CN117230198A - Application of WNT10A and SLCO4A1-AS1 in glioma diagnosis and treatment - Google Patents
Application of WNT10A and SLCO4A1-AS1 in glioma diagnosis and treatment Download PDFInfo
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Abstract
The application belongs to the technical fields of biological medicine and molecular biology, and particularly relates to application of WNT10A and SLCO4A1-AS1 in glioma diagnosis and treatment. The application proves for the first time that the expression of WNT10A and SLCO4A1-AS1 increases with the increase of glioma malignancy, and is inversely related to survival rate; meanwhile, the WNT10A silent slow virus can obviously inhibit the expression level of glioma WNT10A, and the silencing of WNT10A inhibits the proliferation and colony formation of glioma cells, so that the WNT10A silent slow virus can be used as an effective medicament for preventing and/or treating glioma; meanwhile, down-regulation of SLCO4A1-AS1 gene can also effectively inhibit malignant growth and invasion capacity of glioma cells. The application provides a new field of view for searching biomarkers and drug treatment targets for glioma clinical diagnosis and prognosis evaluation, lays a foundation for relevant drug research and development, and has important significance for glioma research and treatment.
Description
Technical Field
The application belongs to the technical fields of biological medicine and molecular biology, and particularly relates to application of WNT10A and SLCO4A1-AS1 in glioma diagnosis and treatment.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the application and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Gliomas are the most common primary intracranial tumor, accounting for 81% of malignant brain tumors. Glioblastomas are the most common gliomas (approximately 45% of all gliomas) and are characterized by poor prognosis, resistance to surgical and chemo-radiochemical regimens, and relative survival rates of only 5% for 5 years. The main reasons for poor prognosis in GBM patients are the malignant phenotypes and characteristics of tumor cells such as high proliferation capacity, invasive growth, intratumoral genetic heterogeneity, and microvascular proliferation. Currently, the standard international regimen for the treatment of GBM is to administer Temozolomide (TMZ) concurrent chemotherapy after maximum surgical resection, in combination with radiation therapy, but the overall therapeutic effect is not ideal. Due to the infiltration of glioma cells into surrounding brain tissue and poor permeability of the blood brain barrier to chemotherapeutic drugs, even with current advanced microsurgical techniques, it is difficult to completely ablate tumors and high resistance and tolerance of glioma cells to therapeutic interventions is easily achieved. Therefore, searching for a novel therapeutic way that effectively inhibits the malignant biological characteristics of gliomas, thereby prolonging the survival time of patients and improving the survival quality of patients is an important challenge facing the field of nerve tumor treatment.
The WNT signaling pathway shows pleiotropic physiological functions, ranging from neurogenesis to stem cell proliferation, and has been implicated in a variety of human cancers, including gliomas. This pathway is particularly relevant in the context of Cancer Stem Cells (CSCs), which have been indicated as key mediators of cancer recurrence and resistance to radiotherapy and chemotherapy. Thus, novel therapeutic strategies directed to specific components of the WNT pathway have been widely explored. The WNT pathway has been found to be associated with poor prognosis and increased risk of colorectal adenoma in patients with gastric and bladder cancer resistance. Applicant previously screened for WNT members specifically expressed in gliomas, with WNT10A as a candidate gene, with the highest difference in significance. Importantly, no studies have been made to date to explore the relevance of WNT10A in human gliomas.
Meanwhile, long-chain non-coding RNAs have been found to have potential relationships with the development and progression of cancer in the past decade. Most of these long non-coding RNAs are endogenous, no less than 200 nucleotides in length, and have limited or no ability to encode proteins. SLCO4A1 antisense RNA 1 (SLCO 4A1-AS 1) is located on the antisense strand of chromosome 20 SLCO4A1, and is markedly overexpressed in several cancer types, associated with poor prognosis. However, its role in gliomas has not been reported.
Disclosure of Invention
In order to overcome the defects in the prior art, the inventor provides the application of WNT10A and SLCO4A1-AS1 in glioma diagnosis and treatment through long-term technical and practical exploration. According to the application, the first study proves that WNT10A and SLCO4A1-AS1 are highly expressed in glioma, the expression quantity of the WNT10A and SLCO4A1-AS1 is related to prognosis of glioma, and meanwhile, malignant growth and invasion capacity of glioma cells can be controlled by regulating and controlling the expression of WNT10A, so that the application of the WNT10A AS a glioma diagnosis/treatment/prognosis marker is expected to be developed.
In order to achieve the technical purpose, the application adopts the following technical scheme:
in a first aspect of the application there is provided the use of a substance for detecting WNT10A and SLCO4A1-AS1 in the manufacture of a product for diagnosing, detecting, monitoring or predicting the progression of glioma.
According to the application, the research proves that the expression level of WNT10A is up-regulated in glioma and is related to tumor molecular classification, and meanwhile, the expression of WNT10A is obviously related to prognosis, and the high expression of WNT10A indicates the poor prognosis of glioma patients. While SLCO4A1-AS1 expression levels were also up-regulated in gliomas and correlated with tumor grade, IDH mutation status, 1p/19q coding status. Further, high expression is predictive of poor prognosis in all glioma patients, LGG and GBM patients. Thus, the WNT10A and SLCO4A1-AS1 may be used AS biomarkers for diagnosing, detecting, monitoring, or predicting the progression of glioma.
