Application of human B3GNT5 gene and related product

A gene and application technology, applied in the application of human B3GNT5 gene and related products, can solve problems such as the lack of B3GNT5 gene

Active Publication Date: 2020-03-31
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no relevant report on the use of B3GNT5 gene in the treatment of liver cancer

Method used

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  • Application of human B3GNT5 gene and related product
  • Application of human B3GNT5 gene and related product
  • Application of human B3GNT5 gene and related product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Example 1 Preparation of RNAi lentivirus for human B3GNT5 gene

[0108] 1. Screening for effective siRNA targets against the human B3GNT5 gene

[0109] Retrieve B3GNT5 (NM_032047) gene information from Genbank; design effective siRNA targets for B3GNT5 gene. Table 1-1 lists the screened effective siRNA target sequences against the B3GNT5 gene.

[0110] Table 1-1 is targeted at the siRNA target sequence of human B3GNT5 gene

[0111] SEQ ID NO TargetSeq(5'-3') 1 TTGGAAGAATGCTACAGAT

[0112] 2. Preparation of lentiviral vector

[0113] Aim at the siRNA target (take SEQ ID NO: 1 as an example) to synthesize a double-stranded DNA Oligo sequence (Table 1-2) with Age I and EcoR I restriction sites at both ends; Dicer acts on the pGCSIL-GFP vector (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd.) to linearize it, and agarose gel electrophoresis identifies the digested fragment.

[0114] Table 1-2 Double-stranded DNA Oligo with sticky end...

Embodiment 2W

[0132] Embodiment 2Western Blotting method detects the silencing efficiency of gene

[0133] 1. Extraction of total cell protein

[0134] 1) The control virus and the RNAi virus targeting the B3GNT5 interference target were used to infect the target cells according to the multiplicity of infection (the multiplicity of infection of BEL-7404 human liver cancer cells was 10, and the multiplicity of infection of SMMC-7721 human non-small cell liver cancer cells was 20 , the following examples all use this multiplicity of infection).

[0135] 2) After 5 days of infection, collect the 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.

[0136] 3) 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 (20 times at 40W, 1 s each time, 2 s apart). ...

Embodiment 3

[0162] Example 3 Real-time fluorescence quantitative RT-PCR method to detect gene silencing efficiency

[0163] The human liver cancer BEL-7404 cells in the logarithmic growth phase were trypsinized to make a cell suspension (the number of cells was about 5×10 4 / ml) were inoculated in a 6-well plate and cultured until the cell confluency reached about 30%. According to the infection multiplicity value, an appropriate amount of the lentivirus prepared in Example 1 was added, the culture medium was replaced after 24 hours of culture, and the cells were collected after the infection time reached 5 days. Total RNA was extracted according to the instruction manual of Invitrogen's Trizol. According to the M-MLV instruction manual of Promega Company, RNA was reverse-transcribed to obtain cDNA (see Table 2-1 for the reverse transcription reaction system, react at 42°C for 1 hour, and then bathe in a water bath at 70°C for 10 minutes to inactivate the reverse transcriptase).

[0164...

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Abstract

The invention, which belongs to the field of biomedical research, particularly relates to application of a human B3GNT5 gene as a target in preparation of a liver cancer treatment drug. The wide and deep research founds that proliferation of liver cancer cells can be effectively inhibited and the apoptosis can be promoted effectively after expression of the human B3GNT5 gene is down-regulated by adopting an RNAi method, so that the growth process of the liver cancer is effectively controlled. The siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus can specifically inhibit the proliferation rate of liver cancer cells, promote apoptosis of the liver cancer cells, inhibit cloning of the liver cancer cells and inhibit growth of the liver cancer, so that the liver cancer is treated. A novel direction is opened up for liver cancer treatment.

Description

technical field [0001] The invention belongs to the field of biomedical research, and specifically relates to the use of human B3GNT5 gene and related products. Background technique [0002] The B3GNT5 gene encodes a member of the β-1,3-N-acetylglucosaminyltransferase family, which links N-acetylglucosamine to lactosylaminoceramide to generate the precursor lactosylceramide (LC3), which is used in Synthesis of type-1 and type-2 lactosamides (doi:10.1074 / jbc.M011369200). This enzyme is essential for the expression of Lewis X epitopes on glycolipid sugars. B3GNT5 and its related glycoside product (LC3) play roles in human malignant diseases, embryonic development and cell differentiation. [0003] B3GNT5 is associated with breast cancer subtype and survival (PMID:25655580). In cisplatin-resistant ovarian cancer cell lines, high expression of B3GNT5 was found by expression profile chip. The expression of B3GNT5 is correspondingly elevated in patients with acute myeloid leuk...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/867A61K45/00A61K31/713A61P35/00C12Q1/6886
CPCC12N15/113C12N15/867A61K45/00A61K31/713A61P35/00C12Q1/6886C12Q2600/136C12Q2600/158C12N2740/15043C12N2800/107C12N2310/14C12N2310/531Y02A50/30
Inventor 陶开山汪建林张洪涛彭伟李艺杰林志斌杨龙
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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