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Application of malignant glioma biomarker

A malignant glioma and target technology, applied in the field of biotechnology, can solve the problems of no related reports

Pending Publication Date: 2020-11-27
THE FIRST AFFILIATED HOSPITAL OF WANNAN MEDICAL COLLEGE YIJISHAN HOSPITAL OF WANNAN MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the expression of RHOJ in malignant glioma and its application in diagnosis, treatment and prognosis have not been reported so far.

Method used

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  • Application of malignant glioma biomarker
  • Application of malignant glioma biomarker
  • Application of malignant glioma biomarker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] We performed RHOJ antibody (Sigma, HPA003050) immunohistochemical staining on the clinical tissue sections collected from patients with malignant glioma: put the collected pathological sections in xylene I for 5 minutes, replace them with xylene II for 5 minutes, and then soak them in absolute ethanol for 3 minutes , 95% ethanol for 3 minutes, 80% ethanol for 3 minutes, 70% ethanol for 3 minutes, tap water for 3 minutes for dewaxing and hydration, placed in antigen retrieval solution (citrate buffer, pH 6.0) and boiled at 95-98°C for 15 minutes, naturally Cool to room temperature for antigen retrieval, wash 3 times with PBST buffer, 3 min each time. Add goat serum blocking solution dropwise, room temperature for 30min. Shake off the blocking solution, add primary antibody (RHOJ, 1:400), overnight at 4°C. Wash 3 times with PBST buffer for 3 min each time, add secondary antibody dropwise, room temperature for 1-2 h, wash 3 times with PBST buffer for 3 min each time, deve...

Embodiment 2

[0022] The siRNA that specifically inhibits RHOJ is based on the RHOJ sequence and the siRNA online design website provided by Invivogen (https: / / link.zhihu.com / ?target=http%3A / / www.invivogen.com / sirnawizard / siRNA.php) Two siRNAs were designed to prevent off-target effects. siRNA was synthesized and provided by Shanghai Gemma Pharmaceutical Technology Co., Ltd.

[0023] Construction of shRNA that specifically inhibits RHOJ and plasmids that overexpress RHOJ. The specific shRNA sequence was obtained from the official website of sigma company, and inserted into the PLKO.1 plasmid vector to construct the shRHOJ expression plasmid. The CDS region of the RHOJ gene was obtained from Pubmed, and primers were designed. The primers were synthesized and provided by GenScript Biotechnology Co., Ltd., and the full-length sequence of RHOJ was obtained by PCR cloning, and inserted into the eukaryotic expression vector plenti-CMV-GFP-puro. Transform the shRHOJ plasmid and the RHOJ expressi...

Embodiment 3

[0041] For Transwell migration assay, the cells were starved overnight in serum-free medium, then trypsinized, suspended in serum-free DMEM medium, and counted to adjust the concentration to 2×10 4 / mL; add 600 μL of DMEM complete medium containing 10% serum to the bottom of the 24-well plate, add 200 μL of cell suspension to the upper chamber, and then put it in the incubator for culture; 24 hours later, carefully take out the small chamber with tweezers, and suck off the upper chamber Liquid, add about 600 μL of 4% paraformaldehyde, fix at room temperature for 30 minutes; suck off the 4% paraformaldehyde in the upper chamber, add about 600 μL of crystal violet, and stain at room temperature for 30 minutes; wash gently with PBS several times, take out the small chamber, and suck out the upper chamber. The liquid in the upper chamber was carefully wiped off with a wet cotton swab; 5 fields of view were randomly selected under a 40x light microscope for observation, counting, and...

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Abstract

The invention relates to application of a malignant glioma biomarker, in particular to application of RHOJ mRNA shown in SEQ ID No:1 and RHOJ protein shown in SEQ ID No:2 as detection targets in preparation of a kit for diagnosing, treating or prognostically judging malignant glioma. We find that silence of RHOJ expression significantly inhibits malignant phenotypes such as glioma cell proliferation and invasion, RHOJ can be used as a molecular marker for malignant glioma diagnosis and prognostic judgment, and treatment strategies such as an RHOJ-targeting antibody or small molecule inhibitorare expected to improve malignant glioma treatment.

Description

technical field [0001] The invention belongs to the fields of biotechnology, pathological examination and oncology, and in particular relates to the application of a malignant glioma biomarker. Background technique [0002] Glioblastoma (GBM) is the most common fatal intracranial primary malignant tumor among central nervous system tumors. The incidence of glioma is 5 / 100,000-8 / 100,000. Second only to pancreatic cancer and lung cancer, it ranks third. Its main features are abundant angiogenesis and diffuse infiltrative growth, difficult to pathologically complete resection, easy recurrence after operation, and resistance to radiotherapy and chemotherapy. Glioma can be divided into grades Ⅰ-Ⅳ according to the pathological malignancy of tumor cells. Low-grade gliomas (LGG) are mainly WHO grade II and III gliomas, while high-grade gliomas (GBM) are mainly WHO grade IV gliomas. With the development of molecular biology and molecular pathological diagnosis, the molecular typ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886G01N33/574G01N33/573A61K45/00A61P35/00
CPCC12Q1/6886G01N33/57407G01N33/57496G01N33/573A61K45/00A61P35/00C12Q2600/158C12Q2600/118G01N2333/914
Inventor 夏洪平江晓春王梅张成飞徐昊珺
Owner THE FIRST AFFILIATED HOSPITAL OF WANNAN MEDICAL COLLEGE YIJISHAN HOSPITAL OF WANNAN MEDICAL COLLEGE
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