CN111979317B - Application of Mllt11 gene in preparation of glioma drug and diagnostic kit thereof - Google Patents
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Abstract
The invention discloses an application of Mllt11 gene in preparation of glioma drugs and diagnostic kits thereof, and belongs to the technical field of precise cancer medical drugs. The invention utilizes immunohistochemistry technology to detect the expression condition of Mllt11 in glioma, and statistically analyzes the relationship between the differential expression condition of Mllt11 protein in proliferating brain tissue and each grade of glioma tissue and the clinical characteristics of patient and the survival time of patient, the result indicates that the expression of Mllt11 is obviously lower than that of normal tissue in glioma tissue, the expression in high grade glioma is obviously lower than that of low grade glioma, and the total survival time of patients in high expression group of Mllt11 is long and the prognosis is better in glioma patients through statistics. Therefore, the Mllt11 can be used as a target point of glioma cell gene diagnosis and treatment, and has wide application in preparing glioma diagnosis kits and drugs for treating glioma.
Description
Technical Field
The invention belongs to the technical field of precise cancer medical drugs, and particularly relates to an application of Mllt11 gene in preparation of a glioma drug and a diagnosis kit thereof.
Background
Glioma occurs in neuroectoderm, the degree of malignancy is high, and blood vessels in tumor tissues are rich, so that glioma grows rapidly, is easy to invade and relapse, has poor prognosis, the 5-year survival rate of adult malignant glioma is lower than 5%, and the incidence rate and the fatality rate of the adult malignant glioma in intracranial primary tumors are the highest. The current standard treatment method for glioma is the combination of surgical resection, radiotherapy and chemotherapy, namely Stupp standard synchronous radiotherapy and chemotherapy and Temozolomide (TMZ) auxiliary chemotherapy are adopted after surgery. Nevertheless, glioma is characterized by invasive growth, no obvious boundary with surrounding normal brain tissue and the like, and is difficult to completely excise in an operation, and the glioma is easy to generate drug resistance to chemotherapeutic drugs, so that the recurrence rate is still high, and the 5-year survival rate of patients is very low. Therefore, it is imperative to find new effective strategies to go out of this dilemma. At present, the biggest bottleneck restricting the development of brain glioma diagnosis and treatment is the deficiency of basic research. As the highest-level command center of the body, the central nervous system tumor has the characteristics of the central nervous system tumor, and the abnormal gene spectrum of the brain glioma is different from that of other system tumors, so that special research is required. With the development of tumor pharmacology and the progress of molecular biology research, molecular targeted therapy becomes a new method for treating malignant tumors except for surgery, radiotherapy and chemotherapy, and unlike traditional chemotherapy, targeted therapy prevents the growth of cancer cells by interfering with specific targeted molecules required by the occurrence of cancer and the growth of tumors, has the advantages of strong specificity, obvious curative effect, small adverse reaction and the like, the development of glioma is a complex process with participation of multiple genes, and each involved gene can become a target point for treatment.
