Application of DDX24 and upstream and downstream molecules RFX8 and Lamb1 thereof in diagnosis and treatment of hepatocellular carcinoma

A DDX24, 1.DDX24 technology, applied in the field of diagnosis and treatment of hepatocellular carcinoma, can solve the problems of lack of disease research and treatment methods, and unclear precise mechanism of action

Active Publication Date: 2020-12-29
THE FIFTH AFFILIATED HOSPITAL SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Our previous study found that mutations in DDX24 (glutamate to lysine at position 271) are associated with "multiorgan venous lymphatic defect syndrome" (MOVLD), a disease involving multiple organs; prior art disclosures, DDX24 may be involved in the occurren

Method used

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  • Application of DDX24 and upstream and downstream molecules RFX8 and Lamb1 thereof in diagnosis and treatment of hepatocellular carcinoma
  • Application of DDX24 and upstream and downstream molecules RFX8 and Lamb1 thereof in diagnosis and treatment of hepatocellular carcinoma
  • Application of DDX24 and upstream and downstream molecules RFX8 and Lamb1 thereof in diagnosis and treatment of hepatocellular carcinoma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Correlation between the expression of DDX24 in hepatocellular carcinoma tissue and the progression of HCC

[0069] 1. Experimental process

[0070] 1.1. Immunohistochemical (IHC) staining

[0071] Liver cancer tissue samples were collected from the Fifth Affiliated Hospital of Sun Yat-sen University. Tissues were fixed overnight in 10% neutral formalin, dehydrated using an increasing concentration gradient, and embedded in low melting point paraffin. Serial 4 micron thick tissue sections were cut and mounted on siliconized glass slides. Immunohistochemical staining used a streptavidin-peroxidase conjugated method. Each tissue section was deparaffinized, rehydrated, immersed in antigen retrieval solution, boiled at 100°C for 10 min, and incubated with peroxidase inhibitors. After blocking non-specific binding with normal goat serum, tissue sections were incubated overnight at 4°C with the following primary antibodies: anti-DDX24 and anti-HPA002554 (Sigma, U...

Embodiment 2

[0099] Example 2 DDX24 regulates the EMT signaling pathway of HCC migration and invasion

[0100] 1. Experimental process

[0101] 1.1. Construction of lentivirus and cell infection

[0102] Cells were infected with a lentiviral vector (U6-sh-DDX24-EGFP-IRES-puromycin) purchased from GeneCopoeia (Rockville, Maryland, USA) to establish DDX24 knockdown or stable overexpression cell lines. HCC cells were infected with recombinant lentiviral transduction units by 0.8 μg / ml polyethylene, and stable cells were selected by 1 ug / ml puromycin two weeks later, and the efficiency of RNA interference was evaluated by qRT-PCR and Western blotting.

[0103] 1.2. RNA sequencing (RNA-seq)

[0104] Total cellular RNA was purified using TRIzol reagent (Invitrogen, Carlsbad, CA). Each set of samples was then used to construct RNA-seq strand-specific libraries and sequenced using the NuGEN Ovation RNA-Seq system. The counts per gene were normalized into FPKM (fragments per kilobase per millio...

Embodiment 3

[0116] Example 3 DDX24 regulates the ERK / AKT signaling pathway of cell proliferation and tumor growth

[0117] 1. Experimental process: with embodiment 1 or embodiment 2.

[0118] 2. Experimental results

[0119] Through CCK-8 and clone formation experiments, it was found that reducing the expression of DDX24 could inhibit the proliferation of liver cancer cells in vitro ( Figure 6 a) and clone formation ( Figure 6 b). We also observed in a xenograft mouse model that DDX24 deficiency significantly suppressed subcutaneous tumor size compared to controls ( Figure 6 c-6e) and weight ( Figure 6 f). In addition, the phosphorylation levels of cell proliferation pathway factors AKT and ERK were also significantly down-regulated in knockdown DDX24 cells ( Figure 6 g). These results suggest that DDX24 regulates HCC proliferation and tumor growth through the ERK / AKT signaling pathway.

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Abstract

The invention belongs to the technical field of medical products, and particularly relates to application of DDX24 and upstream and downstream molecules RFX8 and Lamb1 thereof in diagnosis and treatment of hepatocellular carcinoma. Researches find that compared with paracancerous normal tissues, primary hepatic carcinoma tissues are increased in DDX24 expression level, and the increased level is related to tumor size; and survival analysis shows that RFX8 can up-regulate DDX24 expression at the transcription level, and miR-526a-5p can be directly combined with mRNA of DDX24 and degrade the mRNA of DDX24. According to the application, DDX24 can promote the development of HCC through a downstream LAMB1 pathway, and the research of the pathway is expected to provide a theoretical basis and anexperimental basis for the diagnosis and treatment of HCC.

Description

technical field [0001] The present invention relates to the technical field of medical products, and more specifically, relates to the application of DDX24 and its upstream and downstream molecules RFX8 and Lamb1 to the diagnosis and treatment of hepatocellular carcinoma. Background technique [0002] Hepatocellular carcinoma (HCC) is the most common and extremely aggressive type of malignant primary liver cancer. Although the treatment of HCC has made progress in recent years, the recurrence and metastasis of HCC are still inevitable, and the 5-year survival rate of patients with advanced HCC is only 25%. Therefore, it is a huge challenge to study the pathogenesis of HCC, realize the precise targeted therapy of HCC, improve the accuracy of HCC clinical prognosis prediction, and improve the quality of life of patients. [0003] The DEAD-box family, characterized by the conserved Asp-Glu-Ala-Asp (DEAD) motif, is the largest RNA helicase family found in humans, and it contain...

Claims

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Application Information

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IPC IPC(8): G01N33/574G01N33/573G01N33/68C12Q1/6886C12Q1/6851A61K45/00A61P35/00A61P35/04A61K31/7088A61K31/7105
CPCA61K31/7088A61K31/7105A61K45/00A61P35/00A61P35/04C12Q1/6851C12Q1/6886C12Q2600/118C12Q2600/158G01N33/573G01N33/57438G01N33/57484G01N33/6854G01N2333/78G01N2333/914G01N2800/52C12Q2531/113C12Q2561/113
Inventor 单鸿何欢欢刘天泽甘海润何思蒙
Owner THE FIFTH AFFILIATED HOSPITAL SUN YAT SEN UNIV
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