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Application of human URB1 gene and related products

A gene and application technology, applied in the direction of medical preparations containing active ingredients, genetic engineering, plant genetic improvement, etc., can solve problems such as the lack of URB1 gene

Active Publication Date: 2020-03-31
甘肃省人民医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There is no report about the use of URB1 gene in the treatment of colon cancer

Method used

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  • Application of human URB1 gene and related products
  • Application of human URB1 gene and related products
  • Application of human URB1 gene and related products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0118] Example 1 Preparation of RNAi lentivirus against human URB1 gene

[0119] 1. Screening for effective siRNA targets against human URB1 gene

[0120] Retrieve URB1 (NM_014825) gene information from Genbank; design effective siRNA targets for URB1 gene. Table 1-1 lists the screened effective siRNA target sequences against URB1 gene.

[0121] Table 1-1 siRNA target sequence targeting human URB1 gene

[0122] SEQ ID NO TargetSeq(5'-3') 1 GAACAGATTCACCGTAAAT 2 ACAGCCTTATCCCTTATTT

[0123] 2. Preparation of lentiviral vector

[0124] Synthesize double-stranded DNA Oligo sequences (Table 1-2) containing Age I and EcoR I restriction sites at both ends for siRNA targets (taking SEQ ID NO: 1 and 2 as examples); restrict with Age I and EcoR I The endonuclease acted on the pGCSIL-GFP vector (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd.) to make it linear, and the digested fragment was identified by agarose gel electrophoresis.

[012...

Embodiment 2

[0143] Example 2 Real-time fluorescent quantitative RT-PCR method to detect gene silencing efficiency

[0144] The human colorectal cancer cells HCT 116 cells and RKO cells in the logarithmic growth phase were respectively 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 (both HCT 116 cells and RKO cells are 10, and the following examples follow this infection multiplicity), add an appropriate amount of the lentivirus prepared in Example 1, and replace the medium after culturing for 24 hours. After reaching 5 days, the cells were harvested. 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 h...

Embodiment 3

[0152] Example 3 Western Blotting method to detect gene silencing efficiency

[0153] 1. Extraction of total cell protein

[0154] 1) Infect the target cells (HCT 116 cells and RKO cells) with the control virus and the RNAi virus targeting the URB1 interference target, respectively, according to the multiplicity of infection.

[0155] 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.

[0156] 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).

[0157] 4) Centrifuge at 12000g for 15min at 4°C, take the supernatant and use BCA Protein Assay Kit (manufacturer: Biyuntian, article number: P0010S) to measure the protein concentration.

[0158] 5) Add ...

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Abstract

The invention, which belongs to the field of biomedical research, particularly relates to application of a human URB1 gene as a target in preparation of a colorectal cancer treatment drug. The wide and deep research discovers that proliferation of colorectal cancer cells can be effectively inhibited and apoptosis can be promoted after expression of the human URB1 gene is down-regulated by adoptingan RNAi method, thereby effectively controlling the growth process of the colorectal cancer. The siRNA or a nucleic acid construct containing the siRNA sequence and the lentivirus can specifically inhibit the proliferation rate of colorectal cancer cells, promote apoptosis of the colorectal cancer cells, inhibit cloning of the colorectal cancer cells and inhibit growth of the colorectal cancer, so that the colorectal cancer is treated. A novel direction is opened up for colorectal cancer treatment.

Description

technical field [0001] The invention belongs to the field of biomedical research, and specifically relates to the use of human URB1 gene and related products. Background technique [0002] Ribosome synthesis homolog URB1 (URB1 ribosome biogenesis homolog) is a ribosomal protein, also known as nucleolar ribosome precursor-associated protein 1 (NPA1 Nucleolar Pre-ribosomal-associated protein1). Ribosomes are essential for cell division and proliferation, and inhibition of normal ribosome production can lead to various diseases. Recent studies have found that URB1 can regulate the development of zebrafish digestive organs by intervening downstream of the mTORC1 signaling pathway. The abnormal expression of Urb1 will affect the proliferation of digestive organ cells, and the clinical manifestation is dysplasia of digestive organs. Inhibition of mtor and rptor will result in low expression of Urb1, leading to loss of normal synthesis function of digestive organs (doi:10.1016 / j....

Claims

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Application Information

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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 王涛郭天康
Owner 甘肃省人民医院
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