The molecular pathology diagnosis and the targeted drug development are one of the hot spots of anti-tumor therapy, and a large number of researches and inventions are dedicated to the molecular targeted anti-tumor research so as to improve the anti-tumor effect of the drug and reduce the toxic and side effects of the drug. However, no glioma detection kit with a definite effect is available on the market, and the research and development of glioma targeting drugs mainly focuses on prolonging the survival time of patients. Glioma-associated immunity and targeted therapy are mainly active and passive, and passive immunotherapy includes antibody targeted therapy and adoptive immunotherapy. The targeted drugs of glioma mainly comprise VEGFR targeted drugs, bevacizumab is the targeted drug of brain glioma at present and is approved by FDA, but the target drug is flat in the aspect of prolonging the total survival period; MAPK-important signaling pathway-BRAF inhibitors, in children's low-grade gliomas with fewer gene mutations, BRAF inhibitors in combination with specific drug combinations (e.g., EGFR and Axl inhibitors) may be expected to maximize the benefit of BRAF V600E mutant glioma patients; MET inhibitors inhibit tumor growth of MET mutations. The result of a phase II clinical test of the mTOR inhibitor shows that the condition of adult recurrent low-grade glioma using the mTOR inhibitor tends to be stable, and the PFS in half a year is 84 percent and the PFS in one year is 47 percent. The results of phase III clinical trials of EGFR inhibitors indicate that the survival of newly diagnosed EGFRvIII-expressing glioblastoma patients is not prolonged after use, and that the immune efficacy of the EGFRvIII-expressing glioblastoma patients may need to be achieved by a combination of drugs. With the emergence of rapamycin as a first-generation mTOR inhibitor, a second-generation mTOR inhibitor and the like, the problem of drug resistance is accompanied, and the research of the third-generation mTOR inhibitor brings eosin to the problem of drug resistance. Nature reports the research result that Rapalik-1 can overcome the drug resistance generated by first-generation and second-generation mTOR inhibitors, shows that the Rapalik-1 can cross the blood brain barrier to inhibit three models tested in vivo in glioblastoma, has stronger effectiveness than rapamycin, can play a better treatment effect when being applied to the treatment of patients with brain glioma, and is still in continuous research. Besides the important research in the mature signal pathway, the BCL6 is a marker of prognosis of glioblastoma and a promoter of tumor development, and BCL6 gene silencing inhibits GBM cell growth to induce cell senescence, which is a potential therapeutic target of glioblastoma. Also as the EZH2 gene, it was found that inhibitors thereof are a treatment for pediatric gliomas carrying the potential H3K27M mutation. From the whole tumor field, as the molecular biological mechanism of the tumor is gradually uncovered, in the next decades, the treatment strategy of the tumor will be shifted from taking the tumor as the center to taking the biological immune system and the patient as the center; from the research and treatment progress of glioma, the immune regulation drug can fundamentally improve the immune suppression state of a microenvironment related to tumor cells, so as to promote the tumor cells to be killed by lymphocytes, therefore, the deep research of the immune therapy molecule targeted drug should be strengthened, and in the process of implementing the targeted drug research, special research should be implemented, so as to improve the research value.
Mllt11, located on human chromosome 1, chromosome q21, was originally identified as a mixed lineage leukemia fusion partner in acute myeloid leukemia patients with t (1; 11) (q 21; q23) chromosomal abnormalities in leukemia cells, as a poor prognostic biomarker for pediatric Acute Myeloid Leukemia (AML), adult normal cell hereditary leukemia, and adult myelodysplastic syndrome; mllt11 as a co-stimulatory factor in breast cancer may enhance expression of the STAT3 signaling pathway to promote tumor metastasis; in high-grade glioma, one of the obvious pathological features is tumor angiogenesis, and the angiogenesis factors in the tumor and the surrounding tissues are in a high-level expression state, so if the angiogenesis is reduced, the angiogenesis amount is reduced, and the growth of the tumor can be inhibited. In glioma, a JAK-STAT signal pathway is known as a signal pathway related to angiogenesis, while the action mechanism of Mllt11 in central nervous system malignant tumor and the immune mechanism in glioma microenvironment are still unknown, and the relation between Mllt11 and the prognosis of glioma patients is not reported at home and abroad.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide an application of Mllt11 gene in preparation of a medicament for detecting or treating glioma, and the invention aims to solve another technical problem in preparation of a kit for diagnosis or prognosis of glioma, wherein the Mllt11 gene is used as a marker gene.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
application of Mllt11 Gene (Gene number is Gene ID: 10962) in preparing medicine for detecting glioma.
The Mllt11 Gene is located on human chromosome 1, chromosome q21, and the sequence of the Gene is described in NCBI, and the Gene numbers are Gene ID: 10962. the amino acid sequence is MRDPVSSQYSSFLFWRMPIPELDLSELEGLGLSDTATYKVKDSSVGKMIGQATAADQEKNPEGDGLLEYSTFNFWRAPIASIHSFELDLL.
