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CDC45 as marker of glioblastoma multiforme and application of CDC45 as therapeutic target

A glioblastoma, therapeutic target technology, applied in the biological field, can solve the problem of unclear regulation of downstream proteins, and achieve the effect of promoting proliferation

Inactive Publication Date: 2019-11-19
JINAN UNIVERSITY +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the specific function in GBM and its regulation on downstream proteins are still unclear.

Method used

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  • CDC45 as marker of glioblastoma multiforme and application of CDC45 as therapeutic target
  • CDC45 as marker of glioblastoma multiforme and application of CDC45 as therapeutic target
  • CDC45 as marker of glioblastoma multiforme and application of CDC45 as therapeutic target

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 The high expression of CDC45 in the tissues of patients with glioblastoma is significantly correlated with tumor grade and poor prognosis

[0036] In order to study the clinical manifestations of CDC45 protein in GBM, this example analyzed 148 cases of GBM clinical patient tissue chips by immunohistochemical staining, and determined the expression of CDC45 protein in GBM clinical patient tissues by analyzing the staining of tissue spots. Express the situation.

[0037] Determine the protein expression of different tissue points according to the depth of CDC45 immunohistochemical staining: First, score each tissue point according to the staining situation, such as figure 1 A No staining or very few brown spots is defined as 1, about 20%-50% of the cells are stained as 2, about 50%-75% of the cells are stained as 3, and more than 75% of the cells are stained as 4. The expression of CDC45 and tumor grade in GBM were analyzed according to the immunohistochemical ...

Embodiment 2

[0039] Example 2 Constructing a cell line stably overexpressing CDC45

[0040] In this example, a cell line overexpressing CDC45 was constructed in vitro to verify the functions of CDC45 on proliferation, migration and invasion of GBM cells.

[0041] The method for constructing a CDC45 overexpressing cell line is as follows:

[0042] 1. Primer Design

[0043] Find the mRNA sequence of human CDC45 gene (NM_000963.3) from GenBank (GenBank) and use the Primer blast plug-in in NCBI website to design a pair of primers that can efficiently amplify the CDS sequence of CDC45 gene. DNAman software was used to analyze the restriction enzyme cutting sites of CDC45 and lentiviral overexpression vectors and silencing vectors. Add appropriate restriction enzyme sites to the 5' ends of the upstream and downstream primers of the target gene according to the needs of the experiment, and add corresponding protective bases before the enzyme site.

[0044] Primers are:

[0045] F: CGGAATTCATGTT...

Embodiment 3

[0056] Example 3 CDC45 significantly promotes the proliferation, migration and invasion of GBM cells

[0057] In order to explore the specific function of CDC45 in GBM, the proliferation and growth of CDC45 overexpressed cell lines relative to control cells were detected by CCK8 method, and the migration and invasion ability of cells were detected by Transwell method.

[0058] 1) CCK8 detects cell proliferation

[0059] Take LN229 and U251 cells in good growth state and plate them (96-well plate), with 1000 cells per well for U251 cells and 800 cells per well for LN229 cells (100 μL of complete medium per well, if drug treatment is required, plate Add 50 μL of medium for cells, and add 50 μL of complete medium containing drugs for drug treatment). Set control wells and blank wells, 37°C, 5% CO 2 Cultivate overnight in a constant temperature incubator. 10 μL of CCK8 reaction solution was added to each well, protected from light during the addition process, and placed in a 37...

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PUM

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Abstract

The invention relates to the field of biotechnology, and specifically discloses CDC45 as a marker of glioblastoma multiforme and application of CDC45 as a therapeutic target. In-vitro and in-vivo experiments are combined, it is shown that CDC45 gene has high expression in glioblastoma multiforme(GBM) cells, and has significant relevance to tumor grade and poor prognosis of GBM, and proliferation,migration and invasion of the GBM cells can be promoted significantly through expression of CDC45. Based on the results, the invention further provides that expression products of the CD45 gene can beused as a marker of GBM, CD45 is used as a new target for targeted therapy of GBM, and drugs for down-regulation of the expression amount of CD45 gene are developed, so that a new treatment path andscheme are provided for glioblastoma multiforme.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to CDC45 as a glioblastoma marker and its application as a therapeutic target. Background technique [0002] Glioblastoma (GBM) is the most common and extremely aggressive brain tumor, mainly characterized by short survival (median survival time 12.5 months), high recurrence rate, high tumor heterogeneity and lack of effective treatment means. Standard treatment methods are very prone to drug resistance, which makes the residual tumor cells gradually insensitive to radiotherapy or chemotherapy, and radiotherapy and chemotherapy have huge side effects. On the premise of improving survival with limited prognosis, it is often accompanied by poor quality of life . Therefore, it is urgent to conduct in-depth research on the occurrence and development mechanism of GBM, and to find more accurate and effective therapeutic targets. [0003] CDC45 is a cell cycle protein that regulates...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886G01N33/574G01N33/68A61K45/00A61P35/00
CPCC12Q1/6886G01N33/57407G01N33/57484G01N33/68A61K45/00A61P35/00C12Q2600/158C12Q2600/118C12Q2600/136G01N2333/4739
Inventor 廖化新许雯雯李军旗王茜婷聂颖张建李成明
Owner JINAN UNIVERSITY
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