In a second aspect of the application, a product for diagnosing, detecting, monitoring or predicting the progression of glioma is provided, comprising detecting the expression of WNT10A and SLCO4A1-AS1 in a sample based on a high throughput sequencing method and/or based on a quantitative PCR method and/or based on a probe hybridization method.
In a third aspect of the application, there is provided a system for diagnosing, detecting, monitoring or predicting the progression of glioma, the system comprising:
i) An analysis unit comprising a detection substance selected from the above WNT10A and SLCO4A1-AS1 expression levels in a sample to be tested of a subject, and;
ii) an evaluation unit comprising: determining the disease condition of said subject based on the expression levels of said WNT10A and SLCO4A1-AS1 determined in i).
In a fourth aspect of the present application there is provided the use of a substance which inhibits the expression and/or reduces the activity of WNT10A and SLCO4A1-AS1 in at least one of the following A1) -a 5):
a1 Inhibit glioma cell proliferation or preparing a product that inhibits glioma cell proliferation;
a2 Inhibiting the colony forming ability of glioma cells or preparing a product for inhibiting the colony forming ability of glioma cells;
a3 Inhibiting glioma cell invasion capacity or preparing a product for inhibiting glioma cell invasion capacity;
a4 Inhibit glioma growth or/and invasion or prepare a product for inhibiting glioma growth or/and invasion;
a5 For treating glioma or for preparing a product for treating glioma.
In a fifth aspect of the present application, there is provided a method for preventing and/or treating glioma, which comprises administering to a subject the above-described agent that inhibits the expression and/or reduces the activity of WNT10A and SLCO4A1-AS 1.
Compared with the prior art, the one or more technical schemes have the following beneficial effects:
the technical proposal proves that the expression of WNT10A and SLCO4A1-AS1 increases with the increase of the malignancy degree of glioma for the first time and is inversely related to the survival rate; meanwhile, the WNT10A silent slow virus can obviously inhibit the expression level of glioma WNT10A, and the silencing of WNT10A inhibits the proliferation and colony formation of glioma cells, so that the WNT10A silent slow virus can be used as an effective medicament for preventing and/or treating glioma; meanwhile, down-regulation of SLCO4A1-AS1 gene can also effectively inhibit malignant growth and invasion capacity of glioma cells.
In a word, the technical scheme provides a new field of view for searching biomarkers and drug treatment targets for clinical diagnosis and prognosis evaluation of glioma, lays a foundation for research and development of related drugs, has important significance for research and treatment of glioma, and has good practical application value.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application.
FIG. 1 is a graph showing the expression level and prognostic significance of WNT10A in glioma tissue in example 1, wherein A is the differential expression of WNT10A in glioma and glioblastoma at low levels in the TCGA database, B is the differential expression of WNT10A in glioma and glioblastoma at low levels in the CGGA database, C is the differential expression of WNT10A in glioma at various levels in the TCGA database, D is the differential expression of WNT10A in the CGGA database, E is the differential expression of WNT10A in glioma at various levels in the THE HUMAN PROTEIN ATLAS database and glioblastoma tissue sample at low levels, F is the result of immunohistochemical composition of WNT10A in glioma samples at different levels in our case database, G is the differential expression of WNT10A and stem index SOX2 in glioma samples at different levels in our case database, H is the differential expression of WNT10A in different cell lines, and the differential expression of WNT10A in different cell lines in the TCK is the differential expression profile of WNT10A and the differential expression of WNT 10B in different cell lines, and the differential expression of WNT10A in the TCK is the differential expression profile and the differential expression of WNT10A and the stem index SOX is the differential expression of WNT10A in different cell lines in the TCJ case and the differential expression profile is the differential expression of WNT10 and the differential expression of the GAX is the differential protein. * P <0.05; * P <0.01; * P <0.001.
FIG. 2 is a functional series of the present application showing that WNT 10A-specific knockdown can reduce proliferation and invasion of glioma cells in example 1. A is a growth curve graph of LN229, U118 and GBM#P3 cells based on OD450 in CCK-8 detection, B is a reduction graph of cloning capacity after shWNT10A is transfected into cells, C is a detection graph of EdU proliferation after shWNT10A is transfected into cells, D is a reduction graph of 3D invasion capacity after shWNT10A is transfected into cells, E is an increase graph of apoptosis cells after shWNT10A is transfected into cells, F is a reduction graph of brain invasion capacity after shWNT10A is transfected into cells, G and H are reduction graphs of balling capacity after shWNT10A is transfected into cells, I and J are reduction graphs of stem resistance after shWNT10A is transfected into cells, K is a shWNT10A stable cell in situ xenograft tumor model, biological fluorescence imaging (biolescence imaging, BLI) system is adopted to evaluate tumor growth conditions of mice in a control group and mice in a WNT10A knockdown group, fluorescence intensity is high, L is a control group and a control group is a control group and has a lower survival time, and a group is a lower-phase of immunoknock down tumor is utilized to evaluate the conditions of a low-down tumor cell, and a low-level-down score of the stem cell is utilized to evaluate the tumors of a group. * P <0.05; * P <0.01; * P <0.001.