Application of Mllt11 gene in preparation of a kit for glioma diagnosis.
Application of the Mllt11 gene in preparation of a diagnostic kit for prognosis judgment of glioma.
Furthermore, the expression level of Mllt11 protein is detected, so that the expression level can be used for predicting the prognosis condition of clinical glioma patients.
Application of Mllt11 gene in preparing a medicament for treating glioma.
Furthermore, the medicine for treating glioma is designed by taking the Mllt11 gene as a target spot.
Has the advantages that: compared with the prior art, the invention has the advantages that:
the invention utilizes non-tumor proliferating brain tissue and a glioma patient tissue chip to carry out immunohistochemical staining during 2005-2013 years of university of Nantong subsidiary hospital, and statistically analyzes the relationship between the differential expression condition of Mllt11 protein in proliferating brain tissue and each grade of glioma tissue and the clinical characteristics of patients and the survival time of patients, and the result indicates that the expression of Mllt11 is obviously lower than that of normal tissue in glioma tissue, the expression of Mllt11 is obviously lower than that of low grade glioma in high grade glioma tissue, and the statistics of Mllt11 high expression group patients has long overall survival time and better prognosis in glioma patients. Therefore, the Mllt11 can be used as a target point of glioma cell gene diagnosis and treatment, and has wide application in preparing glioma diagnosis kits and drugs for treating glioma.
Drawings
FIG. 1 is a graph of Mllt11 expression in different grades of glioma; in the figure, a is WHO grade I glioma, b is WHO grade II glioma, c is WHO grade III glioma, and d is WHO grade IV glioma;
FIG. 2 is a statistical graph of the expression level of Mllt11 protein in non-neoplastic brain tissue and different grades of glioma;
FIG. 3 is a graph of the analysis and scoring of Mllt11 expression in tissues of different grade gliomas (WHO grades I, II, III, IV);
FIG. 4 is a graph of survival for patients with high and low expression of Mllt 11.
Detailed Description
The invention is further described with reference to specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. In the following examples, unless otherwise specified, all experimental procedures were carried out according to conventional methods.
Example 1
The main reagents used in the examples were: the two-step method immunohistochemical detection kit comprises: shanghai, Gene science and technology, Inc.; rabbit anti-human Mllt11 monoclonal antibody (for immunohistochemistry, abcam corporation); horseradish peroxidase-labeled goat anti-rabbit secondary antibody (for immunohistochemistry, new company, afzhou); antibody diluent (for immunohistochemistry, bi yun tian biotechnology limited); 0.01mol/L citric acid buffer (pH6.0) (for immunohistochemistry, Fuzhou Mixin Co.); DAB: fuzhou Mixin corporation; xylene, neutral gums, and the like are provided by the pathologist.
The main instruments used in the examples are as follows: tissue chip preparation instrument: beecher Instruments, USA; inverted phase contrast microscopy: olympus, japan; multispectral pathology scanning system: perkin Elmer, USA.
Original source of the sample: 126 glioma tissue section specimens and 25 brain benign disease section specimens, wherein all section tissues are taken from patients treated by 2005-2013 annual hospitalization surgery in the department of pathology of the subsidiary hospital of southern Tong university. All cases were pathohistologically confirmed by two pathologists, patients did not receive immunotherapy, chemotherapy or radiotherapy before surgery, and clinical cases were detailed and complete.
Immunohistochemistry samples: among 126 cases of glioma tissues, 33 cases of low-grade glioma and 93 cases of high-grade glioma; 47 women, 79 men; 81 cases under 60 years old and 45 cases over 60 years old. The 25 cases of benign brain tissues are gliosis, all tissue specimens are fixed by normal 10% neutral formalin and embedded by paraffin, wax blocks have no obvious defects after screening, and the tissue chips with the thickness of 4mm are manufactured by a pathology department and are stored in a refrigerator at 4 ℃ for later use.
1. Fabrication of tissue chips (tissue chips), also known as Tissue Microarrays (TMAs):
(1) marking representative cancer nest areas on the wax stone according to microscopic examination results of the HE stained sections;
(2) mixing paraffin wax and beeswax in the ratio of 1 to prepare a blank receptor wax block; designing 10 multiplied by 7 holes on a wax block, organizing an array with 350 points in total, and then manufacturing a TMA blank wax block by using an organizing chip instrument;
(3) selecting the most representative cancer nest area from the donor wax block on the marked point, and taking tissue blocks with the diameter of 2mm, wherein 1 core is taken in each case;
(4) transferring the tissue core to the hole of the receptor wax block, and taking the corresponding non-tumor hyperplasia tissue as a control;
(5) heating and fusing the tissue array block in a constant-temperature oven at 55 ℃ for 10 minutes, and cooling to room temperature before the tissue array block is melted quickly to fuse the receptor wax block and the donor tissue into a whole;
(6) freezing the tissue chip at 4 deg.C for about 4 hr, and correcting the tissue array block with a full-automatic tissue microtome at 20 mm/r until all tissue chips are completely exposed;
(7) slicing the tissue array block by using a slicer, floating the continuous slices in cold water respectively to naturally unfold the slices, transferring the slices to 45 ℃ warm water for unfolding for about 2 minutes, and pasting the slices on a slide glass treated by the anti-shedding sheet after unfolding for drying;
(8) baking the slices at 60 ℃ for 3 minutes, and continuing baking the slices at 58 ℃ for 16 hours;
(9) and storing the prepared tissue chip in a cutting box, and placing the cutting box in a refrigerating chamber of a refrigerator at 4 ℃ for later use.
2. Immunohistochemical staining (EnVision two-step method)
(1) Dewaxing and hydrating: the tissue chip is firstly put in a thermostat with the temperature of 60 ℃ and is baked for about 6 to 8 hours, so that the xylene is convenient for dewaxing; immersing the dried tissue chip in xylene for 30 minutes 2 times; taking out, and gradient dehydrating with 100% ethanol for 5min, 95% ethanol for 5min, 80% ethanol for 5min, 70% ethanol for 5min, and ddH 2 Washing the tissue chip with O water;
(2) antigen retrieval: placing the tissue chip on a high-temperature resistant slicing frame, placing the tissue chip in a citrate buffer solution with the pH value of 6.0, performing 100% high-temperature repair for 2.5 minutes and 20% high-temperature repair for 15 minutes, naturally cooling to room temperature, washing with PBS for 3 times, each time for 5 minutes, and finally using an immunohistochemical pen to circle out a tissue range;
(3) dropwise adding 30% H 2 O 2 Incubating for 20 minutes in the dark to eliminate the activity of endogenous peroxidase; washing with distilled water, soaking the chip in PBS buffer solution for 5min for 3 times, and taking out for drying;
(4) dripping 10% of confining liquid, placing at room temperature for 20 minutes, and then washing with PBS;
(5) diluting rabbit anti-human Mllt11 monoclonal antibody with primary anti-diluent (dilution ratio is 1: 150), adding 200 μ L onto tissue chip, and standing overnight at 4 deg.C;
(6) taking out the tissue chip on the next day, rewarming for 30 minutes, then putting the tissue chip into PBS buffer solution to soak for 5 minutes for 3 times in total, and then taking out and drying;
(7) dripping 200 mu L of a secondary antibody reinforcing agent on the tissue chip, incubating for 30 minutes at room temperature, placing the tissue chip in a PBS buffer solution for soaking for 5 minutes for 3 times in total, taking out and drying;
(8) dripping 200 mu L of secondary antibody, standing at room temperature for 30 minutes, placing the tissue chip in PBS buffer solution for soaking for 5 minutes for 3 times in total, taking out and drying;
(9) dripping prepared DAB working solution serving as a color developing agent, controlling the color development degree under a light mirror, immediately washing with distilled water after the color development is complete, and stopping the color development;
(10) counterdyeing: placing the glioma tissue section in hematoxylin for counterstaining for 30s, then slowly washing in tap water, then placing in hydrochloric acid-ethanol color separation liquid for about 2-3s, and finally slowly washing for 10 minutes by running water;
(11) and (3) dehydrating: preparing ethanol solutions with different concentration gradients (70% ethanol, 80% ethanol, 95% ethanol, and anhydrous ethanol), sequentially soaking for 3min × 1 times, 5min × 1 times, and 5min × 1 times, slightly drying the ethanol on the slices, and soaking in xylene solution for 5min × 2 times;
(12) sealing: a drop of neutral resin was placed in the center of the glioma section, covered with a glass cover slip and gently pressed, without air bubbles being generated during this process, and placed in a fume hood for air drying.
