Application of substance for reducing USP1 expression in preparation of medicine for treating children T-series acute lymphoblastic leukemia

A technology of acute lymphocytes and expression cassettes, applied in the field of biomedicine

Active Publication Date: 2020-11-13
BEIJING CHILDRENS HOSPITAL AFFILIATED TO CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the mechanism of action of USP1 in the development of T-ALL, especially in children, has not been reported yet.

Method used

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  • Application of substance for reducing USP1 expression in preparation of medicine for treating children T-series acute lymphoblastic leukemia
  • Application of substance for reducing USP1 expression in preparation of medicine for treating children T-series acute lymphoblastic leukemia
  • Application of substance for reducing USP1 expression in preparation of medicine for treating children T-series acute lymphoblastic leukemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Overexpression of the deubiquitinating enzyme USP1 in childhood T-ALL

[0058] 1. mRNA level overexpression

[0059] The NCBI GEO database (https: / / www.ncbi.nlm.nih.gov / geo) downloaded the gene expression profile results (GSE13159) of bone marrow samples from healthy children and children with T-ALL. Using T-Test analysis, we found that in The expression level of USP1 mRNA in children's T-ALL bone marrow samples (n=169) was significantly higher than that in healthy children's bone marrow samples (n=73, pfigure 1 Shown in A.

[0060] 2. Protein level overexpression

[0061] Use a heparin anticoagulant tube to extract 2ml of bone marrow cells from children with T-ALL, add 5ml of erythrocyte lysate and mix well, let it stand for 2 minutes, then centrifuge at 100g for 5 minutes, discard the supernatant and repeat the above steps, and then use normal saline to dilute the bottom of the tube. Wash the cells twice, remove the supernatant, and collect the cells.

...

Embodiment 2

[0064] Example 2: Construction of RNA interference recombinant expression vector of deubiquitinating enzyme USP1

[0065] 1. Selection of RNA interference target sequence

[0066] For the full-length cDNA sequence (SEQ ID NO: 14) of the deubiquitinating enzyme USP1 encoding gene USP1, the following two DNA sequences were selected as target sequences for RNA interference:

[0067] sh-1: the 2574-2594th position of SEQ ID NO: 14 (ie 5'-CCAGTGACCAAACAGGCATTA-3')

[0068] sh-2: the 2030-2050th position of SEQ ID NO: 14 (ie 5'-GCTAGTGGTTTGGAGTTTGAT-3')

[0069] Positions 422-2779 in SEQ ID NO:14 are open reading frames encoding the deubiquitinating enzyme USP1 shown in SEQ ID NO:15.

[0070] 2. Small interfering RNA (siRNA)

[0071] Two siRNAs, siRNA-1 and siRNA-2, were respectively designed for the two target sequences of the deubiquitinating enzyme USP1 in step 1, wherein the target sequence of siRNA-1 was sh-1, and the target sequence of siRNA-2 was sh- 2.

[0072] siRNA-1 ...

Embodiment 3

[0112] Example 3: Deubiquitinase USP1 RNA interference recombinant expression vector lentivirus infection of leukemia cells and Western blot detection of the expression of deubiquitinase USP1 in recombinant leukemia cells

[0113] 1. Deubiquitinating enzyme USP1 RNA interference recombinant expression vector lentivirus infection of leukemia cells

[0114] (1) RNA interference recombinant expression vector lentiviral packaging

[0115] The target gene GV493-USP1-sh-1, GV493-USP1-sh-2 or control sequence GV493-sh-Control plasmid, virus packaging helper plasmids pHelper 1.0 and pHelper2.0 (purchased from Shanghai Jikai Gene Medicine Technology Co., Ltd. ) to co-transfect 293T cells, and extract the supernatant 48-72 hours after the transfection to harvest the virus. Centrifuge at 4°C and 4000g for 10 minutes to remove cell debris; filter the supernatant with a 0.45 μm filter and put it into a 40ml ultracentrifuge tube; balance the samples separately, and put the ultracentrifuge ...

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Abstract

The invention discloses application of a substance for reducing USP1 expression in preparation of a medicine for treating children T-series acute lymphocytic leukemia. The invention discloses short hairpin RNA (Ribonucleic Acid) for forming a stem-loop structure. The short hairpin RNA is shRNA-1 or shRNA-2. The deubiquitination enzyme USP1 has the effects of inhibiting cell apoptosis and promotingcell proliferation in T-series acute lymphocytic leukemia cells, and the invention proves that inhibition of USP1 expression can promote apoptosis of tumor leukemia cells and inhibit proliferation ofleukemia cells. The invention provides a novel way for the treatment of T-series acute lymphocytic leukemia, and the deubiquitinase USP1 is expected to become a potential target for anti-leukemia treatment and has a very wide application prospect in the medical field.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to the application of a substance for reducing the expression of deubiquitinating enzyme USP1 in the preparation of medicines for treating T-lineage acute lymphoblastic leukemia in children. Background technique [0002] Acute lymphoblastic leukemia (ALL) is the most common malignancy in children and the leading cause of childhood disease-related death. T-lineage acute lymphoblastic leukemia (T-ALL) is a very aggressive subtype of ALL, and up to 30% of children with T-ALL are ineffective or relapse. The unknown mechanism of occurrence and development is the bottleneck for further improvement of the cure rate of T-ALL. Therefore, in-depth study of the molecular mechanism of T-ALL in children, especially the development of refractory and relapsed T-ALL, is crucial for exploring new targeted treatment options and improving survival rates. [0003] USP1 (Ubiquitin-specific peptidase 1), that i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/11A61K31/7088A61K31/713A61K45/00A61K31/506A61P35/02
CPCC12N15/113A61K31/7088A61K31/713A61K45/00A61K31/506A61P35/02C12N2310/14C12N2310/531
Inventor 刘曙光高超郑胡镛张瑞东
Owner BEIJING CHILDRENS HOSPITAL AFFILIATED TO CAPITAL MEDICAL UNIV
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