FIG. 3 is a graph showing that WNT10A activates the JNK-cJUN-FOSB pathway in glioblastoma cells through FZD1 in example 1 of the present application. Wherein, the A-F diagram verifies that WNT10A can directly bind with FZD1 to play a role, the G diagram verifies that WNT10A plays a role mainly by activating JNK phosphorylation, the H diagram indicates that downstream play a role by FOSB, the I and J diagrams indicate that knockdown of WNT10A or over-expression of WNT10A can correspondingly influence the expression of FOSB, the K diagram proves that cJUN can act as a transcription factor to influence the expression of FOSB after forming an AP-1 complex, the L diagram indicates that knockdown of WNT10A can influence the activation of FZD1-JNK-Cjun signaling pathway and further influences the expression of FOSB, the M diagram indicates that FZD1 is not activated, the FZD1-JNK-Cjun signaling pathway can not be activated, and the N diagram indicates a mechanism mode diagram of play a role of WNT 10A.
FIG. 4 shows the expression levels of SLCO4A1-AS1 in glioma tissue and normal tissue samples in example 2 of the present application, wherein A is the differential expression of SLCO4A1-AS1 in normal brain tissue and glioblastoma in the combined TCGA and GTEx database, and B is the differential expression of SLCO4A1-AS1 in normal brain tissue and glioblastoma in the GSE44971 database. C and D are the differential expression of SLCO4A1-AS1 in TCGA and CGGA databases in different grades of glioma, E and F are the differential expression of SLCO4A1-AS1 in TCGA and CGGA databases in different ages of glioma. G and H are the differential expression conditions of SLCO4A1-AS1 in TCGA and CGGA databases in different IDH states of glioma, I and J are the differential expression conditions of SLCO4A1-AS1 in TCGA and CGGA databases in different 1P/19q coding states of glioma, and P is less than 0.05; * P <0.01; * P <0.001.
Fig. 5 is a single-and multi-factor regression analysis of SLCO4A1-AS1 in glioma in example 2 of the present application, wherein a and B were single-and multi-factor COX regression analysis performed in TCGA (n=667) and CGGA (n=325) databases, respectively.
FIG. 6 is a graph showing the prognostic significance of SLCO4A1-AS1 in gliomas in example 2 of the present application, wherein FIGS. 6A and 6B are graphs of the prognosis of SLCO4A1-AS1 in all glioma patients, low grade glioma LGG and glioblastoma GBM patients, analyzed in TCGA (n=667) and CGGA (n=325) databases, respectively.
FIG. 7 is a graph showing the proliferation of glioma-reduced cells by SLCO4A1-AS1 knockout in example 2 of the present application; wherein, A is real-time fluorescence quantitative PCR analysis shows that the relative RNA level of SLCO4A1-AS1 is obviously reduced after small interference knockout cells are used, and GAPDH is used AS a control; b is a CCK-8 detection OD 450-based LN229, A172, U118, and GBM#P3 cell growth curve; c is the reduction of the cell clonality after SLCO4A1-AS1 knockout.
FIG. 8 shows the decrease in cell proliferation activity after SLCO4A1-AS1 knockdown in example 2 of the present application; * P <0.05; * P <0.01; * P <0.001.
FIG. 9 is a graph showing the reduction in glioma invasion capacity after transfection of si-SLCO4A1-AS1 into cells in example 2 of the present application. Wherein, A is LN229, A172 and U118 aiming at glioma cells; b is GBM#P3 for glioma cells; * P <0.05; * P <0.01; * P <0.001.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. 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 application 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 application. 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, devices, components, and/or combinations thereof.
In an exemplary embodiment of the present application, there is provided the use of a substance for detecting WNT10A and SLCO4A1-AS1 in the manufacture of a product for diagnosing, detecting, monitoring or predicting the progression of glioma.
According to the application, the research proves that the expression level of WNT10A is up-regulated in glioma and is related to tumor molecular classification, and meanwhile, the expression of WNT10A is obviously related to prognosis, and the high expression of WNT10A indicates the poor prognosis of glioma patients. While SLCO4A1-AS1 expression levels were also up-regulated in gliomas and correlated with tumor grade, IDH mutation status, 1p/19q coding status. Further, high expression is predictive of poor prognosis in all glioma patients, LGG and GBM patients. Thus, the WNT10A and SLCO4A1-AS1 may be used AS biomarkers for diagnosing, detecting, monitoring, or predicting the progression of glioma.
Wherein, WNT10A and SLCO4A1-AS1 are human sources;
the gliomas include low-grade gliomas (grade I-II) and high-grade gliomas (grade III-IV);
and AS described above, WNT10A and SLCO4A1-AS1 up-regulated expression correlated positively with glioma-grade elevation (malignancy), negatively with a better prognosis for glioma patients.
In particular, the methods for diagnosing, detecting, monitoring or predicting progression of glioma include, but are not limited to (early or auxiliary) diagnosis of glioma, evaluation of malignancy, and prognostic evaluation of glioma patient;
the prognostic evaluation includes at least an assessment of the overall survival of a glioma patient.
In yet another embodiment of the present application, a product for diagnosing, detecting, monitoring or predicting the progression of glioma is provided comprising a substance for detecting the expression of WNT10A and SLCO4A1-AS1 in a sample based on a high throughput sequencing method and/or based on a quantitative PCR method and/or based on a probe hybridization method.
