231;全文 * |
| TAK1在卵巢恶性上皮肿瘤中的表达及其对紫杉醇SKOV3和OVCAR3细胞中的化疗增敏研究;李波;《中国博士学位论文全文数据库 医药卫生科技辑》;20171215(第12期);摘要以及第47页第(七)部分至第49页第(十一)部分 * |
| 转化生长因子β 激活酶1 在结肠癌中的表达及临床意义;鞠红艳等;《中国普通外科杂志》;20151031;第24卷(第10期);摘要以及第1和2节 * |
| 转化生长因子β 激活酶1 在食管癌中的表达及其临床意义;乔飞;《实用医学杂志》;20141231;第30卷(第19期);全文 * |
| 转化生长因子β激活激酶１在非小细胞肺癌中的表达及意义;胡娴舒等;《中华实用诊断与治疗杂志》;20160831;第30卷(第8期);全文 * |
| 鞠红艳等.转化生长因子β 激活酶1 在结肠癌中的表达及临床意义.《中国普通外科杂志》.2015,第24卷(第10期), * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109856351A (en) | 2019-06-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhao et al. | Nestin positively regulates the Wnt/β-catenin pathway and the proliferation, survival and invasiveness of breast cancer stem cells | |
| Ye et al. | Exression and PI3K/AKT pathway in gastric cancer and its blockade suppresses tumor growth and metastasis | |
| EP2399129B1 (en) | A method of diagnosis or prognosis of a neoplasm comprising determining the level of expression of a protein in stromal cells adjacent to the neoplasm | |
| Xu et al. | Receptor interactive protein kinase 3 promotes Cisplatin-triggered necrosis in apoptosis-resistant esophageal squamous cell carcinoma cells | |
| Lee et al. | Prognostic impact of the cancer stem cell–related marker NANOG in ovarian serous carcinoma | |
| WO2010096627A1 (en) | Therapeutics and methods for treating neoplastic diseases comprising determining the level of caveolin-1 and/or caveolin-2 in a stromal cell sample | |
| Hu et al. | PIK3CA mutation confers resistance to chemotherapy in triple-negative breast cancer by inhibiting apoptosis and activating the PI3K/AKT/mTOR signaling pathway | |
| He et al. | Estrogen receptor α promotes lung cancer cell invasion via increase of and cross‐talk with infiltrated macrophages through the CCL2/CCR2/MMP9 and CXCL12/CXCR4 signaling pathways | |
| Chang et al. | Parathyroid hormone-like hormone is a poor prognosis marker of head and neck cancer and promotes cell growth via RUNX2 regulation | |
| Li et al. | α1, 6-Fucosyltransferase (FUT8) regulates the cancer-promoting capacity of cancer-associated fibroblasts (CAFs) by modifying EGFR core fucosylation (CF) in non-small cell lung cancer (NSCLC) | |
| Liang et al. | Aryl hydrocarbon receptor nuclear translocator is associated with tumor growth and progression of hepatocellular carcinoma | |
| Cheng et al. | E2F6 functions as a competing endogenous RNA, and transcriptional repressor, to promote ovarian cancer stemness | |
| Hsu et al. | FAM198B is associated with prolonged survival and inhibits metastasis in lung adenocarcinoma via blockage of ERK-mediated MMP-1 expression | |
| Braggio et al. | Autophagy inhibition overcomes sorafenib resistance in S45F‐mutated desmoid tumors | |
| Jian et al. | Phosphatidylethanolamine‐binding protein 4 promotes the epithelial‐to‐mesenchymal transition in non–small cell lung cancer cells by activating the sonic hedgehog signaling pathway | |
| Grellety et al. | Enhancing abiraterone acetate efficacy in androgen receptor–positive triple-negative breast cancer: Chk1 as a potential target | |
| Fu et al. | RYK, a receptor of noncanonical Wnt ligand Wnt5a, is positively correlated with gastric cancer tumorigenesis and potential of liver metastasis | |
| Cao et al. | Hypoxia-related gene FUT11 promotes pancreatic cancer progression by maintaining the stability of PDK1 | |
| Shi et al. | ZCCHC14 regulates proliferation and invasion of non–small cell lung cancer through the MAPK‐P38 signalling pathway | |
| Xie et al. | Evaluation of Cell Surface Vimentin Positive Circulating Tumor Cells as a Diagnostic Biomarker for Lung Cancer Front | |
| Zhang et al. | Rankl expression predicts poor prognosis in gastric cancer patients: results from a retrospective and single-center analysis | |
| Wu et al. | RPL35A is a key promotor involved in the development and progression of gastric cancer | |
| CN109856351B (en) | Method for predicting that TAK1 and medicament synergistically influence apoptosis | |
| US10247732B2 (en) | Materials and methods for diagnosing and predicting the course of prostate cancer | |
| US9897607B2 (en) | Methods, kits, and systems for treatment of metastatic papillary thyroid cancer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |