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Uses and related products of human UBE2S gene

A technology of genes and uses, applied in medical preparations containing active ingredients, genetic engineering, plant genetic improvement, etc.

Pending Publication Date: 2020-06-05
新疆医科大学第三附属医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Ubiquitin-conjugating enzyme E2S (ubiquitin-conjugating enzyme E2S, UBE2S) is the key to extending the ubiquitin chain to the 26S proteasome, and there is no report on the relationship between UBE2S and glioma

Method used

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  • Uses and related products of human UBE2S gene
  • Uses and related products of human UBE2S gene
  • Uses and related products of human UBE2S gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Example 1 Upregulation of UBE2S expression in glioma tissue

[0097] Firstly, the expression level of UBE2S in glioma tissue and normal brain tissue was detected.

[0098] 1. Extraction of total RNA (extraction using Trizol kit from Shanghai Pufei Company)

[0099] (1) Collect the sample and crack it with Trizol.

[0100] Cell samples: collect cells (6-well plate with 80% cell density), centrifuge at 2000rpm for 5min, remove the supernatant, add 1mL Trizol to the cell pellet, mix well, let stand at room temperature for 5min, and then transfer to a new 1.5mL EP tube;

[0101] Tissue samples: Take the tissue samples to be ground out of liquid nitrogen or -80°C refrigerator, put a sterile blade on dry ice, cut the tissue samples into about 3mm×3mm×3mm in size, and place them in a 1.5 mL EP tube. Immerse the working head of the ultra-fine homogenizer in Trizol lysate for 5-10 seconds to inactivate RNase, then grind the tissue; after grinding, centrifuge at 5000 rpm for 3...

Embodiment 2

[0127] Example 2 Construction of UBE2S stable knockdown cell line

[0128] We screened two knockdown knockout targets (SEQ ID NO: 1 and 2), and used shRNA lentivirus to infect the glioma cell line U87 cell line according to the multiplicity of infection.

[0129] 1. Construction and packaging of UBE2S gene RNA interference lentiviral vector

[0130] Using the UBE2S gene as a template, the RNA interference target sequence (SEQ ID NO: 1 and 2) was designed to construct the target gene RNA interference lentiviral vector. After completing the design of the RNA interference target, synthesize a single-stranded DNA oligo containing the interference sequence, anneal and pair to generate a double-stranded DNA; then directly ligate into the cleaved lentiviral vector through the restriction sites at both ends; transfer the ligated product into Prepared E. coli competent cells, positive recombinants were identified by PCR, sent for sequencing verification, and plasmid extraction was per...

Embodiment 3

[0195] Example 3 Western-blot detection of expression level of UBE2S in stable cell lines

[0196] 1. Extraction of total cell protein

[0197]1) Collect stably transfected cell samples, take an appropriate amount of RIPA lysate (Beiyuntian, P0013C), and add PMSF within a few minutes before use, so that the final concentration of PMSF is 1mM.

[0198] 2) Add an appropriate amount of RIPA lysate, and lyse on ice for 10-15 minutes. Scrape the cells and transfer them into a new EP tube, and then ultrasonically disrupt the cells (20b times at 40W, 1s each time, 2s interval).

[0199] 3) Centrifuge at 12000g for 15min at 4°C, take the supernatant and use BCA Protein Assay Kit (manufacturer: Biyuntian, product number: P0010S) to measure the protein concentration.

[0200] 4) Add new lysate to adjust the protein concentration of each sample to be consistent, generally 2 μg / μL. Then add 1 / 5 volume of 6×lodding buffer and mix well, cook in a metal bath at 100°C for 10 minutes, centr...

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Abstract

The present invention relates to the field of biomedical research and particularly to uses of a human UBE2S gene as a target in preparation of glioma therapeutic drugs. Extensive and in-depth researches find that after an RNAi method down-regulates expression of the human UBE2S gene, proliferation of glioma cells can be effectively inhibited, cell apoptosis is promoted and a growth process of gliomas is effectively controlled. A provided shRNA or a nucleic acid construct containing the shRNA sequence and a lentivirus containing the shRNA sequence can specifically inhibit proliferation rate ofthe glioma cells, promoteapoptosis of the glioma cells, inhibit cloning of the glioma cells, inhibit growth of the gliomas, thus treat the gliomas and open up new directions for treatment of the gliomas.

Description

technical field [0001] The invention relates to the field of biomedical research, in particular to the use of human UBE2S gene and related products. Background technique [0002] Glioma is one of the most common primary central nervous system tumors, accounting for about 44% of primary central nervous system tumors, with a high recurrence rate and poor prognosis. In particular, the overall survival (OS) of glioblastoma (GBM) is 16-18 months, and the 5-year survival rate is about 5%. Its 5-year mortality rate ranks second only to pancreatic cancer and lung cancer. three. At present, the pathogenesis of glioma is still unclear. Epidemiological studies have found that ionizing radiation (including radiation therapy and exposure to low-dose radiation such as x-rays, scans and imaging) has a significantly increased risk of glioma. Surgical resection, radiotherapy, chemotherapy and other methods are currently important methods for the clinical treatment of glioma. In recent ye...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61K31/713A61K31/7088A61P35/00C12N15/113C12N15/867
CPCA61K31/7088A61K31/713A61K45/00A61P35/00C12N15/1137C12N15/86C12N2310/14C12N2740/15043C12Y603/02019C12N2310/531
Inventor 郭文佳董晓刚
Owner 新疆医科大学第三附属医院
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