The sample may be a glioma sample of a subject, such as a glioma cell or glioma tissue of a subject.
The product may be a detection reagent, a detection kit, a biosensor, and related detection devices, apparatuses, etc., and is not particularly limited herein.
In yet another embodiment of the present application, there is provided a system for diagnosing, detecting, monitoring or predicting the progression of glioma, the system comprising:
i) An analysis unit comprising a detection substance selected from the above WNT10A and SLCO4A1-AS1 expression levels in a sample to be tested of a subject, and;
ii) an evaluation unit comprising: determining the disease condition of said subject based on the expression levels of said WNT10A and SLCO4A1-AS1 determined in i).
Wherein the WNT10A and SLCO4A1-AS1 are human sources.
The judging of the disease condition of the subject at least comprises evaluating the (early or auxiliary) diagnosis, malignancy degree and prognosis of glioma patients of the subject; further, the prognostic evaluation includes at least an assessment of the overall survival of the glioma patient.
In yet another embodiment of the present application, there is provided the use of a substance that inhibits the expression and/or decreases the activity of WNT10A and SLCO4A1-AS1 in at least one of the following A1) -a 5):
a1 Inhibit glioma cell proliferation or preparing a product that inhibits glioma cell proliferation;
a2 Inhibiting the colony forming ability of glioma cells or preparing a product for inhibiting the colony forming ability of glioma cells;
a3 Inhibiting glioma cell invasion capacity or preparing a product for inhibiting glioma cell invasion capacity;
a4 Inhibit glioma growth or/and invasion or prepare a product for inhibiting glioma growth or/and invasion;
a5 For treating glioma or for preparing a product for treating glioma.
Furthermore, the research of the application shows that after the WNT10A is knocked down, the activation of FZD1-JNK-cJUN signal channels in glioma cells can be effectively inhibited, and further malignant phenotypes such as glioma cell proliferation, invasion and the like are affected.
Substances that inhibit the expression and/or decrease the activity of WNT10A and SLCO4A1-AS1 include, but are not limited to, substances that are directed against RNA interfering molecules or antisense oligonucleotides that inhibit WNT10A and SLCO4A1-AS1, small molecule inhibitors, siRNA, perform lentiviral infection or gene knockdown, and the like.
The product may be a drug or an experimental reagent that may be used for basic research.
According to the application, when the product is a medicament, the medicament further comprises at least one pharmaceutically inactive ingredient.
The pharmaceutically inactive ingredients may be carriers, excipients, diluents and the like which are generally used in pharmacy. Further, the composition can be formulated into various dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc., for oral administration, external use, suppositories, and sterile injectable solutions according to a usual method.
The non-pharmaceutically active ingredients, such as carriers, excipients and diluents, which may be included, are well known in the art and can be determined by one of ordinary skill in the art to meet clinical criteria.
In yet another embodiment of the application, the medicament of the application may be administered to the body in a known manner. Such as systemic delivery via veins. Alternatively via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration may be via single or multiple doses. It will be appreciated by those skilled in the art that the actual dosage to be administered in the present application may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.
In yet another embodiment of the present application, the subject to be administered can be human or non-human mammal, such as mouse, rat, guinea pig, rabbit, dog, monkey, gorilla, etc., preferably human.
In yet another embodiment of the present application, there is provided a method for preventing and/or treating glioma, comprising administering to a subject the above-described agent that inhibits the expression and/or reduces the activity of WNT10A and SLCO4A1-AS 1.
The application is further illustrated by the following examples, which are given for the purpose of illustration only and are not intended to be limiting. If experimental details are not specified in the examples, it is usually the case that the conditions are conventional or recommended by the sales company; the present application is not particularly limited and can be commercially available.
Example 1
1. Materials and methods
1. Tissue sample and database
This example includes glioma samples and normal brain tissue from the Shandong university Qilu hospital neurosurgery. The study protocol was approved by the institutional review board and informed by written consent of each subject. The patient was routinely treated with 3 normal brain tissue, 10 WHO grade II-III, 5 grade IV (GBM) tissue specimens. Glioma specimens were validated and classified by two experienced clinical pathologists according to WHO tumor classification. Clinical information and expression data of glioma samples of the public database are taken from the TCGA and CGGA databases.
2. Cell culture and reagents
Human glioblastoma cells were purchased from cell banks of the national academy of sciences. All cells were cultured using DMEM medium containing 10% fetal bovine serum and incubated at 37 ℃ in a cell incubator containing 5% carbon dioxide.
Knock-down of WNT10A
The lenti-shWNT10A and lenti-Control lentivirus (Geneharma, shanghai; sequences shown in the following Table) constructed and synthesized using lentiviral vectors were used to infect glioma cells according to standard protocols. Subsequent experiments were performed after infection via puromycin screening.
4. Real-time quantitative PCR
RNA was extracted from glioma cells using Trizol reagent (Invitrogen, life Technologies). And reverse transcription is performed. The primers of WNT10A are forward primers: 5'-CTTCCACTGGTGCTGTTTCGT-3' (SEQ ID NO. 3); reverse primer: 5'-TCACTTGCAGACGCTGACCC-3' (SEQ ID NO. 4).
5.Western-blot
The harvested cells were lysed with thermal denaturation in RIPA cell lysis buffer. Protein lysates (20. Mu.g) were analyzed and the proteins were transferred to a polyethylene difluoride film (PVDF). The primary antibody WNT10A, p-JNK, JNK, p-cJUN, cJUN, FOSB, GAPDH was incubated. Specific proteins were detected by enhanced chemiluminescence (ECL, millipore, bredford, USA).
6. Analysis of cell proliferation Capacity-CCK 8
Glioma cells were taken in 96-well cell culture dishes at a density of 3000 cells/well. Cell proliferation was analyzed 24, 48, 72, 96h after transfection with cell count Kit-8 (CCK-8). mu.L of CCK-8 solvent was added to each well and incubated for 1h in a cell incubator. The optical density was then measured at 450nm using Ensight (PerkinElmer) and analyzed to plot cell proliferation curves.
Edu proliferation assay
Glioma cells were taken in 24-well cell culture dishes at a density of 50000 cells/well. After 48 transfection, edU reagent was added, followed by staining according to instructions, photographing under a fluorescence microscope, counting the total number of cells and Edu positive cell number, respectively, and calculating the ratio.
8. Cell colony generation assay
Cells were seeded into 6-well plates at a density of 1000 cells/well. DMEM containing 10% fetal bovine serum was replaced every three days. After 15 days, methanol was fixed, stained with crystal violet for 15 minutes and photographed. Each test was repeated 3 times.
9. Cell balling experiment
Cells were seeded into 12-well plates at a density of 1000 cells/well and cultured using Neurobasal stem cell culture broth. After 10 days, the number of balls was detected by a microscope and counted by photographing. Each test was repeated 3 times.
10. Cell invasion Capacity detection experiment
Glioma balling is the incubation of cells in a balling matrix for 72 hours, implantation of spheroids >2mm in a 96-well plate, and addition of invading glue. Glioma spheroids were photographed every 24 hours with a microscope. Ellipsoids at 0h served as reference points for measuring the area of invasion of the invading cells.
11. Invasion ball-brain-like organ invasion experiment
Our previous work describes a protocol for GBM brain organoid co-culture invasive in vitro system culture of fetal brain organoids in 18 day rats. After 3 weeks of culture, normal brain-like organ cells differentiate to completion, and tumor balls may be exposed to normal brain-like organs. U251-GRP cultured with GFP lentivirus was the last 72-GFP cell (n=3000) cultured on a 96-well plate for 3 days to form tumor balls, and then co-cultured with the mature brain-like organ for 24 hours. The RBs are then processed separately. Relative areas of tumor cell invasion were captured with confocal microscopy at 0, 48 and 96 hours. Image soft analysis GBM cell-related relative area of affected area.
12. In vivo experiments in mice
Establishment of intracranial glioma, GBM#P3-luciferases (1×10) 6 ) Transfected with Lenti-shWNT10A or Lenti-Control virus, then stereotactically implanted into the mouse brain. The growth of intracranial tumors was examined on days 4 and 20 using bioluminescence imaging. Kaplan-Meier survival curves were used to describe time to survival and body weight.
13. Chromatin co-immunoprecipitation-CHIP
Experiments were performed using a Millipore chromatin co-immunoprecipitation kit according to the instructions, with cJUN and IgG antibodies added separately, and the products were quantified using PCR.
14. Immunohistochemistry
Paraffin sections were preheated in 65 ℃ incubator for 60min, dewaxed in xylene I and II for 15min each, and hydrated in pure alcohol I, pure alcohol II, 95% alcohol, 80% alcohol, 70% alcohol for 5min each. After washing with PBS, antigen retrieval was performed with sodium citrate buffer at ph=6.0 at 80 ℃ for 5min and 20 ℃ for 15 min. 3%H 2 O 2 Incubating for 15min to remove endogenous peroxidase activity, and washing with PBS for 5min×3 times; incubating 10% goat serum blocking solution at 37 ℃ for 15min, blocking nonspecific antigen, pouring supernatant, and dripping primary antibody working solution (proliferation marker: ki 67) diluted in proper proportion, and standing overnight at 4 ℃; washing with PBS for 5min×3 times, dripping biotinylated secondary antibody working solution (goat anti-rabbit), and incubating at 37deg.C for 15min; washing with PBS for 5min multiplied by 3 times, dripping working solution of horseradish enzyme labeled streptavidin, and incubating at 37 ℃ for 15min; washing with PBS for 5min×3 times, developing with DAB for 1-3min, and stopping after brown particles are generated; and (5) fully flushing with tap water. Counterstaining with hematoxylin for 3min, washing, differentiating with hydrochloric acid alcohol, and washing with tap water to turn blue. Then dehydrated in alcohol with the concentration of 70%, 80%, 95%, 100% I and 100% II respectively for 5min, and transparent and neutral resin sealing sheets are respectively carried out on xylene I and xylene II for 15 min. And (5) observing and photographing by a microscope after airing and solidifying. .
15. Statistical analysis
ANOVA or t-test was applied using GraphPad Prism 7 software. All experiments were repeated 3 times and mean ± standard error was taken. The Kaplan-Meier survival curve was analyzed using the log-rank test. The chi-square test and fisher's deterministic analysis were applied to determine the relationship between WNT10A expression and clinical pathology. P <0.05 is statistically significant for the differences.