3. Result judgment
The immunohistochemical staining result is judged by scanning the stained tissue chip by a multispectral pathological scanning imaging system (Perkinelmer, USA), setting an analysis software program to automatically read and score, and the staining score is between 0 and 300. The final staining score for Mllt11 protein was the product of staining intensity and area of positive cell staining. The cut points for Mllt11 expression scores were derived by X-tile software. The scores were as follows: 0 to 100 is low expression or no expression, and 101 to 300 is high expression.
The results are shown in fig. 1, 2, 3, where Mllt11 is highly expressed in non-neoplastic brain tissue and low grade glioma tissue, and is less expressed in high grade glioma (fig. 1, 2); analysis of the Mllt11 expression score showed agreement with the immunohistochemical results (FIG. 3).
All data were processed with statistical software SPSS V.20.0 and STATA V.9.0, the metrology data were expressed as mean ± standard deviation, the comparisons between groups were performed using one-way anova, the analysis of the prognostic relationship between Mllt11 expression and glioma patients was performed using Kaplan-Meier survival analysis, and all test results P < O.05 were statistically significant for differences.
The results are shown in fig. 4, the glioma patients with high Mllt11 expression have long overall survival time and good prognosis; the overall survival time of glioma patients with low expression of Mllt11 was short with poor prognosis. The high expression of Mllt11 protein can be used as a prediction index for good prognosis of glioma.
Sequence listing
<110> auxiliary hospital of Nantong university
Application of <120> Mllt11 gene in preparation of glioma drug and diagnostic kit thereof
<130> 1
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 90
<212> PRT
<213> Mllt11(artificial)
<400> 1
Met Arg Asp Pro Val Ser Ser Gln Tyr Ser Ser Phe Leu Phe Trp Arg
1 5 10 15
Met Pro Ile Pro Glu Leu Asp Leu Ser Glu Leu Glu Gly Leu Gly Leu
20 25 30
Ser Asp Thr Ala Thr Tyr Lys Val Lys Asp Ser Ser Val Gly Lys Met
35 40 45
Ile Gly Gln Ala Thr Ala Ala Asp Gln Glu Lys Asn Pro Glu Gly Asp
50 55 60
Gly Leu Leu Glu Tyr Ser Thr Phe Asn Phe Trp Arg Ala Pro Ile Ala
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Ser Ile His Ser Phe Glu Leu Asp Leu Leu
85 90
Claims (4)
- Application of a detection reagent of Mllt11 protein in preparation of a medicine for detecting high-grade glioma.
- Use of a detection reagent for Mllt11 protein in the preparation of a kit for the diagnosis of high-grade glioma.
- Application of a detection reagent of Mllt11 protein in preparation of a diagnostic kit for prognosis judgment of glioma.
- 4. The use according to claim 3, for predicting the prognosis of a patient with clinical glioma by detecting the expression level of Mllt11 protein.
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