2. Experimental results
The expression level of WNT10A is up-regulated in glioma, and is related to tumor molecular classification, and high expression can be used as a marker for poor prognosis of glioma patients
First, the gene expression level differences of WNT family genes in GBM and LGG were analyzed using TCGA, CGGA databases. The results showed that WNT10A, WNT16 expression levels in GBM were significantly elevated, again most significantly differing by WNT10A (shown in fig. 1A-B). Next, we analyzed WNT10A expression levels in different gliomas using TCGA and CGGA databases, and the results showed that WNT10A expression levels were highest in gliomas grade 4 (fig. 1C-D). The THE HUMAN PROTEIN ATLAS database also demonstrates the results of immunohistochemistry and immunofluorescence of samples from our hospital patients (FIGS. 1E-G). We examined the differences in WNT10A levels in glioma cell lines, glioma primary cells and normal astrocytes using RT-qPCR techniques and Western-blot techniques, and found that WNT10A was highly expressed in most glioma cell lines (shown in FIGS. 1H-I). The prognostic value of WNT10A expression in Overall Survival (OS) of glioma patients was examined using Kaplan-Meier survival curves. In the TCGA and CGGA databases, WNT10A expression was significantly correlated with prognosis, and WNT10A high expression was predictive of a poor prognosis (shown in fig. 1J-K). Therefore, WNT10A can be used as a novel glioma prognosis marker, and provides a basis for postoperative survival assessment of patients. Also we found that WNT10A affects glioma dryness (shown in fig. 1L-M), which can promote malignant phenotypes such as glioma proliferation.
2. Verifying the inhibition of the knockdown WNT10A on glioma cell proliferation and invasion
Based on the aberrant upregulation of WNT10A expression in gliomas, suggesting that it may exert oncogene-promoting functions, to assess the role of WNT10A in glioma malignancy biology, lentiviral lenti-shWNT10A was constructed to down-regulate WNT10A expression in glioma cell lines LN229, U118 and gbm#p3. The effect of WNT10A on cell proliferation was determined using CCK-8, colony formation, edU, cell pelleting experiments. CCK-8 results showed that glioma cells from the WNT10A knockdown group proliferated significantly less after 4 days than the control group (FIG. 2A); the result of EdU also confirmed this conclusion (fig. 2C). At the same time, the glioma cell colony forming ability and the spheroidization ability of the WNT10A knockdown group were both inhibited (fig. 2b, g, h). In the invasion capacity test, the invasion capacity of the WNT10A knockdown group of glioma cells was tested again with the brain-like technique at a significantly lower distance to peripheral matrigel invasion than the control group (fig. 2D) at 72 hours, and the conclusion was consistent (fig. 2F). Flow cytometry detection shows that the WNT10A can promote apoptosis of glial cells after knockdown (figure 2E), and the results show that the down-regulation of the WNT10A gene can inhibit malignant growth and invasion capacity of glioma cells.
Influence of WNT10A on intracranial tumor growth
The growth progress of glioma after intracranial seed tumors of nude mice was detected using a bioluminescence imaging (BLI) system. The results showed that the fluorescent intensity was significantly reduced 20 days after the seed tumor in the lenti-shWNT10A group compared with the control group (FIG. 2K). The total survival time of the control group was shorter than that of the lenti-shWNT10A group (P <0.001, FIG. 2L). Immunohistochemical detection of Ki67 cell proliferation index by using mouse brain-stained tissue section shows that the Ki67 histochemical score of the lenti-shWNT10A group is obviously reduced (figure 2M), immunofluorescence shows that SOX2 tumor cell stem index is also obviously reduced (figure 2N), and the result shows that the down-regulation of WNT10A leads to the reduction of the growth speed of glioma cells in vivo.
WNT10A down-regulation of glioblastoma cell FZD1-JNK-cJUN signaling pathway
The cJUN protein belongs to one of activated protein-1 (AP-1) transcription complexes, is a transcription factor with the most transcription activity in the AP-1 complex, and can be used for regulating and controlling biological processes such as cell proliferation, apoptosis and the like by promoting the expression and activation of the cJUN protein through various physical, chemical and biological stimuli and cell stress reactions. High cJUN protein expression has been shown to be closely related to the occurrence and prognosis of a variety of malignant tumors. We predict and verify through protein interaction technology that the receptor FZD1 (FIGS. 3A-F) of WNT10A on the surface of tumor cells shows that after knockdown of WNT10A, the JNK phosphorylation level in tumor cells is reduced, and the JNK phosphorylation level is obviously increased after over-expression of WNT10A, but the signal channels such as beta-Catenin, caMKII and the like closely related to Frizzled receptors are not obviously changed (FIG. 3G). Transcriptome sequencing found that knockdown of WNT10A primarily affected expression of downstream FOSB, which was subsequently verified by RT-qPCR, was consistent with sequencing results (fig. 3H-J), which was an important component of the AP-1 complex, and after JNK phosphorylation was reduced, also affected stability of cJUN protein, which was in turn used as a transcription factor to regulate FOSB expression, which constituted a positive feedback loop, which was verified by CHIP experiments (fig. 3K). And corresponding verification is carried out by adopting a Western-blot technology (shown in fig. 3L-M). Based on the above experiments, we propose the idea that, after WNT10A is knocked down, activation of FZD1-JNK-cJUN signaling pathway in glioma cells is inhibited, thereby affecting malignant phenotypes such as glioma cell proliferation, invasion, etc.
Example 2
1. Materials and methods
1. Tissue sample and database
Clinical information and expression data of glioma samples of the public database are taken from the TCGA, CGGA and GEO databases.
2. Cell culture and reagents
Human glioblastoma cells were purchased from cell banks of the national academy of sciences. All cells were cultured using DMEM medium containing 10% fetal bovine serum and incubated at 37 ℃ in a cell incubator containing 5% carbon dioxide.
SLCO4A1-AS1 knockout
Glioma cells were transfected with high efficiency and low interference according to standard protocols, with the following sequences:
4. real-time quantitative PCR
RNA was extracted from glioma cells using Trizol reagent (Invitrogen, life Technologies). And reverse transcription is performed. The primers of SLCO4A1-AS1 are forward primers: 5'-TGGGCAGAGTGTCGCTG-3' (SEQ ID NO. 7); reverse primer: 5'-GGCATTCAGAGAGTTGCGTTCA-3' (SEQ ID NO. 8).
5. Analysis of cell proliferation Capacity
Glioma cells were uniformly seeded into 96-well plates and divided into three groups (si-NC, si-SLCO4A1-AS1-1, si-SLCO4A1-AS 1-2). After 24, 48, 72, 96 hours of incubation, 10ul of CCK-8 was added to each well, incubated in an incubator for 1-2 hours, and absorbance at 450nm was measured with an microplate reader.
6. Cell colony generation assay
LN229, A172 and U118 cells transfected with si-NC, si-SLCO4A1-AS1-1, si-SLCO4A1-AS1-2 were plated uniformly into 6-well plates, 800 cells per well, cultured in an incubator for about 14 days, and cells were fixed with 4% paraformaldehyde for 20 minutes. Cells were stained with crystal violet for 15 minutes and observed and imaged under a microscope.
EDU experiment
We used the EdU cell proliferation assay kit to perform EdU assays. The control and experimental groups were inoculated in 24 well plates with 2 ten thousand cells per well. After 24h, incubation with EDU was carried out for 2h at 37℃and then fixation with 4% neutral paraformaldehyde fixative at room temperature for 15min at room temperature in the absence of light. Cells were incubated with 0.1mL of 0.5% Triton X-100 permeate for 20 minutes at room temperature. The Click-iT reaction mixture was incubated at room temperature for 30 minutes in the dark. Finally, nuclei were stained with Hoechst 33342 for 15-30 minutes, imaged and counted under a microscope.
8. Cell invasion Capacity detection experiment
Cell invasion was assessed by transwell experiments. LN229, A172, U118 cells after 12-24 hours of transfection were placed in the upper chamber of the 12-well plate, in FBS-free medium. The lower chamber was filled with medium containing 15% Fetal Bovine Serum (FBS). The cells of the upper chamber were rubbed with a cotton swab, stained with 0.1% crystal violet for 10 minutes and counted, and incubated in an incubator at 37℃and 5% CO2 for 24-48 hours, fixed with 4% paraformaldehyde for 30 minutes. In three-dimensional invasion experiments, transfected P3 cells (3000/well) were evenly seeded into 96-well plates. After sphere formation, the challenge gel was added to the wells and incubated in an incubator for 48 hours. The affected sphere was observed under a microscope and imaged. The invasiveness of transfected tumor cells was examined by comparison with the control group.
9. Statistical analysis
ANOVA or t-test was applied using GraphPad Prism 7 software. All experiments were repeated 3 times and mean ± standard error was taken. The Kaplan-Meier survival curve was analyzed using the log-rank test. The chi-square test and fisher's deterministic analysis were applied to determine the relationship between SLCO4A1-AS1 expression and clinical pathology. P <0.05 is statistically significant for the differences.
2. Experimental results
The expression level of SLCO4A1-AS1 was up-regulated in gliomas and correlated with tumor grade, IDH mutation status, 1p/19q coding status
The gene expression level differences of SLCO4A1-AS1 in GBM and normal brain tissues were first analyzed using the TCGA and GTEx combined database. The results showed that SLCO4A1-AS1 expression levels were elevated in gliomas (shown in FIG. 4A) compared to normal brain tissue, AS verified in the GSE44971 database (FIG. 4B). The relationship between SLCO4A1-AS1 expression level and glioma classification was analyzed in further detail (FIGS. 4C, D), and it was revealed that SLCO4A1-AS1 expression level was highest in GBM. The above results indicate an important role for SLCO4A1-AS1 in glioma malignancy classification. Further, the applicant analyzed the relationship between age (FIG. 4E, F), IDH mutation status (FIG. 4G, H), 1p/19q coding status (FIG. 4I, J) and SLCO4A1-AS1 expression level in TCGA and CGGA databases. The results showed significant correlation with high expression of SLCO4A1-AS 1.
SLCO4A1-AS1 high expression can be used AS a marker with poor prognosis for glioma patients
The relationship between SLCO4A1-AS1 and other clinical traits and clinical prognosis was verified using single-factor, multi-factor COX regression analysis, and the results showed that the expression level of SLCO4A1-AS1 was independent of prognosis (shown in FIG. 5). The prognostic value of SLCO4A1-AS1 expression in the Overall Survival (OS) of glioma patients was examined using the Kaplan-Meier survival curve. In TCGA and CGGA databases, SLCO4A1-AS1 expression was significantly correlated with prognosis, with high SLCO4A1-AS1 expression in all glioma patients, LGG and GBM patients, indicating poor prognosis (shown in fig. 6). Therefore, SLCO4A1-AS1 can be used AS a new glioma prognosis marker, and provides a basis for postoperative survival assessment of patients.
3. Verifying the knockout efficiency of SLCO4A1-AS1 in glioma cells and the inhibition effect of knockout SLCO4A1-AS1 on glioma cell proliferation and invasion
Based on the aberrant upregulation of expression of SLCO4A1-AS1 in gliomas, it was suggested that it may play an oncogene role in glioma development and progression, to assess the role of SLCO4A1-AS1 in glioma malignancy biology, small interference was used to knock out expression of SLCO4A1-AS1 in glioma cell lines LN229, a172, U118 and gbm#p3. Real-time quantitative PCR analysis confirmed that SLCO4A1-AS1 expression levels were significantly reduced after knockout of SLCO4A1-AS1 compared to NC groups (FIG. 7A). These results further demonstrate the effectiveness of si-SLCO4A1-AS1 in down-regulating SLCO4A1-AS1 in glioma cell in vitro functional assays. The effect of SLCO4A1-AS1 on cell proliferation was determined using CCK-8, colony formation, and EDU experiments. CCK-8 results showed that glioma cells from the SLCO4A1-AS1 knockout group proliferated significantly less after 4 days than the control group (FIG. 7B); meanwhile, the glioma cell colony forming ability of the SLCO4A1-AS1 knockout group is inhibited (fig. 7C), and the cell proliferation activity of the SLCO4A1-AS1 knockout group is remarkably reduced (fig. 8), and in the invasive ability detection experiment, the glioma cell invasive ability of the SLCO4A1-AS1 knockout group is remarkably reduced (fig. 9). These results indicate that down-regulation of the SLCO4A1-AS1 gene can inhibit malignant growth and invasive capacity of glioma cells.
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (10)
1. Use of a substance that detects WNT10A and SLCO4A1-AS1 for the preparation of a product for diagnosing, detecting, monitoring or predicting the progression of glioma.
2. The use according to claim 1, wherein WNT10A and SLCO4A1-AS1 are of human origin;
the gliomas include low-grade gliomas (grade I-II) and high-grade gliomas (grade III-IV);
up-regulated expression of WNT10A and SLCO4A1-AS1 correlated positively with increased glioma classification (malignancy), and negatively with a better prognosis for glioma patients.
3. The use of claim 1, wherein the means for diagnosing, detecting, monitoring or predicting progression of glioma comprises (early or secondary) diagnosis of glioma, evaluation of malignancy, and prognosis evaluation of glioma patient;
the prognostic evaluation includes at least an assessment of the overall survival of a glioma patient.
4. A product for diagnosing, detecting, monitoring or predicting the progression of glioma, characterized in that said product comprises a substance for detecting the expression of WNT10A and SLCO4A1-AS1 in a sample based on a high throughput sequencing method and/or based on a quantitative PCR method and/or based on a probe hybridization method.
5. The product of claim 4, wherein the sample is a glioma sample from a subject comprising glioma cells or glioma tissue from the subject;
the product is detection reagent, detection kit, biosensor, detection device and equipment.
6. A system for diagnosing, detecting, monitoring or predicting the progression of glioma, said system comprising:
i) An analysis unit comprising a detection substance selected from WNT10A and SLCO4A1-AS1 expression levels in a sample to be tested for a subject, and;
ii) an evaluation unit comprising: determining the disease condition of said subject based on the expression levels of said WNT10A and SLCO4A1-AS1 determined in i).
7. The system of claim 7, wherein WNT10A and SLCO4A1-AS1 are of human origin;
the judging of the disease condition of the subject at least comprises evaluating the (early or auxiliary) diagnosis, malignancy degree and prognosis of glioma patients of the subject; further, the prognostic evaluation includes at least an assessment of the overall survival of the glioma patient.
8. Use of a substance that inhibits the expression and/or decreases the activity of WNT10A and SLCO4A1-AS1 in at least one of the following A1) -a 5):
a1 Inhibit glioma cell proliferation or preparing a product that inhibits glioma cell proliferation;
a2 Inhibiting the colony forming ability of glioma cells or preparing a product for inhibiting the colony forming ability of glioma cells;
a3 Inhibiting glioma cell invasion capacity or preparing a product for inhibiting glioma cell invasion capacity;
a4 Inhibit glioma growth or/and invasion or prepare a product for inhibiting glioma growth or/and invasion;
a5 For treating glioma or for preparing a product for treating glioma.
9. The use according to claim 8, wherein the agent that inhibits the expression and/or reduces the activity of WNT10A and SLCO4A1-AS1 comprises an agent that performs a lentiviral infection or gene knockdown against an RNA interfering molecule or antisense oligonucleotide, a small molecule inhibitor, an siRNA that inhibits WNT10A and SLCO4A1-AS 1.
10. The use according to claim 8, wherein the product is a pharmaceutical or experimental agent for use in basic research.
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